Patent Application
20210010085
Incorporated herein by reference in its entirety is the Sequence Listing submitted via EFS-Web as a text file named SequenceListing.txt., created Sep. 10, 2020 and having a size of 68,024 bytes.
The present invention relates to materials and methods for stratifying and treating cancers and to methods of identifying/selecting patients for treatment of cancer with tyrosine kinase inhibitors.
Cancer is a complex and dynamic disease, and many different ways of analysing and classifying tumours have been developed with the aims of determining the prognosis for the patient, and informing treatment decisions.
Pazopanib is an oral multi-target tyrosine kinase inhibitor (TKI) with a clinical anti-tumour effect that is thought to be exerted through its selective inhibition of VEGFR-mediated angiogenesis as well as direct blockade of growth-promoting receptor tyrosine kinases (RTKs) that include platelet-derived growth factor receptors (PDGFRs), fibroblast growth factor receptors (FGFRs) and KIT1-5. Pazopanib is the first and currently only TKI licensed for the treatment of many subtypes of advanced soft tissue sarcoma (STS) This approval was based on the results of the PALETTE study that randomised 369 patients with pre-treated advanced STS to receive either pazopanib 800 mg once daily or placebo until disease progression32. After a median follow-up of 25 months, a clinically significant improvement in progression-free survival (PFS) was seen in the pazopanib arm (median PFS 4.6 v. 1.6 months; HR 0.31; 95% CI 0.24-0.40; p<0.0001). Despite this evidence of anti-tumour effect, no significant difference in overall survival (OS) was observed between pazopanib and placebo-treated patients. The failure of PFS gain to translate to OS benefit has adversely influenced cost assessment of pazopanib for this indication, leading to funding limitations in certain health economies worldwide6,7.
The development of biomarkers capable of identifying patients most likely to benefit from a therapy is central to the notion of personalised cancer treatment. There is currently an unmet need for predictive biomarkers that are successful in prospectively selecting the subgroup of STS patients most likely to benefit from pazopanib, thus improving the clinical efficiency of the drug. The presence of such a patient subgroup was indicated in a pooled analysis of patients who received the drug within the PALETTE trial or its antecedent EORTC phase II study8. In this retrospective report of unblinded, patient-level data, 76 of 344 analysed patients (22%) experienced PFS greater than 6 months and OS greater than 18 months. No STS histological subtype was identified as being enriched in these outstanding responders. In the single arm phase II trial, prospective stratification of patients into one of four histotype-defined subgroups saw patients with adipocytic tumours fail to meet predefined efficacy cut-off, with a 12 week progression-free survival of 26%9. The leiomyosarcoma, synovial sarcoma and ‘other’ histotype subgroups, however, all showed sufficient evidence of pazopanib response, with these histological subtypes taken forward for phase III investigation. The efficacy of pazopanib in several of the rare STS subtypes encompassed within the heterogeneous ‘other subtypes’ subgroups has been further explored in a number of post-licensing retrospective series10-14. Whilst these studies provide further indication of pazopanib activity across a range of STS diagnoses, none of these rarer subtypes have been found to exhibit particular sensitivity relative to the general STS populations treated in phase II and III studies.
It is possible that there are aspects of tumour biology targeted by pazopanib that are shared by individual cases across different STS subtypes, presenting a potential avenue for biomarker discovery. This is supported by translational research that have identified genomic and gene expression signatures that are able to describe patient subgroups of distinct clinical phenotype both across and within STS subtypes15,16. In a 19 patient cohort of advanced STS treated with pazopanib or related TKIs, Koehler et al found that the presence of TP53 mutations was associated with significantly improved PFS compared to cases with TP53 wildtype tumours17. In a phase I trial of pazopanib in combination with the histone deacetylase inhibitor vorinostat, TP53 hotspot mutations were found in 3 of 11 tested sarcoma patients18. In this study, TP53 mutation was significantly associated with improved disease control and PFS across all tested patients, and also improved OS in a subset with either sarcoma or colorectal cancer. Meanwhile, our laboratory has recently shown that, in malignant rhabdoid tumour cells with basal pazopanib sensitivity, acquired drug resistance is mediated by modulation of PDGFRA and FGFR1 signalling19.
It therefore remains a problem in the art to identify biomarkers for classifying and stratifying patients for susceptibility to pazopanib and other similar tyrosine kinase inhibitors, so that treatment can be tailored to these groups.
The present invention is based on research to identify biomarkers associated with successful treatment with tyrosine kinase inhibitors (TKI) such as Pazopanib.
In doing so, the inventors identified several biomarkers, which could be used independently or in combination to identify patients who would benefit from TKI treatment. The inventors identified biomarkers associated with longer progression-free survival (PFS) and overall survival (OS). In other words, the inventors identified biomarkers for subtypes of cancers which have greater or lesser sensitivity to TKIs such as Pazopanib.
In particular, three groups of biomarkers have been identified for stratifying cancers: 1) baseline expression, e.g. protein expression, of FGFR1 and PDGFRA, for example using immunohistochemistry (IHC), 2) TP53 mutational status, and 3) gene expression levels of genes (shown in List 1) involved in key oncogenic pathways for example as indicated by mRNA transcript abundance levels.
Accordingly, the invention relates to the use of one or more of these groups of biomarkers for stratifying cancers, and selecting or identifying cancers for treatment with TKIs such as Pazopanib. The invention also relates to kits for testing and stratifying cancers, to methods for identifying patients for treatment, and to TKIs for use in methods of treatment of cancer.
The stratification of patients according to the invention involves determining the expression, e.g. protein expression, of FGFR1 and PDGFRA. In some cases, the method of the invention may include following the steps of a decision tree classifier, as depicted in
In addition, the inventors have identified a set of genes shown in table 5 that can be used to stratify PDGFRA-high/FGFR1-low IHC intrinsic resistant poor responder cases from other cases.
In addition, the inventors have developed a gene signature using the genes shown in table 4 that allows for the stratification of patients into the five distinct subgroups identified by the decision tree without the need to apply a decision tree workflow.
In a first aspect the invention provides a method of selecting an individual with cancer for treatment with a TKI. The method may comprise determining the expression levels of PDGFRA and FGFR1 as ‘high’(Hi) or ‘low’(Lo) in a sample of cancer cells from the individual, and selecting an individual for TKI treatment if they have PDGFRA-Hi/FGFR1-Hi PDGFRA-Lo/FGFR1-Lo, or PDGFRA-Lo/FGFR1-Hi expression levels. An individual may be selected for TKI treatment if they have PDGFRA-Lo and/or FGFR1-Hi expression levels. An individual may be selected for TKI treatment if they do not have PDGFRA-Hi/FGFR1-Lo expression levels.
Equally, an individual may be “deselected” for treatment with TKIs (for example selected as more appropriately treated with an alternative therapy choice, such as in one example an anti-PDGFRA antibody (e.g. olaratumab), binding fragment thereof or a pharmaceutical composition comprising said antibody or said binding fragment) if they have PDGFRA-Hi/FGFR1-Lo.
The expression levels of PDGFRA or FGFR1 determined may be the expression levels of FGFR1 and PDGFRA proteins. The expression levels may be determined to be ‘high’ if, using immunohistochemistry (IHC), they have a score of 3 or more, wherein the expression level of PDGFRA and/or FGFR1 is scored using the addition of score for staining intensity and score for proportion of positive tumour cells, wherein for staining intensity scoring 0=absent, 1=weak, 2=moderate and 3=strong, and for proportion of positive tumour cells 0=absent, 1=1-10%, 2=11-50%, 3>50%.
In some embodiments, in the determining step, the protein expression levels of PDGFRA and FGFR1 are determined using immunohistochemistry (IHC).
The methods of selecting an individual with cancer for treatment with a TKI may comprise determining the mutation status of TP53 in a sample of, or a sample derived from, cancer cells from the individual, and selecting the individual for treatment if they have wildtype TP53. The mutation status may be determined using digital PCR, Sanger sequencing or next generation sequencing, for example.
The methods of selecting an individual with cancer for treatment with a TKI may comprise determining, within a sample of cancer cells from the individual, the expression levels of 5 or more of the genes selected from List 1, and selecting an individual for treatment with the TKI based on the expression levels of those genes. For example an individual may be selected for treatment if the expression levels resemble, for example closely resemble those of a subgroup with favourable TKI outcome.
In some embodiments, at least about 40 of the genes listed in List 1 are used in the PARSARC (Pazopanib Activity and Response in SARComa) classification model. In other embodiments, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, or all 229 of the genes listed in List 1 are used in the model. As described in detail herein, the 229 genes listed in List 1 were detected at <10% FDR by SAM analysis. Without wishing to be bound by any particular theory, the present inventors believe that it is the combination of at least 115 of, or even substantially all of, the genes listed in List 1 that affords the most accurate classification of intrinsic subtype and prognostication of outcome or therapeutic response to treatment. Thus, in various preferred embodiments, the methods disclosed herein encompass obtaining the expression profile of a large number of the genes listed in List 1, for example, at least 47, at least 48, at least 49, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 115, at least 120, at least 150, at least 200, or all 229 of the genes listed in List 1. It will also be understood by one of skill in the art that one subset of the genes listed in List 1 can be used to train an algorithm to predict sarcoma subtype or outcome, and another subset of the genes used to characterize an individual subject. Preferably, all 229 genes are used to train the algorithm, and at least 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 of the genes are used to characterize a subject.
The methods may utilize a supervised algorithm to classify future subject samples according to sarcoma biological subtype. A particular exemplary algorithm, referred to herein as the PARSARC classification model, is based on the gene expression profile of a defined subset of genes that has been identified herein as superior for classifying sarcoma biological subtypes, and for predicting risk of relapse and/or response to therapy in a subject diagnosed with sarcoma. The subset of genes, is provided in List 1.
“Gene expression” used herein refers to the relative levels of expression and/or pattern of expression of a gene. The expression of a gene may be measured at the of DNA, cDNA, RNA, mRNA, or combinations thereof. “Gene expression profile” refers to the levels of expression, of multiple different genes measured for the same sample. An expression profile can be derived from a biological sample collected from a subject at one or more time points prior to, during, or following diagnosis, treatment, or therapy for sarcoma (or any combination thereof), can be derived from a biological sample collected from a subject at one or more time points during which there is no treatment or therapy for sarcoma (e.g., to monitor progression of disease or to assess development of disease in. a subject at risk for sarcoma), or can be collected from a healthy subject. Gene expression profiles may be measured in a sample, such as samples comprising a variety of cell types, different tissues, different organs, or fluids (e.g., blood, urine, spinal fluid, sweat, saliva or serum) by various methods including but not limited to next generation sequencing technologies, digital counting (such as nanostring), microarray technologies and quantitative and semi-quantitative RT-PCR techniques.
The expression levels of 10 or more, 15 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 120 or more, 140 or more, 160 or more, 180 or more 200 or more, or substantially all of, or all of the genes in List 1 may be determined, the expression levels of 10 or more, 15 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 110 or more, or substantially all of, or all of the genes in List 1 are determined.
An individual may be selected for treatment if the expression levels of genes in the sample are determined to be similar to, or to resemble, the expression levels of the same genes in a group of patients known to respond well to TKI treatment, wherein the group of patients has cancer (preferably, the same type of cancer as the individual). In other words the patients in the group all have cancer. An individual may be deselected from treatment with a TKI if the expression levels of genes in the sample are determined to be similar to, or to resemble, the expression levels of the same genes in a group of patients known to respond poorly to TKI treatment, wherein the group of patients has cancer (preferably the same type of cancer as the individual).
Accordingly, the methods may further comprise the step of comparing the expression levels of genes in the sample as determined, to the expression levels of the same genes in a group of patients known to respond well to TKI treatment, and to the expression levels of the same genes in a group of patients known to respond poorly to TKI treatment, wherein the group of patients has cancer (preferably, the same type of cancer as the individual), and selecting an individual for treatment with the TKI in step if the expression levels of the genes determined are more similar to the group of individuals which are known to respond well to TKI treatment. It is contemplated herein that the expression levels of said genes (in particular a centroid derived from the expression levels of said genes) may demonstrate predictive ability across cancer types. For example, the marker genes described herein and/or one or more centroids derived from the expression of the genes obtained from sarcoma samples may be amenable to matching a gene expression centroid of, e.g. a renal cancer sample and further more may be predictive of TKI treatment response of the renal cancer subject.
The TKI with which the groups of patients were treated may be any TKI which find use in the present invention. The TKI may be the same as that intended for treatment of the individual. In other words, the individual may be selected for treatment with the same TKI as that with which the groups of patients were treated. Alternatively, the TKI with which the groups of patients were treated may differ from that which is intended for treatment of the individual.
The expression levels determined may be nucleic acid expression levels. These may be determined using an RNA microarray, quantitative PCR or RNA-Seq, for example.
Methods of the invention may involve one, two or all three of the above approaches for selecting individuals for treatment. Accordingly, a method of selecting an individual with cancer may comprise two or more of:
The method may comprise step (a), and if an individual is selected in step (a), then step (b) may be carried out for the individual, and if the individual is selected in step (b) then step (c) may be carried out. An individual may be selected using all three of steps (a) to (c). In some cases, the method may comprise following the steps of the decision tree classifier depicted in
A method of selecting an individual with cancer may comprise two or more of:
The method may comprise step (a), and if an individual is selected in step (a), then step (b) may be carried out for the individual, and if the individual is selected in step (b) then step (c) may be carried out. An individual may be selected using all three of steps (a) to (c). In some cases, the method may comprise following the steps of the decision tree classifier depicted in
A method of selecting an individual with cancer may comprise two or more of:
The method may comprise step (a), and if an individual is selected in step (a), then step (b) may be carried out for the individual, and if the individual is selected in step (b) then step (c) may be carried out. An individual may be selected using all three of steps (a) to (c). In some cases, the method may comprise following the steps of the decision tree classifier depicted in
Any of the methods may comprise the step of obtaining a sample (e.g. a sample of cancer cells) from the individual. In other words the methods may involve the step of obtaining a sample of cancer cells from the individual before the determining step.
Methods of treatment and therapeutic uses are also contemplated.
In addition to aspects described above, the inventors have developed a gene signature for stratification of patients with differential responses to TKIs (e.g pazopanib). In place of determining the expression levels of PDGFRA and FGFR1 as ‘high’ (Hi) or ‘low’ (Lo) in a sample of cancer cells from the individual, in any of the aspects above, a 42 gene signature has been developed which characterises patients that have intrinsic resistance to a TKI as defined by the PDGFRA-high/FGFR1-low IHC status. In other words, instead of determining the expression levels of PDGFRA and FGFR1 as ‘high’ (Hi) or ‘low’ (Lo), the expression levels of 42 genes in table 5, may be used to classify/stratify the patients in all of the aspects described above.
Accordingly, in a second aspect of the invention, a patient may be selected for treatment with a TKI (e.g. pazopanib) based on the expression levels of these genes. For example an individual may be selected for treatment if the expression levels resemble, for example closely resemble those of a subgroup with favourable TKI outcome. In particular, an individual may be selected for treatment if the expression levels of the 42 genes, or a centroid derived from the expression of those genes more closely matches the ‘other’ centroid as shown in table 7. This centroid is representative of cancers having not having PDGFRA-Hi/FGFR1-Lo expression, for example, having PDGFRA-Lo and/or FGFR1-Hi expression, for example having PDGFRA-Hi/FGFR1-Hi, PDGFRA-Lo/FGFR1-Lo, or PDGFRA-Lo/FGFR1-Hi expression.
An individual may be “deselected” for treatment with TKIs (for example selected as more appropriately treated with an alternative therapy choice, such as in one example an anti-PDGFRA antibody (e.g. olaratumab), binding fragment thereof or a pharmaceutical composition comprising said antibody or said binding fragment), based on the expression levels of the 42 marker genes shown in table 5. In particular, an individual may be deselected for treatment (or selected for alternative treatment) if the expression levels of the 42 genes, or a centroid derived from the expression of those genes more closely matches the ‘PDGFRA-Hi/FGFR1-Lo’ centroid as shown in table 7. This centroid is representative of cancers having PDGFRA-Hi/FGFR1-Lo expression.
The comparison with a centroid may be carried out using nearest centroid single sample classification. This takes the gene expression profile of a new sample, and compares it to each of these class centroids and assigns a sample to a subtype based on the nearest centroid. Subgroup prediction is done by calculating the Spearman's rank correlation of each test case to the two centroids, and assigning a sample to a subtype based on closest Eucleadian distance (1-Spearman Correlation) the nearest centroid. Accordingly, Spearman's rank correlation may be used to calculate the distance to a centroid. Similar statistical tests to compare similarity are also known to the skilled person.
The expression levels may be determined in a sample of cancer cells from a patient. Accordingly, the methods of selecting an individual with cancer for treatment with a TKI may comprise determining, within a sample of cancer cells from the individual, the expression levels of 5 or more of the genes selected from table 5, and selecting an individual for treatment with the TKI based on the expression levels of those genes. Alternatively, an individual may be “deselected” from treatment with TKIs, or selected for treatment with an alternative therapy based on the expression levels of those genes. For example an individual be deselected from treatment with a TKI or selected for treatment with an alternative therapy if the expression levels resemble those of a subgroup with intrinsic resistance, or resemble those of a subgroup which have PDGFRA-hi/FGFR1-lo expression.
Accordingly, the invention provides a method of selecting an individual with cancer for treatment with a TKI, the method comprising:
The expression levels of said 20 or more of the genes determined in said step (a) may be compared with:
In some embodiments, at least about 20 of the genes listed in table 5 are used. In other embodiments, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, or all 42 of the genes listed in table 5 are used. For example, at least 20, 25, 30, 35, 40 or all of the genes listed in table 5 are used.
