Patent Application
20230107077
The invention relates to a method of predicting a response of a prostate cancer subject to radiotherapy. Moreover, the invention relates to a diagnostic kit, to a use of the kit in a method of predicting a response of a prostate cancer subject to radiotherapy, to a use of a gene expression profile for each of two or more DNA repair genes in radiotherapy prediction for a prostate cancer subject, and to a corresponding computer program product.
Cancer is a class of diseases in which a group of cells displays uncontrolled growth, invasion and sometimes metastasis. These three malignant properties of cancers differentiate them from benign tumours, which are self-limited and do not invade or metastasize. Prostate Cancer (PCa) is the second most commonly-occurring non-skin malignancy in men, with an estimated 1.3 million new cases diagnosed and 360,000 deaths world-wide in 2018 (see Bray F. et al., “Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries”, CA Cancer
J Clin, Vol. 68, No. 6, pages 394-424, 2018). In the US, about 90% of the new cases concern localized cancer, meaning that metastases have not yet been formed (see ACS (American Cancer Society), “Cancer Facts & FIGS. 2010”, 2010).
For the treatment of primary localized prostate cancer, several radical therapies are available, of which surgery (radical prostatectomy, RP) and radiation therapy
(RT) are most commonly used. RT is administered via an external beam or via the implantation of radioactive seeds into the prostate (brachytherapy) or a combination of both. It is especially preferable for patients who are not eligible for surgery or have been diagnosed with a tumour in an advanced localized or regional stage. Radical RT is provided to up to 50% of patients diagnosed with localized prostate cancer in the US (see ACS, 2010, ibid).
After treatment, prostate cancer antigen (PSA) levels in the blood are measured for disease monitoring. An increase of the blood PSA level provides a biochemical surrogate measure for cancer recurrence or progression. However, the variation in reported biochemical progression-free survival (bPFS) is large (see Grimm P. et al., “Comparative analysis of prostate-specific antigen free survival outcomes for patients with low, intermediate and high risk prostate cancer treatment by radical therapy. Results from the Prostate Cancer Results Study Group”, BJU Int, Suppl. 1, pages 22-29, 2012). For many patients, the bPFS at 5 or even 10 years after radical RT may lie above 90%. Unfortunately, for the group of patients at medium and especially higher risk of recurrence, the bPFS can drop to around 40% at 5 years, depending on the type of RT used (see Grimm P. et al., 2012, ibid).
A large number of the patients with primary localized prostate cancer that are not treated with RT will undergo RP (see ACS, 2010, ibid). After RP, an average of 60% of patients in the highest risk group experience biochemical recurrence after 5 and 10 years (see Grimm P. et al., 2012, ibid). In case of biochemical progression after RP, one of the main challenges is the uncertainty whether this is due to recurring localized disease, one or more metastases or even an indolent disease that will not lead to clinical disease progression (see Dal Pra A. et al., “Contemporary role of postoperative radiotherapy for prostate cancer”,
Transl Androl Urol, Vo. 7, No. 3, pages 399-413, 2018, and Herrera F.G. and Berthold D.R., “Radiation therapy after radical prostatectomy: Implications for clinicians”, Front Oncol, Vol. 6, No. 117, 2016). RT to eradicate remaining cancer cells in the prostate bed is one of the main treatment options to salvage survival after a PSA increase following RP. The effectiveness of salvage radiotherapy (SRT) results in 5-year bPFS for 18% to 90% of patients, depending on multiple factors (see Herrera F.G. and Berthold D.R., 2016, ibid, and Pisansky T.M. et al., “Salvage radiation therapy dose response for biochemical failure of prostate cancer after prostatectomy— A multi-institutional observational study”, Int J Radiat Oncol Biol Phys, Vol. 96, No. 5, pages 1046-1053, 2016).
It is clear that for certain patient groups, radical or salvage RT is not effective.
Their situation is even worsened by the serious side effects that RT can cause, such as bowel inflammation and dysfunction, urinary incontinence and erectile dysfunction (see Resnick M.J. et al., “Long-term functional outcomes after treatment for localized prostate cancer”, N Engl J Med, Vol. 368, No. 5, pages 436-445, 2013, and Hegarty S.E. et al., “Radiation therapy after radical prostatectomy for prostate cancer: Evaluation of complications and influence of radiation timing on outcomes in a large, population-based cohort”, PLoS One, Vol. 10, No. 2, 2015). In addition, the median cost of one course of RT based on Medicare reimbursement is $18,000, with a wide variation up to about $40,000 (see Paravati A.J. et al., “Variation in the cost of radiation therapy among medicare patients with cancer”, J Oncol
Pract, Vol. 11, No. 5, pages 403-409, 2015). These figures do not include the considerable longitudinal costs of follow-up care after radical and salvage RT.
An improved prediction of effectiveness of RT for each patient, be it in the radical or the salvage setting, would improve therapy selection and potentially survival. This can be achieved by 1) optimizing RT for those patients where RT is predicted to be effective (e.g., by dose escalation or a different starting time) and 2) guiding patients where RT is predicted not to be effective to an alternative, potentially more effective form of treatment. Further, this would reduce suffering for those patients who would be spared ineffective therapy and would reduce costs spent on ineffective therapies.
Numerous investigations have been conducted into measures for response prediction of radical RT (see Hall W.A. et al., “Biomarkers of outcome in patients with localized prostate cancer treated with radiotherapy”, Semin Radiat Oncol, Vol. 27, pages 11-20, 2016, and Raymond E. et al., “An appraisal of analytical tools used in predicting clinical outcomes following radiation therapy treatment of men with prostate cancer: A systematic review”, Radiat Oncol, Vol. 12, No. 1, page 56, 2017) and SRT (see Herrera F.G. and Berthold D.R., 2016, ibid). Many of these measures depend on the concentration of the blood-based biomarker PSA. Metrics investigated for prediction of response before start of RT (radical as well as salvage) include the absolute value of the PSA concentration, its absolute value relative to the prostate volume, the absolute increase over a certain time and the doubling time. Other frequently considered factors are the Gleason score and the clinical tumour stage. For the SRT setting, additional factors are relevant, e.g., surgical margin status, time to recurrence after RP, pre-/peri-surgical PSA values and clinico-pathological parameters.
Although these clinical variables provide limited improvements in patient stratification in various risk groups, there is a need for better predictive tools.
A wide range of biomarker candidates in tissue and bodily fluids has been investigated, but validation is often limited and generally demonstrates prognostic information and not a predictive (therapy-specific) value (see Hall W.A. et al., 2016, ibid). A small number of gene expression panels is currently being validated by commercial organizations. One or a few of these may show predictive value for RT in future (see Dal Pra A. et al., 2018, ibid).
In conclusion, a strong need for better prediction of response to RT remains, for primary prostate cancer as well as for the post-surgery setting.
The Affymetrix GeneChip Human Genome U133 Array Set HG-U133A includes over 1,000,000 unique oligonucleotide features covering more than 39,000 transcript variants, which in turn represent greater than 33,000 human genes.
Den R.B. et al., “Genomic