Where fewer than all 42 genes are used, comparison to the centroid shown in table 7 is done using the centroid values shown for the genes used.
As described in detail herein, the 42 genes listed in table 5 were identified using the Classification of Nearest Centroid (CLANC) methodology. Without wishing to be bound by any particular theory, the present inventors believe that it is the combination of at least 35 of, 40 of, or even substantially all of, the genes listed in table 5 that affords the most accurate classification of patients to the PDGFRA-Hi/FGFR1-Lo or ‘other’ subtype, and thus prognostication of outcome or therapeutic response to treatment.
Accordingly, the invention provides a method of selecting an individual with cancer for treatment with a TKI, the method comprising:
It will also be understood by one of skill in the art that one subset of the genes listed in table 5 can be used to train an algorithm to predict ‘PDGFRA-Hi/FGFR1-Lo’ or ‘other’ subtype or outcome, and another subset of the genes used to characterize an individual subject.
Preferably, all 42 genes are used to train the algorithm, and at least 20, 25, 30, 35, 40 or all 42 of the genes are used to characterize a subject.
The methods may utilize a supervised algorithm to classify future subject samples according to sarcoma biological subtype. A particular exemplary algorithm, referred to herein as the PARSARC classification model, is based on the gene expression profile of a defined subset of genes that has been identified herein as superior for classifying biological subtypes, and for predicting risk of relapse and/or response to therapy in a subject diagnosed with sarcoma. The subset of genes is provided in table 5.
Accordingly, the methods may further comprise the step of comparing the expression levels of genes in the sample as determined, to the expression levels of the same genes in a group of patients known to have PDGFRA-Hi/FGFR1-Hi, PDGFRA-Lo/FGFR1-Lo, or PDGFRA-Lo/FGFR1-Hi expression, and to the expression levels of the same genes in a group of patients known not to have PDGFRA-Hi/FGFR1-Lo expression (for example, having PDGFRA-Lo and/or FGFR1-Hi expression, for example having PDGFRA-Hi/FGFR1-Lo expression. The group of patients has cancer (preferably, the same type of cancer as the individual).
In particular, centroids derived from the expression levels of the genes may be used for the comparison.
An individual may be selected for treatment with the TKI if the expression levels of the genes determined are more similar to the group of individuals which are known not to have PDGFRA-Hi/FGFR1-Lo expression (for example, having PDGFRA-Lo and/or FGFR1-Hi expression, for example having PDGFRA-Hi/FGFR1-Hi, PDGFRA-Lo/FGFR1-Lo, or PDGFRA-Lo/FGFR1-Hi expression.
The gene expression levels discussed above, can be used in place of determining PDGFRA and FGFR1 expression in any of the aspects described herein. For example, the methods of the invention may involve one, two or all three of:
The method may comprise step (a), and if an individual is selected in step (a), then step (b) may be carried out for the individual, and if the individual is selected in step (b) then step (c) may be carried out. An individual may be selected using all three of steps (a) to (c). In some cases, the method may comprise following the steps of the decision tree classifier depicted in
Although the expression levels of the genes shown in table 5 may be used in the context of the decision tree, it may be preferable to use the expression levels of these genes (or a sub-group thereof) alone, in order to select individuals for treatment or provide a prognosis in accordance with the invention.
Any of the methods may comprise the step of obtaining a sample (e.g. a sample of cancer cells) from the individual. In other words the methods may involve the step of obtaining a sample of cancer cells from the individual before the determining step.
In addition to aspects described above, the inventors have developed a gene signature for stratification of patients into the distinct subtypes that were distinguished between using the decision tree, without the need to apply the decision tree workflow.
In particular, a gene signature using 225 genes shown in table 4 has been developed which distinguish between 5 groups which correspond to the outcomes of the decision tree as follows:
In the above descriptions, ‘Subgroup A gene expression’ may refer to cancer with a gene expression levels having a closer match to the centroid of subgroup A shown in table 3 than subgroups B or C. ‘Subgroup B gene expression’ may refer to cancer with a gene expression levels having a closer match to the centroid of subgroup A shown in table 3 than subgroups A or C. ‘Subgroup C gene expression’ may refer to cancer with a gene expression levels having a closer match to the centroid of subgroup C shown in table 3 than subgroups A or B.
Accordingly, the 225 genes in table 4 (or a sub-group thereof) may be used to stratify patients into one of 5 groups, which correspond to the output of the decision tree. Similarly to the output of the decision tree, this stratification may be used to select an individual for treatment with a TKI (or for an alternative treatment), or to provide a prognosis.
Accordingly, in a third aspect the invention provides a method of selecting an individual for treatment with a TKI, the method comprising:
At least 40, at least 45, at least 50, at least 6, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 240, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220 or all 225 of the genes in table 4 are used. For example substantially all of or all of the genes in table 4 are used.
Accordingly, the invention provides a method of selecting an individual for treatment with a TKI, the method comprising:
Similarly, an individual may be “deselected” for treatment with TKIs (for example selected as more appropriately treated with an alternative therapy choice, such as in one example an anti-PDGFRA antibody (e.g. olaratumab), binding fragment thereof or a pharmaceutical composition comprising said antibody or said binding fragment), based on the expression levels of the 225 marker genes shown in table 4, or a sub-group thereof. In particular, an individual may be deselected for treatment (or selected for alternative treatment) if the expression levels of the genes, or a centroid derived from the expression of those genes more closely matches any of the IHC+(1), TP53(2), B(4) or C(5) centroids as shown in table 6, than the A(3) centroid.
The comparison with a centroid may be carried out using nearest centroid single sample classification. This takes the gene expression profile of a new sample, and compares it to each of these class centroids and assigns a sample to a subtype based on the nearest centroid. Subgroup prediction is done by calculating the Spearman's rank correlation of each test case to the two centroids, and assigning a sample to a subtype based on closest Eucleadian distance (1-Spearman Correlation) the nearest centroid. Accordingly, Spearman's rank correlation may be used to calculate the distance to a centroid. Similar statistical tests to compare similarity are also known to the skilled person.
The expression levels may be determined in a sample of cancer cells from a patient. Accordingly, the methods of selecting an individual with cancer for treatment with a TKI may comprise determining, within a sample of cancer cells from the individual, the expression levels of 40 or more of the genes selected from table 4, and selecting an individual for treatment with the TKI based on the expression levels of those genes. Alternatively, an individual may be “deselected” from treatment with TKIs, or selected for treatment with an alternative therapy based on the expression levels of those genes. For example an individual be deselected from treatment with a TKI or selected for treatment with an alternative therapy if the expression levels resemble those of a subgroup with intrinsic resistance, or resemble those of a subgroup which have PDGFRA-hi/FGFR1-lo expression.
Where fewer than all 225 genes are used, comparison to the centroid shown in table 6 is done using the centroid values shown for the genes used.
As described in detail herein, the 225 genes listed in table 4 were identified using the Classification of Nearest Centroid (CLANC) methodology. Without wishing to be bound by any particular theory, the present inventors believe that it is the combination of at least 180 or 200 of, or even substantially all of, the genes listed in table 4 that affords the most accurate classification of patients, and thus prognostication of outcome or therapeutic response to treatment.
It will also be understood by one of skill in the art that one subset of the genes listed in table 4 can be used to train an algorithm to predict subtypes (1) to (5) or outcome, and another subset of the genes used to characterize an individual subject. Preferably, all 225 genes are used to train the algorithm, and at least 40 or more of the genes are used to characterize a subject.
The methods may utilize a supervised algorithm to classify future subject samples according to sarcoma biological subtype. A particular exemplary algorithm, referred to herein as the PARSARC classification model, is based on the gene expression profile of a defined subset of genes that has been identified herein as superior for classifying biological subtypes, and for predicting risk of relapse and/or response to therapy in a subject diagnosed with sarcoma. The subset of genes, is provided in table 4.
Accordingly, the methods may further comprise the step of comparing the expression levels of genes in the sample as determined, to the expression levels of the same genes in a group of patients known to correspond to groups (1)-(5) in the decision tree. The characteristics of these groups are set out above.
In other words, the expression levels of the 40 or more of the genes determined in said step may be compared with:
An individual may be selected for treatment with the TKI if the expression levels of the genes determined are most similar to the group of individuals which are known to be in subgroup (3), that is have (a) subgroup A gene expression, and (b) TP53 wildtype, and (c) not PDGFRA-Hi/FGFR1-Lo expression (e.g. having PDGFRA-Lo and/or FGFR1-Hi expression, e.g. PDGFRA-Hi/FGFR1-Hi or PDGFRA-Lo/FGFR1-Lo or PDGFRA-Lo/FGFR1-Hi).
An individual may be selected for treatment if the expression levels of at least 40 of the 225 genes in table 4 in the sample are determined to be similar to, or to resemble, or have the closest match to the expression levels of the same genes (for example as represented by a centroid) in a group of patients known to respond well to TKI treatment, wherein the group of patients has cancer (preferably, the same type of cancer as the individual). In other words the patients in the group all have cancer.
An individual may be deselected from treatment with a TKI if the expression levels of at least 40 of the 225 genes in table 4 in the sample are determined to be similar to, or to resemble, or have the closest match to the expression levels of the same genes (for example as represented by a centroid) in a group of patients known to respond poorly to TKI treatment, wherein the group of patients has cancer (preferably the same type of cancer as the individual). The gene expression levels discussed above, can be used in place of the decision tree or in place of combinations of the tests for stratifying patients.
Any of the methods may comprise the step of obtaining a sample (e.g. a sample of cancer cells) from the individual. In other words the methods may involve the step of obtaining a sample of cancer cells from the individual before the determining step.
Any of these selection methods may be used to inform treatment choices, in the methods of treatment disclosed herein.
The methods described above may be described as methods of detecting inherent resistance to a TKI in a cancer in an individual. In these methods, the same determining and optionally comparing steps may be carried our as in the methods of selection. In place of step (b) selecting an individual for treatment, the methods of detecting inherent resistance to a TKI comprise step (b) identifying a cancer as having inherent resistance to a TKI.
A cancer is identified as having inherent resistance if it would not be selected for treatment according to the methods described herein. For example: (1) if the cancer has one or more of:
In a fourth aspect the invention provides a tyrosine kinase inhibitor for use in a method of treating cancer in an individual, wherein the individual has been selected for treatment according to the method of the first, second or third aspects of the invention.
In particular, the invention provides a tyrosine kinase inhibitor (TKI) for use in a method of treating cancer in an individual, said method comprising:
In particular, the invention provides a tyrosine kinase inhibitor (TKI) for use in a method of treating cancer in an individual, said method comprising:
In particular, the invention provides a tyrosine kinase inhibitor (TKI) for use in a method of treating cancer in an individual, said method comprising:
In a fifth aspect, the invention provides a method of treating cancer with a TKI in an individual in need thereof, wherein the individual has been selected for treatment according to the method of the first, second or third aspects of the invention.
In particular, the method may comprise:
In particular, the method may comprise:
In particular, the method may comprise:
In a sixth aspect, the present invention provides use of a TKI in the manufacture of a medicament for treating cancer in an individual, wherein the individual has been selected for treatment according to the method of the first, second or third aspects of the invention. In particular, the use may comprise use of a TKI in the manufacture of a medicament for use in a method of the fifth aspect of the invention.
According to any one of the aspects of the invention the cancer may be selected from: soft tissues sarcoma (STS), metastatic renal cell carcinomas (mRCC), gastrointestinal stromal tumour (GIST), hepatocellular carcinoma (HCC), neuroendocrine tumour (NET), medullary thyroid cancer (MTC), non-squamous non-small cell lung cancer (non-squamous NSCLC), and chronic myeloid leukaemia (CML). In particular the cancer may be STS. In particular the cancer may be advanced STS. The cancer in the individual and in the groups of patients for whom the TKI response is known may be selected from these cancer types.
As described in detail herein, particular biomarker signatures identified herein are predictive of treatment outcomes for pazopanib therapy. The present inventors believe that the methods and kits of the present invention are similarly predictive of treatment outcomes with other TKIs, and without wishing to be bound by any particular theory, the inventors believe that, in particular, TKIs having similar pharmacological action in terms of kinases targeted will be especially applicable to the present invention. Based on an analysis of overlapping molecular targets shared by pazopanib, regorafenib and sorafenib (see
In particular, according to any of the aspects of the invention the TKI may be selected from: Pazopanib, Regorafenib, Sorafenib, Sunitinib, Lenvatinib, Axitinib, Nintedanib, and Ponatinib, and pharmaceutically acceptable salts thereof. In particular, the TKI may be Pazopanib. While any pharmaceutically acceptable salt is contemplated herein, particular examples of salt forms of TKIs, which are contemplated in accordance with the present invention, include:
In particular, the TKI is Pazopanib or a pharmaceutically acceptable salt thereof and the cancer is soft tissue sarcoma, for example advanced STS.
In a seventh aspect, the invention provides a kit for use in identifying a cancer suitable for treatment with a TKI. In line with the first aspects of the invention, the kit may have reagents, probes and/or instructions for detecting at least one of:
For example, the kit may have probes for detecting the expression levels of 5 or more, 10 or more, 15 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 110 or more, 115 or more, 120 or more, 140 or more, 160 or more, 180 or more 200 or more, or substantially all of, or all of the genes in List 1.
The kit may also have probes for detecting the expression levels of PDGFRA and FGFR1. The kit may also have probes for detecting the mutation status of TP53.
In some cases in accordance with the seventh aspect of the present invention, the kit takes the form of a companion diagnostic comprising:
In line with the second aspect of the invention, the kit may have reagents, probes and/or instructions for detecting the expression levels of at least 20 of the genes in table 5, and optionally: (a) the mutation status of TP53; and (b) the expression levels of 5 or more of the genes in List 1.
For example, the kit may have probes for detecting the expression levels of at least about 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, or all 42 of the genes listed in table 5.
The kit may also have probes for detecting the mutation status of TP53. The kit may have probes for detecting the expression levels of 5 or more, 10 or more, 15 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 110 or more, 115 or more, 120 or more, 140 or more, 160 or more, 180 or more 200 or more, or substantially all of, or all of the genes in List 1.
In some cases in accordance with the seventh aspect of the present invention, the kit takes the form of a companion diagnostic comprising:
The companion diagnostic may comprise (i) and (ii) in a single package or in separate or associated packages.
In line with the third aspect of the invention, the kit may have reagents, probes and/or instructions for detecting the expression levels of at least 40 of the genes in table 4.
For example, the kit may have probes for detecting the expression levels of at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 240, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220 or all 225 of the genes in table 4.
In some cases in accordance with the seventh aspect of the present invention, the kit takes the form of a companion diagnostic comprising:
The companion diagnostic may comprise (i) and (ii) in a single package or in separate or associated packages.
In an eighth aspect, the invention provides a method of determining a prognosis of TKI treatment response in an individual.
In line with the first aspect of the invention, the method may comprise one or more of:
An individual may be determined to have a good prognosis following TKI treatment if they have one or more of:
The individual may be determined to have a good prognosis if they meet all of the criteria or (a), (b) and (c) for which they were tested. In other words, an individual may not have a good prognosis if they do not meet any one of the criteria for which they are tested.
An individual may be determined to have a poor prognosis if they have one or more of:
In line with the second aspect of the invention, the method may comprise determining the expression levels of 20 or more genes from table 5, and optionally:
An individual may be determined to have a good prognosis following TKI treatment if they have a cancer having expression levels of 20 or more of the genes in table 5 a closer match to a second reference centroid corresponding to the expression profile of said 20 or more genes determined in a second group of subjects known not to have PDGFRA-Hi/FGFR1-Lo expression, than a first reference centroid corresponding to the expression profile of said 20 or more genes determined in a first group of subjects known to have PDGFRA-Hi/FGFR1-Lo expression, and optionally:
An individual may be determined to have a poor prognosis if they have a cancer having expression levels of 20 or more of the genes in table 5 a closer match to a first reference centroid corresponding to the expression profile of said 20 or more genes determined in a first group of subjects known to have PDGFRA-Hi/FGFR1-Lo expression, than a second reference centroid corresponding to the expression profile of said 20 or more genes determined in a second group of subjects known not to have PDGFRA-Hi/FGFR1-Lo expression.
In line with the third aspect of the invention, the method may comprise determining the expression levels of 40 or more genes from table 4.
An individual may be determined to have a good prognosis following TKI treatment if they have a cancer having the expression levels of the 40 or more genes in table 4 a closer match to a third reference centroid than said first, second, fourth or fifth reference centroids, wherein the reference centroids are:
An individual may be determined to have a poor prognosis if they have a cancer having the expression levels of the 40 or more genes in table 4 a closer match to a first, second, fourth or fifth reference centroid than a third reference centroids, wherein the reference centroids are:
In a ninth aspect, the present invention provides an anti-PDGFRA antibody, binding fragment thereof or a pharmaceutical composition comprising said antibody or said binding fragment for use in a method of treating cancer in an individual who has not been selected for treatment with a TKI according to the first, second or third aspects of the invention.
In line with the first aspect of the invention, the individual may have been selected as having one or more of:
In certain embodiments, the anti-PDGFRA antibody may comprise olaratumab.
In line with the second aspect of the invention, the individual may have been selected if they have a cancer having expression levels of 20 or more of the genes in table 5 a closer match to a first reference centroid corresponding to the expression profile of said 20 or more genes determined in a first group of subjects known to have PDGFRA-Hi/FGFR1-Lo expression, than a second reference centroid corresponding to the expression profile of said 20 or more genes determined in a second group of subjects known not to have PDGFRA-Hi/FGFR1-Lo expression.
In line with the third aspect of the invention, the individual may have been selected if they have a cancer having the expression levels of the 40 or more genes in table 4 a closer match to a first, second, fourth or fifth reference centroid than a third reference centroids, wherein the reference centroids are:
In certain embodiments, the anti-PDGFRA antibody may comprise olaratumab.
The markers, methods of measuring them, cancers, tyrosine kinase inhibitors and other details of the invention are described below. These details are applicable to all of the aspects of the invention.
In the context of the present invention the ‘markers’ or ‘biomarkers’ allow stratification of cancers based on their association with a patient outcome. The biomarkers may include expression level of particular genes, expression levels of particular proteins and mutational status of particular genes.
The markers may be detected by conventional means in a sample containing cancer cells or cancer cell material or components (e.g. nucleic acids and/or proteins), obtained from an individual. Accordingly, the methods and uses disclosed herein may involve the step of determining the presence or absence of, expression level of or mutation status of a biomarker. The methods and uses may base a prognostic or diagnostic decision on the presence or absence of, expression level of or mutation status of a biomarker as already determined. The methods and uses may involve the step of determining the presence or absence of, expression level of or mutation status of a biomarker in a sample of cancer cells obtained from an individual.
In some embodiments the methods may comprise the step of obtaining a sample of cancer cells or cancer cell material or components (e.g. nucleic acids and/or proteins) from an individual. The obtained sample may then be tested as described.
The present inventors have found that high levels of PDGFRalpha (herein “PRGFRA”, also known as PDGFRa or PDGFRα) and low levels of FGFR1 expression in cancer cells are associated with a worse outcome when treated with a TKI, as compared to other PDGFRA/FGFR1 expression profiles. This expression profile may be described as an “FGFR1-Lo/PDGFRA-Hi”, or “PDGFRA-Hi/FGFR1-Lo”. In particular patient groups with a PDGFRA-Hi/FGFR1-Lo expression profile have lower overall survival (OS) and progression-free survival (PFS) than other patients.
Cancers with other expression profiles i.e. not PDGFRA-Hi/FGFR1-Lo (e.g. PDGFRA-Lo and/or FGFR1-Hi, e.g. PDGFRA-Hi/FGFR1-Hi, PDGFRA-Lo/FGFR1-Lo or PDGFRA-Lo/FGFR1-Hi) are therefore more suitable for treatment with a TKI as they have better patient outcomes. Accordingly, in some embodiments an individual may be selected for treatment with a TKI if they have a cancer that does not have not PDGFRA-Hi/FGFR1-Lo expression (e.g. having PDGFRA-Lo and/or FGFR1-Hi, e.g. PDGFRA-Hi/FGFR1-Hi, PDGFRA-Lo/FGFR1-Lo or PDGFRA-Lo/FGFR1-Hi expression).
Wherever cancers or individuals having PDGFRA-Hi/FGFR1-Hi or PDGFRA-Lo/FGFR1-Lo or PDGFRA-Lo/FGFR1-Hi are referred to herein, cancers or individuals not having PDGFRA-Hi/FGFR1-Lo expression can be used in the same way. Accordingly the embodiments disclosed herein in relation to PDGFRA-Hi/FGFR1-Hi or PDGFRA-Lo/FGFR1-Lo or PDGFRA-Lo/FGFR1-Hi can be equally applied to cancers or individuals not having PDGFRA-Hi/FGFR1-Lo (e.g. having PDGFRA-Lo and/or FGFR1-Hi). Similarly, an individual may be deselected for TKI treatment, or considered for another therapy if they have a cancer which has PDGFRA-Hi/FGFR1-Lo expression.
In the present invention, references to PDGFRα denote the receptor tyrosine kinase (RTK) platelet-derived growth factor alpha. PDGFRa is a cell surface tyrosine kinase receptor.
The HUGO Gene Symbol report for PDGFRα can be found on the world wide web at genenames.org/cgi-bin/gene_symbol_report?hgnc_id=8803 which provides links to the human PDGFRA nucleic acid and amino acid sequences, as well as reference to the homologous murine and rat proteins. The human form has the HGNC ID: 8803, and the ensemble gene reference ENSG00000134853. The uniprot reference is P16234.
References to FGFR1 denote the fibroblast growth factor receptor 1. FGFR1 is a cell surface tyrosine kinase receptors.
The HUGO Gene Symbol report for FGFR1 can be found on the world wide web at genenames.org/cgi-bin/gene_symbol_report?hgnc_id=HGNC:3688 which provides links to the human FGFR1 nucleic acid and amino acid sequences, as well as reference to the homologous murine and rat proteins. The human form has the HGNC ID: 3688, and the ensemble gene reference ENSG00000077782. The uniprot reference is P11362.
The methods and uses disclosed herein may involve the step of determining the expression level of FGFR1 and PDGFRA or basing a prognostic or diagnostic decision on the expression level of FGFR1 and PDGFRA already determined. The methods and uses may involve the step of determining the expression level of FGFR1 and PDGFRA in a sample of cancer cells obtained from the individual.
In accordance with the invention the expression levels of FGFR1 and PDGFRA may be determined at the protein level or the nucleic acid level. In other words gene expression or protein expression levels of FGFR1 and PDGFRA may be determined.
Protein expression levels may be determined in a sample containing cancer cells obtained from an individual. Protein expression levels may be determined by any available means, including using immunological assays. For example, expression levels may be determined by immunohistochemistry (IHC), Western blotting, ELISA, immunoelectrophoresis, immunoprecipitation and immunostaining. Using any of these methods it is possible to determine the relative expression levels of PDGFRA and FGFR1 proteins.
Protein expression levels may be determined for example using specific binding agents capable of binding to FGFR1 or PDGFRA. A type of specific binding agent is an antibody, capable of specifically binding to FGFR1 or PDGFRA.
The antibody or other specific binding agent may be labelled to enable it to be detected or capable of detection following reaction with one or more further species, for example using a secondary antibody or binding agent that is labelled or capable of producing a detectable result, e.g. in an ELISA type assay. As an alternative a labelled binding agent may be employed in a Western blot to detect FGFR1 or PDGFRA protein.
In particular PDGFRA and FGFR1 expression levels may be determined in a sample of cancer cells, for example using immunohistochemical (IHC) analysis.
IHC analysis can be carried out using paraffin fixed samples or fresh frozen tissue samples, and generally involves staining the samples to highlight the presence, intensity and proportion of cells which express the target protein.
Using IHC, tumour/cancer specimens can be stained and scored for intensity and for proportion of positive tumour cells. For example, for intensity scoring 0=absent, 1=weak, 2=moderate, and 3=strong. For the proportion of positive tumour cells 0=absent, 1=1-10%, 2=11-50%, 3>50%. According to this system, sections with a score of 3 (intensity score+proportion score) may be counted as ‘high’ (Hi). Cumulative scores of <3 may be counted as ‘low’ (Lo). Accordingly, a cancer having PDGFRA-Hi/FGFR1-Hi, PDGFRA-Lo/FGFR1-Lo or PDGFRA-Lo/FGFR1-Hi or PDGFRA-Hi/FGFR1-Lo expression may be a cancer which meets the definition of Hi or Lo according to this IHC scoring system.
Expression levels may be measured using different techniques as described herein, but even if another measurement technique is used in the methods of the invention, expression may still be considered as Hi or Lo using the IHC scoring. In other words, the IHC score system above may be used to define the threshold between Hi and Lo expression, even if IHC itself is not used in the methods of the invention.
Representative examples of scored tumour tissue can be seen in supplementary
Alternatively or additionally, the determination of PDGFRA and FGFR1 expression levels may involve determining the presence or amount of PDGFRA and FGFR1 mRNA in a sample. Methods for doing this are well known to the skilled person. By way of example, they include using PCR involving one or more primers based on each of a PDGFRA and FGFR1 nucleic acid sequence to determine the level of PDGFRA and FGFR1 transcript is present in a sample.
Determining PDGFRA and FGFR1 mRNA levels may carried out by extracting RNA from a sample of cancer cells and measuring PDGFRA and FGFR1 expression specifically using quantitative real time RT-PCR. Alternatively or additionally, the expression of PDGFRA and FGFR1 could be assessed using RNA extracted from a sample of cancer cells for an individual using microarray analysis, which measures the levels of mRNA for a group of genes using a plurality of probes immobilised on a substrate to form the array.
Suitable kits for measuring the expression levels of these markers are described elsewhere herein. Expression levels (e.g. mRNA levels) may involve measuring expression (e.g. mRNA level) of PDGFRA and/or FGFR1 relative to the expression level (e.g. mRNA level) of one or more (e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 18 or 20 or more) “housekeeping” genes. In this context, a housekeeping gene may be any gene for which the expression level is unaffected or largely unaffected by cancer subtype. In particular, suitable housekeeping genes may be selected from those referred to in Supplementary Methods 4 herein, i.e. ACAD9, AGK, AMMECR1L, C10orf76, CC2D1B, CNOT10, CNOT4, COG7, DDX50, DHX16, DNAJC14, EDC3, EIF2B4, ERCC3, FCF1, FTSJ2, GPATCH3, HDAC3, MRPS5, MTMR14, NOL7, NUBP1, PIAS1, PIK3R4 and PRPF38A.
As discussed elsewhere herein, the genes in table 5 (or a sub-set thereof) may be used in place of PDGFRA/FGFR1 expression to stratify patients, and identify those with inherent resistance to TKIs (e.g. pazopanib).
Tumour protein p53 (‘TP53’ or ‘p53’) is a tumour suppressor protein encoded by the TP53 gene in humans. Mutation of TP53 was found to be associated with improved PFS in a retrospective study of 19 patients of mixed STS treated with an anti-angiogenic TKI (95% Pazopanib)17.
In a phase 1 trial of Pazopanib combined with the histone deacetylase inhibitor vorinostat, hotspot TP53 mutations were discovered in 11 of 36 tested patients (3 of 11 sarcoma patients)18. TP53 mutation was significantly associated with improved rates of disease control and progression-free survival across all tested patients, and also with improved median overall survival in a subset of tested patients with either sarcoma or colorectal cancer. This study involved tiny numbers of TP53 mutant sarcomas, and the results are not conclusive, with the association between TP53 and TKIs requiring further investigation.
Using TP53 exon sequencing, the present inventors have found that the mutational status of TP53 was associated with patient outcomes for OS (overall survival) and PFS (progression-free survival). In particular, they found that wildtype TP53 was associated with longer OS and PFS, while TP53 mutations were associated with shorter OS and PFS.
Wildtype TP53 refers to TP53 which does not have non-synonymous mutations. In particular wildtype TP53 may have no non-synonymous mutations in the exons or splice sites.
TP53 mutant refers to TP53 having non-synonymous mutations. A TP53 mutation may be a described as a mutation in TP53 that is associated with cancer. A TP53 mutation may be in a protein coding region or in a splice site for example.
Mutations of TP53 include insertions, inversions, deletions, and/or point mutations.
Mutations in TP53 can be detected using sequencing technologies such as Sanger sequencing. The International Agency for Research on Cancer compile a database of TP53 mutations relating to cancer. These mutations may be detected using the protocol and probes they suggest.
Generally PCR can be used to amplify gene sequences from genomic DNA prepared from a sample of cancer cells. Primers for amplification of exons within TP53 are generally available. The amplified DNA can be sequenced, for example using Sanger sequencing, and mutations identified. Other sequencing techniques may be used, including next generation sequencing (NGS) methods. NGS offers the speed and accuracy required to detect mutations in cancer, either through whole-genome sequencing (WGS) or by focusing on specific regions or genes using whole-exome sequencing (WES) or targeted gene sequencing. Examples of NGS techniques include methods employing sequencing by synthesis, sequencing by hybridisation, sequencing by ligation, pyrosequencing, nanopore sequencing, or electrochemical sequencing.
Additional methods to detect the mutation include matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) spectrometry, restriction fragment length polymorphism (RFLP), high-resolution melting (HRM) curve analysis, and denaturing high performance liquid Chromatography (DHPLC). Other PCR-based methods for detecting mutations include allele specific oligonucleotide polymerase chain reaction (ASO-PCR) and sequence-specific primer (SSP)-PCR. Mutations of may also be detected in mRNA transcripts through, for example, RNA sequence or reverse transcriptase PCR. Mutations may also be detected in the protein through, for example, peptide sequencing by mass spectrometry.
In certain embodiments, determining whether the individual has a mutated TP53 cancer is performed on genomic nucleic acid extracted from a sample of cells obtained from the cancer, from a sample of cancer cells circulating in blood and/or from circulating tumour DNA (ctDNA) in blood or plasma. Techniques for enriching a blood or plasma sample for circulating tumour DNA (e.g. based on fragment size) have been described. Moreover, sequencing techniques for identifying cancer-associated mutations in ctDNA have been described (e.g. based on digital PCR, targeted deep sequencing, nested real-time PCR, and the like). See, for example, PLoS Med. 2016 December; 13(12): e1002198.
Mutant allele specific probes may also be used to detect mutated TP53. These may be nucleic acid probes. For example, a series of allele-specific probes may be used to detect TP53 mutations. Such probes may be used in PCR. Mutation detection using Nanostring technology, e.g. as described on the world wide web at nanostring.com/application/files/9514/9636/4522/GLNS_PM0005_PB_nCounter_Vantage_3D_DNA_SNV_Solid_Tumor_Panel.pdf, are specifically contemplated herein.
It is also possible to detect TP53 mutations using mismatch detection methods to detect mismatches in the DNA or mRNA using probes. It is possible to detect protein TP53 mutations using immunostaining, for example IHC, with antibodies specific to mutant alleles of p53 protein.
Suitable kits for determining the TP53 mutation status are described elsewhere herein.
The HUGO Gene Symbol report for TP53 can be found on the world wide web at genenames.org/cgi-bin/gene_symbol_report?hgnc_id=HGNC:11998 which provides links to the human TP53 nucleic acid and amino acid sequences, as well as reference to the homologous murine and rat proteins. The human form has the HGNC ID: 11998, and the ensemble gene reference ENSG00000141510. The uniprot reference is P04637.
There is growing evidence of shared aspects of molecular pathology that can stratify patients with mixed STS subtypes into groups of similar phenotype. The French Sarcoma Group reported an expression signature of 67 genes related to genomic instability and mitosis that was able to identify subgroups with distinct metastasis-free survival within 2 independent cohorts of mixed STS subtypes15. The so-called CINSARC (Complexity Index in SARComa) is now under prospective investigation as a possible predictive biomarker for neoadjuvant chemotherapy in unselected STS (NCT02789384). Meanwhile, molecular correlates with the long-observed variation in clinical behaviour within histological subtypes have been described in leiomyosarcoma through RNA-Seq gene expression profiling16.
Given that pazopanib directly inhibits a number of growth-promoting RTKs, expression levels of these targets in tumour cells are attractive candidates for evaluation as predictive biomarkers. In a mRCC phase II trial of the combination of interferon-alpha with sorafenib, a multi-targeted kinase inhibitor with target selectivity that overlaps with that of pazopanib, Ho et al. demonstrated using in-situ hybridisation that higher FGFR1 transcript levels was associated with shorter PFS40.
In contrast, it has recently been shown that malignant rhabdoid tumour cells that display high levels of PDGFRA and FGFR1 expression are sensitive to pazopanib treatment in vitro19. It appears likely that the effect of relative expression of these and other RTKs in modulating downstream signalling pathways and influencing drug sensitivity are complex and variably controlled at epigenetic, transcriptional and post-translational levels.
The inventors have investigated additional factors which allow further stratification of individuals with cancer. Clusters of patients with distinct gene expression signatures in the cancer cell samples were identified. These clusters were used to identify the genes which can be used to distinguish between groups of patients with differing prognosis following TKI treatment.
Identification of biological subtype samples and genes: An expanded cancer-pathway associated gene set, comprised primarily of genes found 13 cancer pathways, was initially used to identify biological subtypes. The 13 cancer pathways are:
Twenty two sarcomas with IHC-WT across 770 genes were analyzed by consensus clustering (CC). The CC algorithm statistically identifies significant/unique groups by testing the null hypothesis that a group of samples is from a single cluster, where a cluster is characterized as a multivariate normal distribution. CC was run to identify between 2 to 8 subgroups, with a permutation of 100 times and stopping when the test was no longer significant (p >0.001). Using a supervised approach, a minimized gene set was derived from these biological class labelled samples based on a non-parametric approach to identify the most significant differential genes across these three subgroups.
Using multivariable cox regression model, we confirmed the significant association of these 3 biological subgroups with progression free survival and overall survival respectively (p<0.001)
List 1: 229 genes identified at a false discovery rate (FDR)<10% in multiclass SAM analysis that identifies clinical outcome subgroups A, B and C in unbiased consensus clustering of 22 IHCneg (PDGFRA-Hi/FGFR1-Hi, PDGFRA-Lo/FGFR1-Hi, PDGFRA-Lo/FGFR1-Lo), P53 wt cases. Each gene is identified below by name with the Entrez number for each shown in brackets.
As identified by multiclass Significance Analysis of Microarrays (SAM), these genes in List 1 are differentially expressed in subgroups of individuals which have different patient outcomes. In other words, the markers are differentially expressed between patients that are more or less susceptible to TKI treatment.
These markers can therefore be used to distinguish between subgroups of patients which have different PFS and OS outcomes after TKI treatment. The markers can be used to identify cancers as sensitive to TKI treatment or resistant to TKI treatment.
The markers are generally involved in key oncogenic pathways. In the methods and uses of the invention, the expression levels of 5 or more, 10 or more, 15 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 110 or more, 115 or more, 120 or more, 140 or more, 160 or more, 180 or more 200 or more, or substantially all of, or all of the genes in List 1 may be determined. For example, the expression levels of at least 41, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, or all 229 the genes in List 1 may be determined.
In particular, the genes in List 1 are used to stratify cancers that are already known (a) not to have PDGFRA-Hi and FGFR1-Lo expression and (b) having TP53 wildtype, into subgroups.
The below techniques can be used for determination of expression levels of genes in List1, and also tables 4 and 5.
Reference to determining the expression level refers to determination of the expression level of an expression product of the gene. Expression level may be determined at the nucleic acid level or the protein level.
The gene expression levels determined may be considered to provide an expression profile. By “expression profile” is meant a set of data relating to the level of expression of one or more of the relevant genes in an individual, in a form which allows comparison with comparable expression profiles (e.g. from individuals for whom the prognosis is already known), in order to assist in the determination of prognosis and in the selection of an individual for treatment with a TKI.
The determination of gene expression levels may involve determining the presence or amount mRNA in a sample of cancer cells. Methods for doing this are well known to the skilled person. Gene expression levels may be determined in a sample of cancer cells using any conventional method, for example using nucleic acid microarrays or using nucleic acid synthesis (such as quantitative PCR). For example, gene expression levels may be determined using RNA microarrays. The nucleic acid quantification methods discussed elsewhere herein, for example in relation to FGFR1 and PDGFRA may also be applied to the genes in List 1 and tables 4 and 5.
Alternatively or additionally, the determination of gene expression levels may involve determining the protein levels expressed from the genes in a sample containing cancer cells obtained from an individual. Protein expression levels may be determined by any available means, including using immunological assays. For example, expression levels may be determined by immunohistochemistry (IHC), Western blotting, ELISA, immunoelectrophoresis, immunoprecipitation and immunostaining. Using any of these methods it is possible to determine the relative expression levels of the proteins expressed from the genes listed in List 1 and tables 4 and 5.
Methods of determining protein expression levels are discussed in relation to the determination of FGFR1 and PDGFRA expression levels and such methods may also be used here, for example using specific binding agents capable of binding each of the proteins expressed from the genes listed in List 1 and tables 4 and 5.
Suitable kits for measuring the expression levels of these markers are described elsewhere herein.
Gene expression levels (from List 1 or a subset thereof, or from table 4 or a subset thereof) may be compared with the expression levels of the same genes in cancers from a group of patients known to respond well (having a good prognosis, or good OS and PFS) to TKI treatment, or be sensitive to TKI treatment. Gene expression levels may be compared to the expression levels of the same genes in cancers from a group of patients known to respond poorly to TKI treatment (having a poor prognosis, or poor OS and PFS) or be resistant to TKI treatment.
The patients/individuals to which the comparison is made may be referred to as the ‘control group’. Accordingly, the determined gene expression levels may be compared to the expression levels in a control group of individuals having cancer. The comparison may be made to expression levels determined in cancer cells of the control group. The comparison may be made to expression levels determined in samples of cancer cells from the control group. The cancer in the control group may be the same type of cancer as in the individual. For example, if the expression is being determined for an individual with soft tissue sarcoma (STS), the expression levels may be compared to the expression levels in the cancer cells of patients with soft tissue sarcoma.
Other factors may also be matched between the control group and the individual and cancer being tested. For example the stage of cancer may be the same. For example, if the individual being tested has advanced soft tissue sarcoma, the expression levels of the genes may be compared to the expression levels of the same genes in advanced soft tissue sarcomas in a group of patients known to have a good prognosis after treatment with a TKI and/or a group of patients known to have a poor prognosis after treatment with a TKI.
Additionally the control group may have been treated with the same TKI. For example, if the TKI is pazopanib, all of the patients in the control group(s) may have been treated with pazopanib.
Accordingly, an individual may be stratified or grouped according to their similarity of gene expression with the group with good or poor prognosis.
As used herein, a group of patients known to respond poorly to TKI treatment may be those known to have a poor outcome following TKI treatment as defined elsewhere herein. A group of patients known to respond well to TKI treatment may be those known to have a good outcome following TKI treatment as defined elsewhere herein, or a group having a superior outcome to those found to have a poor outcome following TKI treatment.
As described in further detail in Example 1 herein, in order to create a test to identify biological subgroups, we used consensus clustering (CC) to objectively separate the 22 tumours into stable biological groups. The goal of the consensus clustering was to search for a partition of the 22 tumours into 2 or at most 8 groups using the expression of the 730 signature genes which had already been found to be associated with 13 canonical cancer pathway. We used hierarchical consensus clustering with 1-pearson correlation to identify robust unsupervised clusters by performing 200 iterations subsampling 80% of the samples each round. We identified 3 clearly separated biological groups, namely “A”, “B” and “C”. Using Multi-class Significant Analysis of Microarray (Tusher PNAS 2001, PMID:11309499), we identified a list of 229 genes with significant differential expressed among the three subgroups (see List 1). Using the Prediction Analysis of Microarray (PAM) algorithm, we built a standardised centroid for each of the biological subgroup (see Table 3 herein). The centroid was the average gene expression for each gene in each subtype (or “class”, i.e. A or B or C) divided by the within-class standard deviation for that gene. Nearest centroid single sample classification takes the gene expression profile of a new sample, and compares it to each of these class centroids and assigns a sample to a subtype based on the nearest centroid. Subgroup prediction is done by calculating the Spearman's rank correlation of each test case to the three centroids, and assigning a sample to a subtype based on closest Eucleadian distance (1-Spearman Correlation) the nearest centroid.
The dominance of a particular biological subgroup for an individual tumour may be determined in an analogous fashion to that widely used for identifying intrinsic breast cancer subtypes using the PAM50 genes and Nanostring technology and a closest centroid approach (see, e.g., Parker et al. JCO, PMID: 19204204; Tibshirani 2002 PNAS PMID:12011421).
In some embodiments, the present invention provides methods for classifying, prognosticating, or monitoring sarcoma in subjects. In particular, data obtained from analysis of gene expression may be evaluated using one or more pattern recognition algorithms. Such analysis methods may be used to form a predictive model, which can be used to classify test data.
For example, one convenient and particularly effective method of classification employs multivariate statistical analysis modelling, first to form a model (a “predictive mathematical model”) using data (“modelling data”) from samples of known subgroup (e.g., from subjects known to have a particular sarcoma biological subgroups: A, B and C as defined by the genes in List 1 and the centroids given in table 3; or from subjects known to have a particular sarcoma biological subgroups based on the genes listed in table 4 and the centroids given in table 6), and second to classify an unknown sample (e.g., “test sample”) according to subgroup. Pattern recognition methods have been used widely to characterize many different types of problems ranging, for example, over linguistics, fingerprinting, chemistry and psychology. In the context of the methods described herein, pattern recognition is the use of multivariate statistics, both parametric and non-parametric, to analyse data, and hence to classify samples and to predict the value of some dependent variable based on a range of observed measurements. There are two main approaches. One set of methods is termed “unsupervised” and these simply reduce data complexity in a rational way and also produce display plots which can be interpreted by the human eye. However, this type of approach may not be suitable for developing a clinical assay that can be used to classify samples derived from subjects independent of the initial sample population used to train the prediction algorithm.
The other approach is termed “supervised” whereby a training set of samples with known class or outcome is used to produce a mathematical model which is then evaluated with independent validation data sets. Here, a “training set” of gene expression data is used to construct a statistical model that predicts correctly the “subgroup” of each sample. This training set is then tested with independent data (referred to as a test or validation set) to determine the robustness of the computer-based model. These models are sometimes termed “expert systems,” but may be based on a range of different mathematical procedures such as support vector machine, decision trees, k-nearest neighbour and naïve Bayes. Supervised methods can use a data set with reduced dimensionality (for example, the first few principal components), but typically use unreduced data, with all dimensionality. In all cases the methods allow the quantitative description of the multivariate boundaries that characterize and separate each subtype in terms of its intrinsic gene expression profile. It is also possible to obtain confidence limits on any predictions, for example, a level of probability to be placed on the goodness of fit (see, for example, Kowalski et al., 1986). The robustness of the predictive models can also be checked using cross-validation, by leaving out selected samples from the analysis.
The PARSARC classification model described herein is based on the gene expression profile for a plurality of subject samples using the genes listed in List 1. The plurality of samples includes a sufficient number of samples derived from subjects belonging to each subgroup class. By “sufficient samples” or “representative number” in this context is intended a quantity of samples derived from each subtype that is sufficient for building a classification model that can reliably distinguish each subgroup from all others in the group. A supervised prediction algorithm is developed based on the profiles of objectively-selected IHCnegTP53 wt (non-PDGFRA-Hi/FGFR1-Lo; TP53 wt) prototype samples for “training” the algorithm. The samples are selected and subtyped using an expanded gene set, such as that of the genes of List 1.
A similar approach may be applied to the genes listed in table 4.
Alternatively, the samples can be subtyped according to any known assay for classifying sarcoma subgroups. After stratifying the training samples according to subtype, a centroid-based prediction algorithm is used to construct centroids based on the expression profile of the intrinsic gene set described in List 1. An exemplary centroid for each of three tumour subtypes A-C is shown in Table 3 herein.
Alternatively, after stratifying the training samples according to subtype, a centroid-based prediction algorithm is used to construct centroids based on the expression profile of the intrinsic gene set described in table 4. An exemplary centroid for each of five tumour subtypes is shown in Table 6 herein.
“Translation” of the descriptor coordinate axes can be useful. Examples of such translation include normalization and mean-centering. “Normalization” may be used to remove sample-to-sample variation. Some commonly used methods for calculating normalization factor include: (i) global normalization that uses all genes on the microarray or nanostring codeset; (ii) housekeeping genes normalization that uses constantly expressed housekeeping/invariant genes; and (iii) internal controls normalization that uses known amount of exogenous control genes added during hybridization (Quackenbush (2002) Nat. Genet. 32 (Suppl.), 496-501). In one embodiment, the genes listed in List 1 can be normalized to control housekeeping genes. Exemplary housekeeping genes include MRPL19, PSMC4, SF3A1, PUM1, ACTB, GAPD, GUSB, RPLPO, and TFRC. It will be understood by one of skill in the art that the methods disclosed herein are not bound by normalization to any particular housekeeping genes, and that any suitable housekeeping gene(s) known in the art can be used. Many normalization approaches are possible, and they can often be applied at any of several points in the analysis. In one embodiment, microarray data is normalized using the LOWESS method, which is a global locally weighted scatterplot smoothing normalization function. In another embodiment, qPCR data is normalized to the geometric mean of set of multiple housekeeping genes.
“Mean-centering” may also be used to simplify interpretation for data visualisation and computation. Usually, for each descriptor, the average value of that descriptor for all samples is subtracted. In this way, the mean of a descriptor coincides with the origin, and all descriptors are “centered” at zero. In “unit variance scaling,” data can be scaled to equal variance. Usually, the value of each descriptor is scaled by 1/StDev, where StDev is the standard deviation for that descriptor for all samples. “Pareto scaling” is, in some sense, intermediate between mean centering and unit variance scaling. In pareto scaling, the value of each descriptor is scaled by 1/sqrt(StDev), where StDev is the standard deviation for that descriptor for all samples. In this way, each descriptor has a variance numerically equal to its initial standard deviation. The pareto scaling may be performed, for example, on raw data or mean centered data.
“Logarithmic scaling” may be used to assist interpretation when data have a positive skew and/or when data spans a large range, e.g., several orders of magnitude. Usually, for each descriptor, the value is replaced by the logarithm of that value. In “equal range scaling,” each descriptor is divided by the range of that descriptor for all samples. In this way, all descriptors have the same range, that is, 1. However, this method is sensitive to presence of outlier points. In “autoscaling,” each data vector is mean centered and unit variance scaled. This technique is a very useful because each descriptor is then weighted equally, and large and small values are treated with equal emphasis. This can be important for genes expressed at very low, but still detectable, levels.
In one embodiment, data is collected for one or more test samples and classified using the PARSARC classification model described herein. When comparing data from multiple analyses (e.g., comparing expression profiles for one or more test samples to the centroids constructed from samples collected and analyzed in an independent study), it will be necessary to normalize data across these data sets. In one embodiment, Distance Weighted Discrimination (DWD) is used to combine these data sets together (Benito et al. (2004) Bioinformatics 20(1): 105-114, incorporated by reference herein in its entirety). DWD is a multivariate analysis tool that is able to identify systematic biases present in separate data sets and then make a global adjustment to compensate for these biases; in essence, each separate data set is a multi-dimensional cloud of data points, and DWD takes two points clouds and shifts one such that it more optimally overlaps the other.
In some embodiments described herein, the prognostic performance of the PARSARC IHC, TP53 mutational status and biological and/or other clinical parameters is assessed utilizing a Cox Proportional Hazards Model Analysis, which is a regression method for survival data that provides an estimate of the hazard ratio and its confidence interval. The Cox model is a well-recognized statistical technique for exploring the relationship between the survival of a patient and particular variables. This statistical method permits estimation of the hazard (i.e., risk) of individuals given their prognostic variables (e.g., intrinsic gene expression profile with or without additional clinical factors, as described herein). The “hazard ratio” is the risk of death at any given time point for patients displaying particular prognostic variables.
An individual grouped with the good prognosis group, may be identified as having a cancer that is sensitive to TKI treatment, they may also be referred to as an individual that responds well to TKI treatment. An individual grouped with the poor prognosis group, may be identified as having a cancer that is resistant to TKI treatment, they may also be referred to as an individual that responds poorly to TKI treatment.
Where the individual is grouped with the good prognosis group, the individual may be selected for treatment with the TKI. Where the individual is grouped with the poor prognosis group, the individual may be deselected for treatment with the TKI.
Whether a prognosis is considered good or poor may vary between cancers and stage of disease. In general terms a good prognosis is one where the OS and/or PFS is longer than average for that stage and cancer type. A prognosis may be considered poor if PFS and/or OS is lower than average for that stage and type of cancer. The average may be the mean OS or PFS.
For example, a prognosis may be considered good if the PFS is >6 months and/or OS >18 months. Similarly PFS of <6 months or OS of <18 months may be considered poor. In particular PFS of >6 months and/or OS of >18 months may be considered good for advanced cancers.
In particular PFS of >6 months and/or OS of >18 months may be considered good for pazopanib treatment, for example of soft tissue sarcoma (STS), in particular advanced STS.
In general terms, a “good prognosis” is one where survival (OS and/or PFS) of an individual patient can be favourably compared to what is expected in a population of patients within a comparable disease setting. This might be defined as better than median survival (i.e. survival that exceeds that of 50% of patients in population).
In particular, PFS >4 months and/or OS >12 months may be considered good following pazopanib treatment for patients with advanced soft tissue sarcomas, based on median survival in the pazopanib arm in the PALLETTE trial. Very good survival in the same population might be considered PFS >6 months and/or OS >18 months.
“Predicting the likelihood of survival of a sarcoma patient” is intended to assess the risk that a patient will die as a result of the underlying sarcoma.
“Predicting the likelihood of progression-free survival” is intended to mean that the patient neither dies nor experiences radiological disease progression by RECIST criteria.
“Predicting the response of a sarcoma patient to a selected treatment” is intended to mean assessing the likelihood that a patient will experience a positive or negative outcome with a particular treatment.
As used herein, “indicative of a positive treatment outcome” refers to an increased likelihood that the patient will experience beneficial results from the selected treatment (e.g. reduction in tumour size, ‘good’ prognostic outcome, improvement in disease-related symptoms and/or quality of life).
“Indicative of a negative treatment outcome” is intended to mean an increased likelihood that the patient will not receive the aforementioned benefits of a positive treatment outcome.
The present inventors have found that the FGFR1/PDGFRA expression, TP53 mutation status and gene expression signatures using the genes in List 1, or a sub-set thereof have more predictive power for response to TKI when used in combination. In one particular example, they may be combined in a decision tree classifier as depicted in
Accordingly, in the methods of the invention, any of the three methods may be used individually or in combination.
For example, the TP53 mutational status and the expression levels of FGFR1 & PDGFRA may both be determined. These two tests were found to have an additive predictive ability for progression-free and overall survival.
For example, for an individual with a cancer determined to have the PDGFRA-Hi/FGFR1-Lo phenotype, an alternative therapy to the TKI may be considered. For cancers with other PDGFRA/FGFR1 expression profiles, further tests may be carried out. In other words in cancers determined not to have PDGFRA-Hi/FGFR1-Lo expression, e.g. to have PDGFRA-Hi/FGFR1-Hi, PDGFRA-Lo/FGFR1-Lo, PDGFRA-Lo/FGFR1-Hi further test may be carried out.
The mutation status of TP53 may be determined for a cancer determined not to have PDGFRA-Hi/FGFR1-Lo expression be PDGFRA-Hi/FGFR1-Hi, PDGFRA-Lo/FGFR1-Lo or PDGFRA-Lo/FGFR1-Hi.
Similarly, for cancers determined to be TP53 wildtype, further tests can be carried out. For example, PDGFRA/FGFR1 expression levels can be determined.
Accordingly, cancers which are determined not to have PDGFRA-Hi/FGFR1-Lo expression (e.g. as having PDGFRA-Hi/FGFR1-Hi, PDGFRA-Lo/FGFR1-Lo or PDGFRA-Lo/FGFR1-Hi) and TP53 wildtype may be identified as having a relatively good prognosis following TKI treatment and be selected for treatment with a TKI.
Cancers determined not to have PDGFRA-Hi/FGFR1-Lo expression (e.g. to be PDGFRA-Hi/FGFR1-Hi, PDGFRA-Lo/FGFR1-Lo or PDGFRA-Lo/FGFR1-Hi) and TP53 mutated may be deselected from TKI treatment. Cancers determined to be PDGFRA-Hi/FGFR1-Lo and TP53 wildtype may be deselected from TKI treatment. In other words if a cancer is determined to be TP53 mutated, and/or PDGFRA-Hi/FGFR1-Lo it may be determined to have a relatively poor prognosis following treatment with a TKI and deselected for treatment with a TKI.
The TP53 and FGFR1/PDGFRA statuses may also be combined with the levels of gene expression of the genes in List 1 to further select patients with cancers suitable for TKI (eg. Pazopanib) treatment. In particular, for a cancer which determined not to have PDGFRA-Hi/FGFR1-Lo expression (e.g. to have PDGFRA-Hi/FGFR1-Hi, PDGFRA-Lo/FGFR1-Lo or PDGFRA-Lo/FGFR1-Hi) and TP53 wildtype, the gene expression levels of genes selected from those in List 1 may be determined.
Any combination of the tests is possible. For example individuals may be selected or deselected for treatment with the TKI, stratified, or given a prognosis based on expression of PDGFRA and FGFR1 and the expression levels of genes selected from List 1. For example, individuals may be selected or deselected for treatment with the TKI, stratified, or given a prognosis based on expression of PDGFRA and FGFR1 and TP53 mutation status. For example individuals may be selected or deselected for treatment with the TKI, stratified, or given a prognosis based on TP53 mutation status and the expression levels of genes selected from List 1. For example individuals may be selected or deselected for treatment with the TKI, stratified, or given a prognosis based on expression of PDGFRA and FGFR1, TP53 mutation status, and the expression levels of genes selected from List 1.
In particular, an individual may be selected for treatment with a TKI or determined to have a good prognosis following TKI treatment if they meet one, two or all of the criteria:
In particular, for part (c), an individual may be selected for treatment with a TKI or determined to have a good prognosis if they are identified as sensitive to TKI treatment based on the expression levels of genes selected from List 1. For example, an individual may be selected for treatment with a TKI if the expression profile of the genes of List 1 measured for the sample from the individual are a closest match to the centroid of subgroup A relative to the centroids of subgroup B and C. The centroids may be pre-determined centroids such as the exemplary centroids shown in Table 3 or otherwise pre-determined and, for example, retrieved from an electronic data record or the centroids may be established de novo by making use of a training set of gene expression profiles from a plurality of subjects known to have responded well to TKI therapy, and from a plurality of subjects known to have responded poorly to TKI therapy, for a cancer of interest.
In particular, an individual may be deselected from treatment with a TKI or determined to have a poor prognosis following TKI treatment if they meet one, two or all of the criteria:
In particular, for part (c), an individual may be deselected for treatment with a TKI or determined to have a poor prognosis if they are identified as resistant to TKI treatment based on the expression levels of genes selected from List 1. For example, an individual may be deselected for treatment with a TKI if the expression profile of the genes of List 1 measured for the sample from the individual are a closest match to the centroid of subgroup B or C relative to the centroid of subgroup A. The centroids may be pre-determined centroids such as the exemplary centroids shown in Table 3 or otherwise pre-determined and, for example, retrieved from an electronic data record or the centroids may be established de novo by making use of a training set of gene expression profiles from a plurality of subjects known to have responded well to TKI therapy, and from a plurality of subjects known to have responded poorly to TKI therapy, for a cancer of interest.
Details of the protocols are given elsewhere herein.
In some embodiments the determining steps are carried out in series. After a determining step an individual with cancer may be selected for a further determining step using the criteria above, or may be deselected. This makes a ‘decision tree’ allowing efficient selection of patients for treatment with a TKI. For example, the decision tree shown in
For example the PDGFRA and FGFR1 expression status may be determined in a sample of cancer cells from an individual. If the expression levels are PDGFRA-Hi/FGFR1-Lo, the individual is deselected or determined to have a poor prognosis.
If the expression levels are not PDGFRA-Hi/FGFR1-Lo (e.g. PDGFRA-Hi/FGFR1-Hi, PDGFRA-Lo/FGFR1-Lo or PDGFRA-Lo/FGFR1-Hi) then the individual is selected to determine the TP53 mutation status. If the TP53 is mutated, the individual may be deselected or determined to have a poor prognosis.
If TP53 is wildtype, then the individual is selected to determine the expression levels of 5 or more of the genes in List 1. The patient may be selected or deselected based on the expression profile. In particular, the gene expression profile measured for a sample from the individual may be assessed for closeness of fit to gene expression centroids of subgroups differing in respect of their TKI treatment outcome for the cancer of interest. For example, the gene expression profile measured for a sample from the individual may be assessed for closeness of fit to gene expression centroids shown in Table 3.
Similarly, the TP53 mutation status may be first determined. If the TP53 is mutated, the individual may be deselected or determined to have a poor prognosis. If TP53 is wildtype, then the individual is selected to determine PDGFRA and FGFR1 expression status. If the expression levels are PDGFRA-Hi/FGFR1-Lo, the individual is deselected or determined to have a poor prognosis. If the expression levels are not PDGFRA-Hi/FGFR1-Lo (e.g. PDGFRA-Hi/FGFR1-Hi, PDGFRA-Lo/FGFR1-Lo or PDGFRA-Lo/FGFR1-Hi) then the individual is selected to determine the expression levels of genes selected from List 1. The patient may be selected or deselected based on the expression profile of these genes.
In some embodiments the gene expression profile is only determined for an individual having a cancer which has been determined not to have PDGFRA-Hi/FGFR1-Lo expression (e.g. PDGFRA-Hi/FGFR1-Hi, PDGFRA-Lo/FGFR1-Lo or PDGFRA-Lo/FGFR1-Hi) and TP53 wildtype. Testing of PDGFRA & FGFR1 expression and TP53 mutation status may be sequential or in parallel.
In some embodiments the gene expression profile, PDGFRA & FGFR1 expression and TP53 mutation status are all determined in parallel.
Tyrosine kinase inhibitors which can be used for the treatment of cancer find use in the present invention, in particular TKIs with a similar activity profile to Pazopanib.
These include the small molecule inhibitors Pazopanib (CAS number 444731-52-6), Regorafenib (CAS number 755037-03-7), Sorafenib (CAS number 284461-73-0), Sunitinib (CAS number 341031-54-7), Lenvatinib (CAS number 417716-92-8), Axitinib (CAS number 319460-85-0), Nintedanib (CAS number 656247-18-6), and Ponatinib (CAS number 943319-70-8), and pharmaceutically acceptable salts thereof.
Any one of these TKIs may be used in accordance with the present invention. In a preferred embodiment the TKI is Pazopanib.
Salts or derivatives of the exemplary inhibitors may be used for the treatment of cancer. As used herein “derivatives” of the therapeutic agents includes salts, coordination complexes, esters such as in vivo hydrolysable esters, free acids or bases, hydrates, prodrugs or lipids, coupling partners.
Salts of the compounds of the invention are preferably physiologically well tolerated and non-toxic. Many examples of salts are known to those skilled in the art. Compounds having acidic groups, such as phosphates or sulfates, can form salts with alkaline or alkaline earth metals such as Na, K, Mg and Ca, and with organic amines such as triethylamine and Tris (2-hydroxyethyl) amine. Salts can be formed between compounds with basic groups, e.g., amines, with inorganic acids such as hydrochloric acid, phosphoric acid or sulfuric acid, or organic acids such as acetic acid, citric acid, benzoic acid, fumaric acid, or tartaric acid. Compounds having both acidic and basic groups can form internal salts.
Esters can be formed between hydroxyl or carboxylic acid groups present in the compound and an appropriate carboxylic acid or alcohol reaction partner, using techniques well known in the art.
Derivatives which as prodrugs of the compounds are convertible in vivo or in vitro into one of the parent compounds. Typically, at least one of the biological activities of compound will be reduced in the prodrug form of the compound, and can be activated by conversion of the prodrug to release the compound or a metabolite of it.
Other derivatives include coupling partners of the compounds in which the compounds is linked to a coupling partner, e.g. by being chemically coupled to the compound or physically associated with it. Examples of coupling partners include a label or reporter molecule, a supporting substrate, a carrier or transport molecule, an effector, a drug, an antibody or an inhibitor. Coupling partners can be covalently linked to compounds of the invention via an appropriate functional group on the compound such as a hydroxyl group, a carboxyl group or an amino group. Other derivatives include formulating the compounds with liposomes.
The cancers which are stratified and treated according to the present invention are any of the cancers treatable using the TKIs. Accordingly, cancers to be treated or stratified according to the present invention include:
Soft tissues sarcomas (STS), for example advanced soft tissue sarcomas, metastatic renal cell carcinomas (mRCC), gastrointestinal stromal tumour (GIST), hepatocellular carcinoma (HCC), neuroendocrine tumour (NET), medullary thyroid cancer (MTC; also known as medullary thyroid carcinoma), non-squamous non-small cell lung cancer (NSCLC), and chronic myeloid leukaemia (CML).
In particular the cancer may be STS, for example advanced STS.
The treatment may be the first, second or third line treatment.
If the cancer is a soft tissues sarcoma (STS), the methods disclosed herein may be employed to determine suitability for treatment with Pazopanib or Regorafenib, in particular Pazopanib.
If the cancer is a metastatic renal cell carcinoma (mRCC), the methods disclosed herein may be employed to determine suitability for treatment with Pazopanib, Sorafenib, Sunitinib, Lenvatinib or Axitinib. In particular the methods may be used to determine suitability of Pazopanib for treatment of metastatic renal cell carcinoma.
If the cancer is a gastrointestinal stromal tumour (GIST), the methods disclosed herein may be employed to determine suitability for treatment with Regorafenib or Sunitinib. The methods may be used to determine suitability of Regorafenib as a third-line treatment for GIST.
If the cancer is a hepatocellular carcinoma (HCC), the methods disclosed herein may be employed to determine suitability for treatment with Sorafenib.
If the cancer is a neuroendocrine tumour (NET), the methods disclosed herein may be employed to determine suitability for treatment with Sunitinib.
If the cancer is a medullary thyroid cancer (MTC), the methods disclosed herein may be employed to determine suitability for treatment with Lenvatinib.
If the cancer is a non-squamous non-small cell lung cancer (non-squamous NSCLC), the methods disclosed herein may be employed to determine suitability for treatment with Nintedanib. The methods may be used to determine suitability of Nintedanib as a second-line treatment for non-squamous NSCLC.
If the cancer is a chronic myeloid leukaemia (CML), the methods disclosed herein may be employed to determine suitability for treatment with Ponatinib. The methods may be used to determine suitability of Ponatinib to treat solid tumour CML.
In particular the cancer may be a soft-tissue sarcoma and the TKI may be Pazopanib. For example the cancer may be an advanced soft tissue sarcoma.
Methods of stratification, identification and treatments disclosed herein particularly apply to soft-tissue sarcomas and treatment with pazopanib. In particular the soft-tissue sarcoma is advanced STS and the TKI is pazopanib.
The methods disclosed herein may be applied to advanced cancers. Generally ‘advanced’ cancers are not amenable to curative surgery, because they are locally advanced, locally recurrent or metastatic. For example, advanced STS is STS that is not amenable to curative surgery.
It is also contemplated that the methods disclosed herein would be useful in early stage disease, for example pre-operatively.
The individuals to be treated, stratified or tested for selection in accordance with the present invention may, in some cases, not have previously been treated with the TKI, e.g. pazopanib. However, in certain cases, the individual may have been treated with, or may be undergoing treatment with, a TKI (e.g. pazopanib). In such cases, the methods of the present invention may find use in, for example, monitoring treatment and/or predicting the future course of continuing treatment with a TKI (e.g. pazopanib).
The individual to be treated is an animal, preferably a mammal, in particular a human.
Any individual that is not selected for treatment with a TKI, or who is given a poor prognosis with TKI treatment, may be ‘deselected’ from treatment with a TKI, or selected for an alternative treatment as discussed elsewhere herein.
The aspects of the invention relating to prognosis, treatment, selection of patients for treatment and devices suitable for use in these methods are discussed in more detail below. The details about particular TKIs, cancers and methods of carrying out tests on cancer cell samples as described above apply to all of these aspects.
A “test sample” as used herein may, in some cases, be a cell or tissue sample (e.g. a biopsy), a biological fluid, an extract (e.g. a protein or DNA extract obtained from the subject). In particular, the sample may be a tumour sample, a blood sample (including plasma or serum sample), a cerebrospinal fluid sample, or a non-tumour tissue sample. The sample may be one which has been freshly obtained from the subject or may be one which has been processed and/or stored prior to making a determination (e.g. frozen, fixed or subjected to one or more purification, enrichment or extractions steps). In some cases, the sample may be obtained directly from the tumour, obtained from circulating cancer cells and/or circulating tumour DNA.
In one aspect, the invention relates to methods for determining a prognosis, and in particular to methods of identifying individuals with a poor prognosis or good prognosis following TKI treatment. Such a prognosis may help determine whether a TKI inhibitor should be administered.
The invention also relates to methods for stratification or grouping of individuals with cancer according to their prognoses following treatment with a TKI. The method may involve stratifying individuals into a sub-group having poor prognoses or good prognoses.
The invention also relates to methods of selecting individuals for treatment with a tyrosine kinase inhibitor. An individual determined to have a good prognosis following TKI treatment may be selected for treatment with a TKI. An individual determined to have a poor prognosis following TKI treatment may be deselected for treatment with a TKI.
These methods may be described as in vitro methods.
The methods may be useful for determining the likelihood of an individual responding to treatment with a TKI and for helping to determine appropriate treatments for individuals with cancer.
The methods may be useful for identifying individuals with cancer having inherent resistance to a TKI, e.g. pazopanib.
The markers identified by the present inventors are markers of patient outcomes. They can be used to predict prognosis following treatment with a tyrosine kinase inhibitor. In particular, the inventors have identified markers of progression-free survival (PFS) and overall survival (OS).
PFS is the time from first dose of TKI until radiological disease progression or death from any cause. OS is the time from first dose with a TKI until death from any cause. PFS and OS are generally expressed in months.
In this context the poor and good prognosis are relative. Whether a prognosis is considered good or poor may very between cancers and stage of disease. In general terms a good prognosis is one where the OS and/or PFS is longer than average for that stage and cancer type. A prognosis may be considered poor if PFS and/or OS is lower than average for that stage and type of cancer. The average may be the mean OS or PFS.
For example, a prognosis may be considered good if the PFS is >6 months and/or OS >18 months. Similarly PFS of <6 months or OS of <18 months may be considered poor. In particular PFS of >6 months and/or OS of >18 months may be considered good for advanced cancers.
In particular PFS of >6 months and/or OS of >18 months may be considered good for pazopanib treatment, for example of soft tissue sarcoma (STS), in particular advanced STS.
For example, a prognosis may be considered good if the PFS is >6 months and/or OS >18 months. Similarly PFS of <6 months or OS of <18 months may be considered poor. In particular PFS of >6 months and/or OS of >18 months may be considered good for advanced cancers.
In particular PFS of >6 months and/or OS of >18 months may be considered good for pazopanib treatment, for example of soft tissue sarcoma (STS), in particular advanced STS.
In general terms, a “good prognosis” is one where survival (OS and/or PFS) of an individual patient can be favourably compared to what is expected in a population of patients within a comparable disease setting. This might be defined as better than median survival (i.e. survival that exceeds that of 50% of patients in population).
In particular, PFS >4 months and/or OS >12 months may be considered good following pazopanib treatment for patients with advanced soft tissue sarcomas, based on median survival in the pazopanib arm in the PALLETTE trial. Very good survival in the same population might be considered PFS >6 months and/or OS >18 months.
Any of the methods may use one or more of: 1) expression of FGFR1 and PDGFRA, 2) TP53 mutational status, and 3) expression profiles for genes involved in key oncogenic pathways (those shown in list 1).
The methods may make use of the expression profiles of genes shown in table 5.
The methods may make use of the expression profiles of genes shown in table 4.
The same methods and markers may be applied to determine whether a cancer in an individual is likely to be sensitive to TKI treatment. In other words, markers of a good prognosis following TKI treatment are also markers of a cancer that is sensitive to TKI treatment. Markers of a poor prognosis following TKI treatment are also markers of a cancer that is resistant to TKI treatment. Accordingly, the methods of determining a prognosis may also be considered methods of determining sensitivity of a cancer to TKI treatment.
The methods may comprise the step of determining the expression levels of PDGFRA and FGFR1 as ‘high’(Hi) or ‘low’(Lo) in a sample of cancer cells from an individual. The individual is selected for treatment or determined to have a good prognosis or stratified as having a good prognosis if they have:
The individual may be selected for treatment or determined to have a good prognosis or stratified as having a good prognosis if they have PDGFRA-Lo and/or FGFR1-Hi expression levels. Accordingly, the criteria of PDGFRA-Lo and/or FGFR1-Hi may be applied to any of the applications and methods disclosed herein in place of the PDGFRA-Hi/FGFR1-Hi or PDGFRA-Lo/FGFR1-Lo, or PDGFRA-Lo/FGFR1-Hi criterion.
The methods may also use the mutation status of TP53. Accordingly, the methods may comprise the step of determining the mutation status of TP53 in a sample of cancer cells from an individual. The individual is selected for treatment or determined to have a good prognosis or stratified as having a good prognosis if they have wildtype TP53.
The methods may also use gene expression profiles. Accordingly, the method may comprise the step of determining the expression levels in a sample of cancer cells from the individual of 5 or more of the genes selected from List 1. The individual is selected for treatment or determined to have a good prognosis or stratified as having a good prognosis based on the expression levels of those genes. In particular, the individual is selected for treatment or determined to have a good prognosis or stratified as having a good prognosis if the expression profile of the genes of List 1 measured in a sample obtained from the individual is a closest match for the centroid of subgroup A as defined herein (e.g. the exemplary centroids shown in Table 3). These gene expression profiles may be applied to cancers that are already determined (a) not to be PDGFRA-Hi/FGFR1-Lo, and (b) TP53 wildtype.
As mentioned elsewhere these methods may be combined for further selection and to give a better indication of patient outcome.
Accordingly the methods may comprise the steps of:
The methods may comprise the steps of:
The patient/individual may only selected for treatment with the TKI or determining a good prognosis following treatment with a TKI if the selection criteria are met for the determining steps carried out in the method.
A patient/individual may be selected for treatment with the TKI or determined to have a good prognosis following treatment with a TKI if they have PDGFRA-Hi/FGFR1-Hi, PDGFRA-Lo/FGFR1-Lo, or PDGFRA-Lo/FGFR1-Hi expression levels and they have wildtype TP53. A patient may be selected for treatment with the TKI or determined to have a good prognosis following treatment with a TKI if they have PDGFRA-Hi/FGFR1-Hi, PDGFRA-Lo/FGFR1-Lo, or PDGFRA-Lo/FGFR1-Hi expression levels and based on the expression levels of 5 or more of the genes in List 1. A patient may be selected for treatment with the TKI or determined to have a good prognosis following treatment with a TKI if they have wildtype TP53 and based on the expression levels of 5 or more of the genes in List 1.
A patient may be selected for treatment with the TKI or determined to have a good prognosis following treatment with a TKI if they have PDGFRA-Hi/FGFR1-Hi, PDGFRA-Lo/FGFR1-Lo, or PDGFRA-Lo/FGFR1-Hi expression levels, wildtype TP53, and based on the expression levels of 5 or more of the genes in List 1.
Patients who do not meet one more of the criteria may be deselected from treatment with the tyrosine kinase inhibitor.
In some embodiments the determining steps are carried out in series. After a determining step an individual with cancer may be selected for a further determining step using the criteria above, or may be deselected. This makes a ‘decision tree’ allowing efficient selection of patients for treatment with a TKI. For example, the decision tree shown in
Alternatively, the methods may make use of the expression profiles of genes shown in table 5. The method may comprise determining the expression levels of 20 or more genes from table 5, and optionally:
An individual may be determined to have a good prognosis following TKI treatment if they have a cancer having expression levels of 20 or more of the genes in table 5 a closer match to a second reference centroid corresponding to the expression profile of said 20 or more genes determined in a second group of subjects known not to have PDGFRA-Hi/FGFR1-Lo expression, than a first reference centroid corresponding to the expression profile of said 20 or more genes determined in a first group of subjects known to have PDGFRA-Hi/FGFR1-Lo expression, and optionally:
An individual may be determined to have a poor prognosis if they have a cancer having expression levels of 20 or more of the genes in table 5 a closer match to a first reference centroid corresponding to the expression profile of said 20 or more genes determined in a first group of subjects known to have PDGFRA-Hi/FGFR1-Lo expression, than a second reference centroid corresponding to the expression profile of said 20 or more genes determined in a second group of subjects known not to have PDGFRA-Hi/FGFR1-Lo expression.
The methods may make use of the expression profiles of genes shown in table 4. The method may comprise determining the expression levels of 40 or more genes from table 4.
An individual may be determined to have a good prognosis following TKI treatment if they have a cancer having the expression levels of the 40 or more genes in table 4 a closer match to a third reference centroid than said first, second, fourth or fifth reference centroids, wherein the reference centroids are:
An individual may be determined to have a poor prognosis if they have a cancer having the expression levels of the 40 or more genes in table 4 a closer match to a first, second, fourth or fifth reference centroid than a third reference centroids, wherein the reference centroids are:
More details of the determining steps and combinations thereof are given elsewhere herein.
In any of the methods described herein the determining steps may be historical, and the methods may make use of expression levels that have already been determined.
Treatment of Cancer with TKIs
In one aspect the present invention provides methods and medical uses for the treatment of cancers with TKIs. According to these methods and treatments the cancers are ones which are identified herein as being sensitive to treatment with TKI.
Accordingly, a tyrosine kinase inhibitor for use in a method of treating cancer in an individual is provided, wherein the cancer has PDGFRA-Hi/FGFR1-Hi, PDGFRA-Lo/FGFR1-Lo, or PDGFRA-Lo/FGFR1-Hi expression levels; and/or wildtype TP53; and/or has been identified as sensitive to TKI treatment based on the expression levels of 5 or more of the genes in List 1.
Also provided is the use of a TKI in the manufacture of a medicament for treating a cancer in an individual wherein the cancer has been identified as having PDGFRA-Hi/FGFR1-Hi, PDGFRA-Lo/FGFR1-Lo, or PDGFRA-Lo/FGFR1-Hi expression levels, and/or wildtype TP53, and/or has been identified as sensitive based on the expression levels of 5 or more of the genes in List 1.
Also provided is a method of treating a cancer in an individual comprising administration of a TKI, wherein the cancer has PDGFRA-Hi/FGFR1-Hi, PDGFRA-Lo/FGFR1-Lo, or PDGFRA-Lo/FGFR1-Hi expression levels, and/or wildtype TP53, and/or has been identified as sensitive based on the expression levels of 5 or more of the genes in List 1.
Also provided is a tyrosine kinase inhibitor for use in a method of treating cancer in an individual is provided, wherein the cancer has been identified as sensitive to TKI treatment based on the expression levels of 20 or more of the genes in table 5. Also provided is a tyrosine kinase inhibitor for use in a method of treating cancer in an individual is provided, wherein the cancer has been identified as sensitive to TKI treatment based on the expression levels of 40 or more of the genes in table 4.
Also provided is the use of a TKI in the manufacture of a medicament for treating a cancer in an individual wherein the cancer has been identified as sensitive to TKI treatment based on the expression levels of 20 or more of the genes in table 5. Also provided is the use of a TKI in the manufacture of a medicament for treating a cancer in an individual wherein the cancer has been identified as sensitive to TKI treatment based on the expression levels of 40 or more of the genes in table 4.
Also provided is a method of treating a cancer in an individual comprising administration of a TKI, wherein the cancer has been identified as sensitive to TKI treatment based on the expression levels of 20 or more of the genes in table 5. Also provided is a method of treating a cancer in an individual comprising administration of a TKI, wherein the cancer has been identified as sensitive to TKI treatment based on the expression levels of 40 or more of the genes in table 4.
The methods and treatments disclosed herein may involve the steps of determining whether a patient is suitable for treatment.
The methods and treatments may relate to treatment of an individual who has been pre-selected for treatment using the methods described herein. For example, a tyrosine kinase inhibitor is provided for use in a method of treating cancer in an individual, wherein the individual has been selected for treatment as described herein.
Also provided is the use of a tyrosine kinase inhibitor in the manufacture of a medicament for treating cancer in an individual, wherein the individual has been selected for treatment as described herein.
Also provided is a method of treating cancer in an individual in need thereof with a tyrosine kinase inhibitor, wherein the individual has been selected for treatment as described herein.
While the individuals or patients for treatment may have been pre-selected, the methods and uses may also comprise the active steps of selecting an individual for treatment.
The uses and methods may comprise the step of determining if the cancer is susceptible to TKI treatment using one or more of 1) baseline expression of FGFR1 and PDGFRA, for example using immunohistochemistry (IHC), 2) TP53 mutational status, and 3) mRNA transcript abundance for genes involved in key oncogenic pathways (shown in List 1).
The methods may involve the step of obtaining a sample of cancer cells from the individual, and selecting the individual for treatment based on the tests disclosed herein.
For example, the methods may involve the steps of:
For example, a patient may be selected for treatment if they have PDGFRA-Hi/FGFR1-Hi, PDGFRA-Lo/FGFR1-Lo, or PDGFRA-Lo/FGFR1-Hi expression levels, and/or wildtype TP53, and/or based on the expression levels of 5 or more of the genes in List 1.
The selection criteria are set out in more detail elsewhere herein, as are the methods and techniques for carrying out the determining steps.
The methods may involve the step:
The individual to be treated is preferably a mammal, in particular a human.
The treatments disclosed may be described including the step of administering the TKI to the individual, e.g. in a therapeutically effective amount. Treatment of an individual with cancer may also be described as treatment of a patient in need thereof.
The TKIs disclosed herein for the treatment of cancer, may be administered alone, but it is generally preferable to provide them in pharmaceutical compositions that additionally comprise with one or more pharmaceutically acceptable carriers, adjuvants, excipients, diluents, fillers, buffers, stabilisers, preservatives, lubricants, or other materials well known to those skilled in the art and optionally other therapeutic or prophylactic agents. Examples of components of pharmaceutical compositions are provided in Remington's Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins.
The term “pharmaceutically acceptable” as used herein includes compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgement, suitable for use in contact with the tissues of a subject (e.g. human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Each carrier, excipient, etc. must also be “acceptable” in the sense of being compatible with the other ingredients of the formulation.
The active agents disclosed herein for the treatment of cancer are preferably for administration to an individual in a “prophylactically effective amount” or a “therapeutically effective amount” (as the case may be, although prophylaxis may be considered therapy), this being sufficient to show benefit to the individual. For example, the agents (inhibitors) may be administered in amount sufficient to delay tumour progression, or prevent tumour growth and/or metastasis or to shrink tumours. For example, the agents may be administered in an amount sufficient to induce apoptosis of cancer cells.
The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 20th Edition, 2000, Lippincott, Williams & Wilkins. A composition may be administered alone or in combination with other treatments, either simultaneously or sequentially, dependent upon the condition to be treated.
The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing the active compound into association with a carrier, which may constitute one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active compound with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.
The agents disclosed herein for the treatment of deficient cancer may be administered to a subject by any convenient route of administration, whether systemically/peripherally or at the site of desired action, including but not limited to, oral (e.g. by ingestion); topical (including e.g. transdermal, intranasal, ocular, buccal, and sublingual); pulmonary (e.g. by inhalation or insufflation therapy using, e.g. an aerosol, e.g. through mouth or nose); rectal; vaginal; parenteral, for example, by injection, including subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, and intrasternal; by implant of a depot, for example, subcutaneously or intramuscularly.
Formulations suitable for oral administration (e.g., by ingestion) may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active compound; as a powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion; as a bolus; as an electuary; or as a paste.
Formulations suitable for parenteral administration (e.g., by injection, including cutaneous, subcutaneous, intramuscular, intravenous and intradermal), include aqueous and non-aqueous isotonic, pyrogen-free, sterile injection solutions which may contain anti-oxidants, buffers, preservatives, stabilisers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents, and liposomes or other microparticulate systems which are designed to target the compound to blood components or one or more organs. Examples of suitable isotonic vehicles for use in such formulations include Sodium Chloride Injection, Ringer's Solution, or Lactated Ringer's Injection. Typically, the concentration of the active compound in the solution is from about 1 ng/ml to about 10 μg/ml, for example from about 10 ng/ml to about 1 μg/ml. The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets. Formulations may be in the form of liposomes or other microparticulate systems which are designed to target the active compound to blood components or one or more organs.
Compositions comprising agents disclosed herein for the treatment of cancer may be used in the methods described herein in combination with standard chemotherapeutic regimes or in conjunction with radiotherapy. Examples of other chemotherapeutic agents include Amsacrine (Amsidine), Bleomycin, Busulfan, Capecitabine (Xeloda), Carboplatin, Carmustine (BCNU), Chlorambucil (Leukeran), Cisplatin, Cladribine (Leustat), Clofarabine (Evoltra), Crisantaspase (Erwinase), Cyclophosphamide, Cytarabine (ARA-C), Dacarbazine (DTIC), Dactinomycin (Actinomycin D), Daunorubicin, Docetaxel (Taxotere), Doxorubicin, Epirubicin, Etoposide (Vepesid, VP-16), Fludarabine (Fludara), Fluorouracil (5-FU), Gemcitabine (Gemzar), Hydroxyurea (Hydroxycarbamide, Hydrea), Idarubicin (Zavedos). Ifosfamide (Mitoxana), Irinotecan (CPT-11, Campto), Leucovorin (folinic acid), Liposomal doxorubicin (Caelyx, Myocet), Liposomal daunorubicin (DaunoXome®) Lomustine, Melphalan, Mercaptopurine, Mesna, Methotrexate, Mitomycin, Mitoxantrone, Oxaliplatin (Eloxatin), Paclitaxel (Taxol), Pemetrexed (Alimta), Pentostatin (Nipent), Procarbazine, Raltitrexed (Tomudex®), Streptozocin (Zanosar®), Tegafur-uracil (Uftoral), Temozolomide (Temodal), Teniposide (Vumon), Thiotepa, Tioguanine (6-TG) (Lanvis), Topotecan (Hycamtin), Treosulfan, Vinblastine (Velbe), Vincristine (Oncovin), Vindesine (Eldisine) and Vinorelbine (Navelbine).
Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the formulation used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician.
In general, a suitable dose of the active compound is in the range of about 100 μg to about 250 mg per kilogram body weight of the subject per day. Where the active compound is a salt, an ester, prodrug, or the like, the amount administered is calculated on the basis of the parent compound, and so the actual weight to be used is increased proportionately.
The invention also provides kits for use in the methods described herein. In other words, the invention provides a kit for stratifying individuals with cancer, for identifying a cancer suitable for treatment with a TKI, for determining a prognosis, and for determining if a cancer is likely to be sensitive to treatment with a TKI.
The kit may comprise specific binding agents for detecting the biomarkers. These specific binding agents may also be referred to as probes.
In particular, the kit may contain probes for detecting 5 or more, 10 or more, 15 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 120 or more, 140 or more, 160 or more, 180 or more 200 or more, or substantially all of, or all of the gene expression products of the genes in List 1. For example, the kit may contain nucleic acid probes which specifically bind to the mRNA expression products of the genes in List 1. The device (kit) can quantify the gene expression level of the genes in List 1.
The binding agents may be immobilised on one or more solid supports, for example on a microarray chip.
The kit may also have probes for expression analysis of PDGFRA and FGFR1. The kits may have probes for detection of PDGFRA and FGFR1 nucleic acids or proteins. For example, specific binding proteins such as antibodies may be used for the detection of PDGFRA and FGFR1 proteins, or specific nucleic acid probes may be used for the detection of PDGRA and FGFR1 gene or mRNA transcript.
The kit may also comprise probes for determining TP53 mutation status. For example, the kit may comprise probes specific for mutations in TP53.
Accordingly, the kit may comprise probes for determining TP53 mutation status, determining PDGFRA and FGFR1 expression and determining the expression of at least 5 genes selected from List 1.
The probes may all be used in a single device, for example on a single microarray. The kit may thus allow simultaneous determination of TP53 status, PDGFRA and FGFR1 expression and expression levels of 5 or more of the genes in List 1. In other words, the kit may allow determination of TP53 status, PDGFRA and FGFR1 expression and expression levels of 5 or more of the genes in List 1 in a single assay, or on a single microarray.
Suitable kits for mutation status, protein quantification and gene expression include nCounter® Vantage 3D™ Solid Tumor Assays (nanoString Technologies).
In addition, the kit may comprise one or more binding agents capable of binding specifically to an expression product of a control gene which is not differentially expressed between individuals affected and unaffected by the cancer of interest. The level of expression from this control gene may be measured in order to assist in quantification of the expression products of the genes of List 1, and/or for quality assurance of an assay performed using the kit. Preferably a control gene is chosen which is constitutively expressed in the cells of the biological sample (i.e. always expressed, at substantially the same level, under substantially all conditions). Such genes are often referred to as “housekeeping” genes. Exemplary housekeeping genes include any one or more of the housekeeping genes described in Supplementary methods 4 herein (ACAD9, AGK, AMMECR1L, C10orf76, CC2D1B, CNOT10, CNOT4, COG7, DDX50, DHX16, DNAJC14, EDC3, EIF2B4, ERCC3, FCF1, FTSJ2, GPATCH3, HDAC3, MRPS5, MTMR14, NOL7, NUBP1, PIAS1, PIK3R4 and PRPF38A).
The kit may comprise further binding agents capable of binding to expression products of other biomarker genes or control genes. However, in preferred embodiments, the kit comprises binding agents for expression products of less than 1000 different genes, e.g. less than 500 different genes, less than 400, less than 300, less than 250, less than 200, or less than 160 different genes. For example, the kit may comprise comprises binding agents for expression products of the genes listed in List 1 and/or PDGFRA & FGFR1, and/or TP53, and no more than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800 or 900 additional genes expression products.
Alternatively, the kit may have probes for detecting the expression levels of at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, or all 42 of the genes listed in table 5. For example, the kit may contain nucleic acid probes which specifically bind to the mRNA expression products of the genes in table 5. The device (kit) can quantify the gene expression level of the genes in table 5.
Alternatively, the kit may have probes for detecting the expression levels of at least 40, at least 45, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 240, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220 or all 225 of the genes in table 4. For example, the kit may contain nucleic acid probes which specifically bind to the mRNA expression products of the genes in table 4. The device (kit) can quantify the gene expression level of the genes in table 4.
These kits may contain binding agents/probes for control genes as described above.
The kit is suitable for use in the methods of the invention described in this specification, and may comprise instructions for performing one or more methods of the invention.
In certain embodiments, the kit of the invention takes the form of a companion diagnostic and includes (in addition to the components described above) with it, or is intended to be provided alongside, a TKI or pharmaceutical composition or dosage form comprising a TKI.
Embodiments of the present invention will now be described by way of example and not limitation with reference to the accompanying figures. However various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.
The present invention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or is stated to be expressly avoided. These and further aspects and embodiments of the invention are described in further detail below and with reference to the accompanying examples and figures.
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“and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example “A and/or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.
The following is presented by way of example and is not to be construed as a limitation to the scope of the claims.
Collection and analysis of anonymised archival FFPE tissue and associated clinical data was approved in as a sub-study protocol amendment to the Royal Marsden-sponsored Elucidation of a Molecular signature of Pazopanib Response in Advanced soft tissue Sarcoma including solitary fibrous tumours (EMPRASS) study. (RMH Committee for Clinical Research reference 4107, NHS Research Ethic Committee reference 14/WA/0164). This cohort is referred to as RMH-SARC in this manuscript.
Patients were retrospectively identified for inclusion by search of institutional database and electronic patient records compiled during routine clinical practice. Eligibility criteria for inclusion were: i) histopathological diagnosis of soft tissue tumour as confirmed by contemporaneous report by specialist sarcoma histopathologist; ii) received at least one dose of pazopanib for treatment of unresectable or advanced STS; iii) available FFPE tumour specimen, obtained from patient prior to first dose of pazopanib. Treatment and response monitoring was as per standard institutional practice, with pazopanib at 800 mg once daily until disease progression, intolerable toxicity or significant clinical deterioration. Dose interruption and/or reduction were instigated based on standard institutional guidelines and the discretion of the treating physician. Baseline clinico-pathological characteristics and survival data were collected on retrospective review of contemporaneous electronic medical records. All related radiological imaging was retrospectively reviewed and disease response assessed according to RECIST 1.1. This manuscript is written according to the Reporting Recommendations for Tumor Marker Prognostic Studies (REMARK) guidelinesl5.
Available pre-pazopanib FFPE tumour specimens were identified and retrieved from an institutional diagnostic archive, with the specimen taken closest to pazopanib initiation processed in cases where several pre-treatment specimens were available. Newly sectioned haematoxylin and eosin (H&E) stained slides were reviewed to confirm viable tumour content. With reference to H&E, blocks containing tumour material of sufficient size were marked in three spatially discrete areas of representative viable tumour tissue. 1 mm diameter core biopsies were taken from each marked site and re-embedded lengthways into a new paraffin block to form a tissue microarray (TMA). Following coring, 4×10 μm sections were cut and, where necessary, macrodissected to enrich for >75% viable tumour content. Sections were then used for tumour DNA and total RNA extraction using All Prep DNA/RNA FFPE kit (Qiagen, Hilden, Germany) following vendor's standard protocol. DNA and RNA concentrations were measured using Qubit fluorometric quantitation (Thermo Fisher Scientific, Waltham, Mass., USA). RNA Integrity Number and percentage of total RNA <300 bp in size was measured using 2100 Bioanalyzer system (Agilent, CA, USA). RNA and DNA samples were stored at −80° C. until use in downstream analyses.
Serial 4 μm sections were cut from TMA and from specimens not included in the TMA due to inadequate size. Tumour cell plasma membrane and cytoplasmic staining for PDGFRA (Cell Signalling, clone D1E1E) and FGFR1 (Epitomics, 2144-1) was assessed by immunohistochemistry by researchers blinded to associated outcome data (see Supplemental Methods for reagent and method details). IHC staining was scored in terms of intensity (0=absent, 1=weak, 2=moderate, 3=strong) and proportion of positive tumour cells (0=absent, 1=1-10%, 2: =11-50%, 3: >50%). The summation of the two scores give values ranging from 0 to 6 (Supplemental
Extracted tumour DNA was used as a template for amplification and Sanger sequencing of exons 2-11 of TP53 as per International Agency for Research on Cancer (IARC) protocol16 (see Supplemental Methods for primer design and PCR experimental conditions). PCR products were Sanger sequenced (Eurofins Genomics, Ebersberg, Germany). Sequences were aligned to reference human TP53 sequence (GrCH38.p7) and analysed for variants using CLC Sequence Viewer v7.7 (Qiagen).
Expression of 730 genes, representing 13 major cancer pathways including key driver genes was assessed using nCounter PanCancer Pathways panel (NanoString Technologies, Seattle, Wash., USA). 150 ng total RNA was used as input for hybridisation and digital analysis as per manufacturer's instructions using nCounter Dx analysis system (NanoString Technologies). In cases with high RNA degradation, loading adjustments of up to 300 ng were made. Expression data was processed as follows: a) background correction was done by subtracting the geometric mean of the negative control probes, b) normalised by positive control normalization factor calculated as geometric mean of the positive controls followed by normalisation with the housekeeping genes. Expression values were then Log 2 transformed and subjected to gene-based centring.
In order to identify biological subgroups within a subset of 22 patients defined by the absence of identified IHC or TP53 sequencing-based markers, consensus clustering (CC) was used to objectively separate the tumours into stable biological subgroups17. The goal of CC was to search for a partition of the 22 tumours into at least 2 or, at most, 8 groups using expression of the 730 cancer pathway-associated genes. CC with 1-Pearson was used to identify robust unsupervised clusters by performing 200 iterations subsampling 80% of the samples each round. Having identified initially five clusters that were consolidated into three clearly separated subgroups, Multiclass Significance Analysis of Microarrays (SAM)18 was used to identify a subset of genes with significant differential expression (false discovery rate ≤5%) among the three subgroups. Functional enrichment analysis of these gene subsets was performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID. Reference (training) gene expression profiles datasets for each of the three subgroups were built using gene subsets identified by SAM analysis. These gene subsets were also used to calculate a standardised centroid representing each of the three subgroups based on the Prediction Analysis of Microarray (PAM) algorithm. The nearest centroid single sample classification was used to assign individual tumour case to one of three subgroups from independent cohorts. The algorithm compared the individual cases' gene expression profile to each of three class centroids and was assigned to a subgroup based on the closest Euclidean distance to the centroid.
In order to assess whether gene expression data alone could be used to identify subgroups of distinct outcomes following pazopanib therapy, CC was also performed as above to partition all 38 patients into at least 2 or, at most, 8 groups using expression of the 730 cancer pathway-associated genes. Hierarchical clustering of the 38 patients using expression data for genes annotated as involved in each of 13 canonical cancer pathways was performed in order investigate for enrichment of biological processes in identified patient subgroups.
Independent Evaluation of identified biomarkers in TCGA-SARC dataset RNA sequencing (RNA-Seq) and accompanying clinical data for 261 cases of mixed STS were downloaded from The Cancer Genome Atlas (TCGA-SARC; accessed 27 Feb. 2017). The abundance of transcripts was estimated using an Expectation-Maximization algorithm implemented in the software package RSEM8 v1.1.13. Quality control of RNA-Seq data was performed as described in TCGA, and RSEM data was upper quartile normalized and Log 2 transformed. When comparing data from multiple analyses (e.g. comparing expression profiles for one or more test samples to the centroids constructed from samples collected and analyzed in an independent study), it is necessary to normalize data across these data sets. Distance Weighted Discrimination (DWD) was used to combine TCGA-SARC and RMH-SARC data sets together to adjust for systematic biases between these two separate datasets23.
Each eligible case with available gene expression data was assigned to one of three subgroups on the basis of distance from centroids defined within the RMH-SARC cohort. High and low expression levels of FGFR1 and PDGFRA were defined using a cutoff at the first tertile of normalized gene expression values. Associated TP53 mutational status (defined as exonic non-synonymous single nucleotide variant or small indel) from DNA sequencing data for the cohort was downloaded from cBioPortal (accessed 22 Apr. 2017).
Normalised gene expression profile data by 3′End RNA-sequencing (3SEQ) from a cohort of 99 cases of leiomyosarcoma (LMS) (GSE45510; accessed Sep. 3, 2017) was obtained16, referred to here as Stanford-LMS. DWD was used to combine Stanford-LMS and RMH-SARC datasets together to adjust for systematic biases between these two separate datasets. Each case was assigned to one of three subgroups on the basis of distance from centroids defined within our RMH-SARC cohort. Descriptive statistics was done to compare the frequency of our subgroups within each of the molecular LMS subgroups as described by Guo et al16. Hierarchical clustering of Stanford-LMS using our list of significant differential genes identified in RMH-SARC was used to illustrate the gene expression pattern.
The stepwise primary objectives were to assess whether a surrogate of two immunohistochemical markers (FGFR1 and PDGFRA) and TP53 mutation status had statistical significant prognostic information for advanced STS. In this event, the two biomarkers panel were tested to determine if there is added statistically significant prognostic information to standard clinicopathological variables in multivariable comparisons. The secondary analyses included identification of biological subgroups based on gene expression profiles, and evaluation of the significance of these biological subgroups association with patient outcome. Progression free survival (PFS—defined as time in months from first dose of pazopanib to radiological disease progression or death from any cause) was the primary outcome endpoint, with overall survival (OS—defined as a time in months from first dose of pazopanib to death from any cause) as the secondary outcome endpoint. Data cut-off for survival follow-up was 30 Nov. 2016. Statistical analyses were performed by two senior statisticians. The Kaplan-Meier method was used to estimate PFS and OS, and the log-rank test to compare survival in different strata. Multivariable cox regression model was used to estimate the significance adjusted for the standard clinicopathological variables (including age, tumour grade, performance status and histological subtype). Proportional hazard assumption was tested using Schoenfeld residuals, and where deemed appropriate, Restricted Mean Survival was used. Interaction tests between FGFR1 and PDGFRA expression to predict for survival were evaluated for PFS and OS respectively. Likelihood ratio tests based on proportional hazards regression were used to test the prognostic information of all biomarkers. The quantification of the amount of prognostic information provided by one biomarker was assessed by the likelihood ratio χ2 value (LRχ2), and the additional information of one biomarker to biomarker score was measured by the increase of the likelihood ratio χ2 value (ΔLRχ2) obtained from the proportional hazards model.
Retrospective retrieval and analysis of anonymous archival FFPE tissue was approved in a protocol amendment to the Elucidation of a Molecular signature of Pazopanib Response in Advanced soft tissue Sarcoma including solitary fibrous tumours (EMPRASS) study, a Royal Marsden sponsored-single arm translational phase II study (CCR 4107, REC 14/WA/0164).
This is a summary of independent research supported by the National Institute for Health Research (NIHR) Biomedical Research Centre at The Royal Marsden NHS Foundation Trust and The Institute of Cancer Research, the Liddy Shriver Sarcoma Initiative and The Royal Marsden Charity. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health.
DAKO link automated stainer was used for all IHC processing. Tissue sections were deparaffinised with xylene then rehydrated with graded ethanol (100%, 95% to 80%). Antigen retrieval was performed using DAKO FlexEnvision kit (K8002) with either microwave baking for 18 mins in citrate at pH6 (PDGFRa) or pressure cooking for 2 mins in citrate pH6 (FGFR1). Slides were incubated with primary antibodies (FRFR1—E pitomics 2144-1, 1:50 dilution; PDGFRA—Cell Signalling clone D1E1E, 1:250) for 60 minutes at room temperature and visualised using DAKO FlexEnvision (Rabbit/Mouse) kit (K8002), followed by application of DAB, resulting in visible brown colouration reaction at site of target antigen. Finally, nuclear counterstaining with haematoxylin was performed prior to coverslipping. Positive controls were normal breast (PDGFRA) and appendix (FGFR1). Negative control was through omission of primary antibody.
1SEQ ID Nos: are listed next to each sequence.
Between October 2009 and September 2016, 99 patients with advanced soft tissue sarcoma were treated with pazopanib at the Royal Marsden Hospital. Of these, 46 patients had retrievable FFPE tumour material that had been sampled prior to initiation of pazopanib. On examination of these materials, tissue that was adequate for downstream analysis was available for 38 patients (
Baseline clinic-pathological characteristics are summarised in Table 1.
Average age was 54.4 years (range 19.8-81.2). Median number of prior lines of therapy was 1.5 (interquartile range (IQR) 0-2). All patients with documented performance status were ECOG 0-2. All but one patient had metastatic disease, with a median of 2 organ sites involved by disease (IQR1.75-3.25). Sixteen distinct STS subtypes were represented within our cohort, with leiomyosarcoma the most common subtype (11 cases). Solitary fibrous tumour (SFT) was the second most represented subtype (7 cases)—the over-representation of this rare subtype resulted from the stated focus of the EMPRASS study on SFT and subsequent pursuit of tissue blocks from referring centres. All but two of 38 cases were intermediate or high histological grade—of the two cases with low histological grade by FNLCC criteria, one was unresectable solitary fibrous tumour of the retroperitoneum whilst the other was a metastatic case of extraskeletal myxoid chondrosarcoma, an STS subtype not typically allocated grade on the basis of low grade morphological appearances that are incongruent with a more aggressive clinical phenotype. In this case, grade 1 was allocated on basis of FNLCC differentiation score of 2, <10 mitoses/high power field and no necrosis present.
At data cut-off on 30 Nov. 2016 (median follow-up 26.2 months), 35 of 38 patients (92%) had experienced a progression-free survival event and 31 (82%) had died. Median PFS for the cohort was 3.7 months (IQR 1.8-6.9), median OS was 9.5 months (IQR 3.9-19.6). Following retrospective review of imaging series by RECIST 1.1 criteria, 1/38 (2.6%) patient experienced objective radiological response, 20/38 (52.6%) had stable disease and 17/38 (44.7%) progression as best response. For patients with partial response or stable disease, median PFS was 6.4 months (IQR 3.7-12.9).
We analysed pre-pazopanib tumour material to categorise cases as having either high or low tumour expression of FGFR1 and PDGFRA by IHC, and as either TP53 mutated or wildtype tumours through Sanger sequencing of TP53 coding exons. 20/38 cases had high FGFR1 expression, 17/38 had high PDGFRA expression, and 10/38 tumours harboured non-synonymous mutations of TP53. These mutations were primarily missense single nucleotide variants (SNV) within the DNA binding domain of the gene, although single examples of small frameshift deletion, SNV at splice donor site and a 272 bp intragenic inversion were found (Supplemental
FGFR1 expression level was not significantly associated with post-pazopanib PFS or OS. High PDGFRA was associated with worse OS (HR 2.08; 95% CI 1.01-4.35; p=0.04) but no difference in PFS. The interaction test between FGFR1 and PDGFRA expression status for PFS was statistically significant (p=0.001), suggesting that the effect of PDGFRA expression on progression depends on FGFR1 and vice versa. To examine the clinical value of combining the FGFR1 and PDGFRA expression status, patients were stratified into 4 subgroups as follows: FGFR1-Hi/PDGFRA-Hi, FGFR1-Hi/PDGFRA-Lo, FGFR1-Lo/PDGFRA-Lo and FGFR1-Lo/PDGFRA-Hi. In univariate analysis, patients with FGFR1-Lo/PDGFRA-Hi tumours (henceforth designated F-Lo/P-Hi) had significantly associated with worse PFS (HR 9.64; 95% CI 3.58-25.94; p<0.0001) and OS (HR 6.70; 95% CI 2.51-17.91; p<0.0001) when compared to patients with tumours exhibiting one of the other three FGFR1/PDGFRA combinations (hereafter designated IHCneg) (Table 2; Supplemental
Only one of the seven F-Lo/P-Hi cases harboured a TP53 mutation. In the remaining 31 IHCneg cases, TP53 mutation was associated with significantly worse PFS (HR 2.34; 95% CI 1.03-5.34; p=0.04) and OS (HR 3.47; 95% CI 1.44-8.39; p=0.003) when compared to those with wildtype TP53 (TP53 wt) (Supplemental
The independent association of F-Lo/P-Hi status and TP53 mutation status with poor outcome was evaluated in multivariable Cox proportional hazard models adjusted for clinico-pathological factors (age, tumour grade, performance status, tumour histological subtype) (Table 2). F-Lo/P-Hi status (IHCneg vs F-Lo/P-Hi: PFS HR 12.54; 95% CI 3.86-40.72; p<0.001) and TP53 mutation (TP53 wt vs mutation: PFS HR 3.97; 95% CI 1.45-10.86; p=0.007) were independently associated with significantly higher risk of progression. F-Lo/P-Hi status (OS HR 22.11; 95% CI 5.90-82.81; p<0.001) and TP53 mutation (OS HR 7.90; 95% CI 2.56-24.41; p<0.001) also demonstrated independent association with OS. Higher histological grade (HR 3.51; 95% CI 1.40-8.79; p=0.007) and performance status (HR 8.23; 95% CI 2.54-26.69; p<0.001) were also independently associated with worse OS but not with PFS. Histological subtype did not demonstrate independent association with either PFS or OS6,7. Taken together, these data show that both F-Lo/P-Hi IHC status and TP53 mutational status separately identify subgroups of patients with poor outcome following pazopanib, and that this prognostic information is independent of STS histological subtype or other clinico-pathological factors.
indicates data missing or illegible when filed
Analysis of Cancer Pathway-Related Gene Expression Reveals Subgroups with Distinct Pazopanib Outcomes
To gain a better understanding of the underlying biology driving improved pazopanib outcome in the remaining 22 IHCnegTP53 wt patients within our cohort, we performed expression analysis of cancer pathway-related genes in these cases. Consensus clustering demonstrated optimal separation of the cases into five biologically-defined clusters, labelled as biological subgroups 1-5 (
Noting that all six cases of LMS within the 22 patients clustered together in subgroup B (
Integration of Molecular Risk Classifiers into a Clinical Decision Tree Model
Based on the observation that our cohort could be stratified into subgroups of contrasting post-pazopanib outcome through the sequential assessment of FGFR1/PDGFRA IHC, TP53 mutational status and then gene expression analysis, we conceived a clinical decision tree based on this approach for molecular risk classification (
Multivariable comparison is shown in
Recognising that the PARSARC classifier could potentially reflect general prognostic associations in STS rather than a pazopanib-specific effect, we sought to assess whether the classifier was indeed associated with differential OS in an independent cohort of patients with STS who had not received pazopanib. While no such cohort with annotated FGFR1 and PDGFRA protein expression is publicly available, a 261 case STS cohort annotated with genomic and mRNA transcript data is publically available from TCGA, referred here as TCGA-SARC. Due to lack of IHC data, we first sought to assess whether quantitation of FGFR1 and PDGFRA mRNA transcripts can be used as a surrogate marker to recapitulate the group defined by F-Lo/P-Hi IHC in our RMH-SARC cohort. We also assessed the degree to which FGFR1 and PDGFRA mRNA transcript abundance reflected IHC scoring of these proteins in our cohort. Cases with high FGFR1 or PDGFRA protein expression on IHC had significantly higher gene transcript levels than those with low protein expression (Supplementary
We assessed whether F-Lo/P-Hi gene expression, TP53 mutation and/or gene expression-defined subgroups A-C had prognostic associations in a STS cohort which had not received pazopanib therapy using the TCGA-SARC dataset. After exclusion of 7 cases who had received pazopanib, gene expression data and TP53 sequencing data were available for 250 and 232 patients respectively. Having defined a high/low expression cut-off at the 1st tertile of normalised expression scores for FGFR1 and PDGFRA, 49/250 (19.6%) of TCGA patients exhibited low FGFR1 and high PDGFRA expression. No significant difference in overall survival was seen between these patients and those without the F-Lo/P-Hi signature (
Collectively, this analysis finds that while the molecular risk classifiers can be used to categorize subgroups demonstrating significantly different prognosis in our pazopanib-treated cohort, we did not observe significant different prognosis of these subgroups when applied in an independent mixed STS cohort of patients who had not received pazopanib therapy.
In this retrospective study of a heterogeneous cohort of STS patients treated with pazopanib, we performed targeted molecular analysis including assessing expression levels of pazopanib targets FGFR1 and PDGFRA, TP53 mutational analysis and cancer pathway-related gene expression profiling. To our knowledge, this is the largest molecular study of pazopanib-treated STS to date and the first to provide an in-depth examination of multiple aspects of molecular pathology within the same cohort.
When assessing tumour specimens sampled prior to initiation of pazopanib therapy, the combination of low FGFR1 and high PDGFRA protein expression on IHC was associated with very poor PFS and OS following pazopanib therapy. Furthermore, the presence or absence of TP53 mutation in the IHC negative tumours was able to identify two subgroups of contrasting outcomes, with cases harbouring TP53 mutations experiencing worse PFS and OS compared to TP53 wildtype cases. Finally, consensus clustering of gene expression data was able to further stratify the remaining patients with IHCnegTP53 wt tumours resulting in three clinical subgroups with patients in subgroup A associated with the best outcomes. Inclusion of F-Lo/P-Hi IHC status, TP53 mutational status and gene expression subgroup allocation into multivariable analysis produced the best performing predictive model. Taken together, we propose that these data form the basis for a clinical decision making tree that could potentially assist in identifying suitable candidates for pazopanib therapy from an unselected advanced STS population (
Pazopanib shows selectivity for both FGFR1 and PDGFRA which are RTKs with documented capacity to activate multiple canonical oncogenic pathways24-26. We recently reported that malignant rhabdoid tumour cells with high expression of both FGFR1 and PDGFRα are sensitive to pazopanib, and that downregulation of PDGFRA expression was associated with the development of an acquired pazopanib resistance that could be overcome through the addition of a selective FGFR1 inhibitor27. In our cohort, there was significant interaction between protein expression levels of FGFR1 and PDGFRA in predicting PFS, suggesting that the poor prognosis F-Lo/P-Hi IHC subgroup may reflect a currently undefined oncogenic pathway biology that confers primary pazopanib resistance. Pathway enrichment analysis of gene expression data from the full 38 patient cohort finds an upregulation of genes involved in the JAK-STAT signalling pathway in 6 of 7 IHC-positive cases, reflecting a potential role of this pathway in pazopanib resistance (supplementary
The presence of TP53 mutation provided independent prognostic information in multivariable analysis for PFS and OS in our 38 patient cohort. Additionally, in patients without the poor prognosis F-Lo/P-Hi signature, presence of TP53 mutation identified a further poor prognosis subgroup that had significantly worse PFS and OS compared to IHCnegTP53 wt patients. This finding is in contrast to the reported findings of a study by Koehler et al where the presence of TP53 mutation as detected with targeted next-generation gene sequencing was associated with favourable PFS following pazopanib therapy compared to patients with TP53 wildtype tumours (HR0.38; 95CI 0.09-0.83; p=0.036)17. The reason for the inconsistency of the associations of TP53 mutation with pazopanib-related between the two studies is unclear. Both studies included patients from a broad range of STS subtypes, with few patients representing each subtype, and so random error in patient selection may have introduced biological differences between the two study cohorts. Whilst LMS was the most represented subtype in both studies, SFT accounted for 0 of 19 patients in Koehler's study as opposed to 7 of 38 patients in our study, with TP53 mutation found in 3 of 7 SFT cases. The functional impact of the TP53 mutations in our cohort has not been established. Koehler et al did not state the specific mutations detected in their cohort, although note that all were predicted to be loss of function. As it is recognised that specific point mutations of TP53 can result in loss or gain of function28 it is possible that variation in the functional impact of TP53 mutation between studies resulted in opposite clinical phenotypes in relation to pazopanib. Differences in TP53 sequencing methods used in the two studies could also have contributed to the discrepancy—the higher rate of TP53 mutation seen in Koehler's study (10 of 19 patients vs. 10 of 38 patients) may indicate the greater sensitivity of their next-generation sequencing over our Sanger sequencing in terms of ability to detect low level TP53-mutant clones. The role of TP53 mutation as a marker for both pazopanib therapy and overall prognosis in advanced STS requires further investigation—of note, in our cohort the presence of TP53 mutation was almost mutually exclusive to the F-Lo/P-Hi IHC subgroup, with TP53 mutation detected in only 1 of 7 cases with the poor-outcome-related IHC signature.
Gene expression analysis has been widely used in translational cancer research as a means of identifying tumour subgroups of distinct clinical behaviour and underlying biology. A number of reported studies have demonstrated that different STS histological subtypes have distinct, subtype-specific gene expression profiles29-31 . On analysing gene expression data from our heterogeneous 38 patient cohort, cases of the same STS subtype clustered together in a manner consistent with these previously reported findings and thus providing validation to our data (supplementary
There are several limitations to our study. Our single institution cohort, although the largest tissue-based study of a pazopanib-treated cohort to date, is small and has been assessed retrospectively, producing vulnerability to systematic and random biases. Clinical annotation was based on retrospective review of contemporaneous medical documentation, where the absence of a prospective protocol will have contributed to variations in management such as decisions regarding dose reductions/interruptions, timing of radiological assessment and cessation of therapy. The included patients represented a broad range of STS subtypes, representing heterogeneous biology and reflecting daily clinical practice. Further heterogeneity was introduced by the study of archival tumour tissue which variably represented primary, recurrence or metastatic lesions that were taken only days prior to pazopanib commencement in some cases, whilst in other cases the archival sample originated several years earlier with several lines of intervening systemic therapy delivered. Some patients died during or shortly after completion of pazopanib therapy, whilst others went on to receive varied post-pazopanib therapies. Despite these limitations, we have been able to identify molecular signatures that identify patient subgroups with a significantly distinct post-pazopanib outcome. The heterogeneity of disease and specimen studied is representative of a typical scenario faced by oncologists considering prescribing pazopanib for patients with advanced STS, and supports the potential usefulness of our candidate decision tree. Whilst we found no such associations in the TCGA STS cohort not defined by pazopanib exposure, it cannot be established in our retrospective cohort whether the association between the identified molecular readouts and outcome is specific to pazopanib treatment, rather than a more general prognostic association unrelated to drug exposure. Our findings should be considered as hypothesis generating, with analysis of carefully selected pazopanib-naïve control cohorts and/or prospective assessment of the identified molecular signature required to provide greater insight into any predictive relationship with pazopanib.
Advanced STS remains associated with poor prognosis and limited lines of effective treatment36. The recruitment of heterogeneous ‘all-comer’ cohorts to phase III drug trials in STS continues to contribute to the frequent failure to translate early efficacy signals into definitive evidence of survival benefit37. Whilst pazopanib received regulatory approval on the basis of PFS advantage over placebo in a mixed STS cohort, the drug's clinical effectiveness is limited by the lack of predictive biomarkers for benefit. In a retrospective, heterogeneous advanced STS cohort, we have identified a method of molecular classification of tumours that identifies patient subgroups with distinct PFS and OS following pazopanib therapy. If successfully validated, our proposed clinical decision tree would assist in the prospective identification a group less likely to benefit from pazopanib for whom alternative drugs or best supportive care should be considered. Notably, the PDGFRa-targeting monoclonal antibody olaratumab recently received accelerated FDA approval for the 1st line treatment of advanced STS in combination with doxorubicin on the basis of marked OS benefit in a randomised phase II study—this drug would represent an avenue of interest in the F-Lo/P-Hi PDGFRA-overexpressing, poor prognosis patient group that we have identified38. Furthermore, recently published results of a randomised phase II trial of regorafenib, a TKI with target selectivity overlap with pazopanib, indicates efficacy in several STS subtypes but not adipocytic tumours39. The similarity of these clinical data with those of pazopanib raises the question of whether the molecular signature we have identified can also provide risk classification for treatment with regorafenib and other related TKIs. Our study presents a basis for development of biomarkers that may employ simple IHC or genotyping approaches or more sophisticated companion diagnostic assays that can identify STS patients most likely to benefit from pazopanib and other related TKIs.
Patients were identified by retrospective search of prospectively compiled institutional database and electronic patient records. Eligibility criteria for inclusion were: i) histopathological diagnosis of soft tissue tumour as confirmed by contemporaneous report by specialist sarcoma histopathologist; ii) received at least one dose of Pazopanib for treatment of unresectable or advanced STS; iii) available FFPE tumour specimen, obtained from patient prior to first dose of Pazopanib. Treatment and response monitoring was as per local practice, with Pazopanib at 800 mg once daily until disease progression, intolerable toxicity or significant clinical deterioration. Dose interruption and/or reduction were used as per treating physician's judgement. Baseline clinico-pathological characteristics and survival data was collected on retrospective review of contemporaneous electronic medical records. All related radiological imaging was retrospectively reviewed and disease response assessed according to RECIST 1.1.
Available pre-Pazopanib FFPE tumour specimens were identified and retrieved from our institutional diagnostic archive. Where more than one pre-treatment specimen was identified, the one taken closest to Pazopanib start date was processed. Newly sectioned H&E slides were reviewed to confirm viable tumour content. With reference to H&E, blocks containing tumour material of sufficient size were marked in three spatially discrete areas of representative viable tumour tissue. Sections were then used for total RNA extraction using All Prep DNA/RNA FFPE kit (Qiagen, Hilden, Germany) following vendor's standard protocol. RNA concentrations were measured using Qubit fluorometric quantitation (Thermo Fisher Scientific, Waltham, Mass., USA). RNA Integrity Number and percentage of tRNA <300 bp in size was measured using 2100 Bioanalyzer system (Agilent, CA, USA). RNA were stored at −80c until use in downstream analyses.
Expression of 730 genes, representing 13 major cancer pathways including key driver genes was assessed using nCounter PanCancer Pathways panel (NanoString Technologies, Seattle, Wash., USA). 150 ng total RNA was used as input for hybridisation and digital analysis as per manufacturer's instructions using nCounter Dx analysis system (NanoString Technologies). In cases with high RNA degradation, loading adjustments of up to 300 ng were made. Expression data was processed as follows: a) background correction was done by subtracting the geometric mean of the negative control probes, b) normalised by positive control normalization factor calculated as geometric mean of the positive controls followed by normalisation with the housekeeping genes. Expression values were then Log 2 transformed and subjected to gene-based centring.
A 225-gene subtype predictor was developed using cancer pathway-related gene expression profiles from Nanostring using 38 prototype samples obtained at Royal Marsden Hospital (RMH-SARC). The Classification of Nearest Centroid (CLANC) and cross-validation (random 10% left out in each of 10 cycles) were used to assess the robustness of the minimized gene set for reproducibility of classification19. The 225 genes selected genes contributing to distinguishing the different subtypes are provided in Table 6. The final algorithm consists of centroids constructed as described for the PAM algorithm20 and distances calculated using Spearman's rank correlation (or similar statistical tests to compare similarity). The centroids of the training set using the 225-gene classifier and their contributions are provided in Table 6.
Gene-Expression Based Algorithm to Identify PDGFRA-High/FGFR1-Low IHC Patients which are Intrinsically Resistant to Pazopanib.
A 42-gene predictor for pazopanib resistance was developed based on the 225-gene algorithm. The list of 42 genes provided in Table 7 was selected based on their relative importance contributing to identify the tumours resistant to pazopanib therapy (as defined by PDGFRA-high/FGFR1-low IHC) from others. The final algorithm consists of the two centroids constructed for pazopanib resistant cases and others (Table 7), and the distance respectively calculated using Spearman's rank correlation (or similar statistical tests to compare similarity). A test sample will be assigned to resistant type based on the following formula
A=[Correlation coefficient to Resistant −Correlation coefficient to others]
Building on earlier reports, we assembled a clinically annotated tumour cohort from patients with STS treated with pazopanib at the Royal Marsden Hospital (RMH-SARC). Eligible patients were identified through retrospective search of hospital medical and histopathology records. Eligibility for inclusion was defined as: i) histopathological diagnosis of soft tissue tumour as confirmed by contemporaneous report by specialist sarcoma histopathologist; ii) received at least one dose of pazopanib for treatment of unresectable or advanced STS; and iii) available adequate FFPE tumour specimen, obtained from patient prior to first dose of pazopanib. In total, 38 cases that met eligibility criteria were identified, with collected tumour specimen and associated clinical data included in the analyses described below (clinic-pathological characteristics summarised in Table 1 in example 1, above). Average age was 54.4 years (range 19.8-81.2). Median number of prior lines of therapy was 1.5 (interquartile range (IQR) 0-2). All patients with documented performance status were ECOG 0-2. All but one patient had metastatic disease, with a median of 2 organ sites involved by disease (IQR 1.75-3.25). 16 distinct STS subtypes were represented within our cohort, with leiomyosarcoma and solitary fibrous tumour the most represented subtypes (11 and 7 cases respectively). All but two tumours were intermediate or high grade on archival pre-treatment specimen—of the two cases with low histological grade, one was extraskeletal myxoid chondrosarcoma with metastatic disease, and the other was unresectable solitary fibrous tumour of the retroperitoneum.
Progression free survival (PFS), defined as time in months from first dose of pazopanib to radiological disease progression or death from any cause, was the primary clinical outcome endpoint. Overall survival (OS), defined as a time in months from first dose of pazopanib to death from any cause, was the secondary clinical outcome endpoint. The Kaplan-Meier method was used to estimate PFS and OS, and the log-rank test to compare survival in different strata. Multivariable cox regression model was used to estimate the significance adjusted for the standard clinic-pathological variables (including age, tumour grade and performance status). Interaction tests for biomarkers, FGFR and PDGFR, for survival effect were performed for association with PFS and OS. Proportional hazard assumption was tested using Schoenfeld residuals. Restricted mean progression free and overall all survival estimates were also calculated by the biomarkers strata.
At the time of data collection (median follow-up 26.2 months), 35 of 38 patients (92%) had experienced a progression-free survival event and 31 (82%) had experienced an overall survival event. Median PFS for the cohort was 3.7 months (IQR 1.8-6.9), median OS was 9.5 months (IQR 3.9-19.6 m). Following radiology review of imaging series, 1/38 (2.6%) patient experienced objective radiological response, 20/38 (52.6%) had stable disease and 17/38 (44.7%) progression as best response. For patients with partial response or stable disease, median PFS was 6.4 m (IQR 3.7-12.9).
Our overarching goal in this study was to identify a gene signature that allows for the stratification of patients into the five distinct subgroups without the need to apply the previously reported decision tree workflow. In addition, we sought to identify a set of genes that is capable of identifying PDGFR-high/FGFR1-low IHC intrinsic resistant poor responder cases from other subgroups. Using the Classification of Nearest Centroid (CLANC) methodology described in the methods section, we identified a set of 225 genes that is capable of classifying patients into one of the 5 previously described subgroups (Table 4).
We also identified a 42 gene predictor to classify patients that have intrinsic resistance to pazopanib as defined by the PDGFRA-high/FGFR1-low IHC status (Table 5).
All references cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.
The specific embodiments described herein are offered by way of example, not by way of limitation. Any sub-titles herein are included for convenience only, and are not to be construed as limiting the disclosure in any way.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1712871.1 | Aug 2017 | GB | national |
| 1808941.7 | May 2018 | GB | national |
The present application is a § 371 of International Patent Application No. PCT/EP2018/071758, filed Aug. 10, 2018, which claims priority from GB Application No. 1712871.1, filed Aug. 10, 2017 and GB Application No. 1808941.7, filed May 31, 2018. The entire disclosure of each of the aforesaid applications is incorporated by reference in the present application.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2018/071758 | 8/10/2018 | WO | 00 |
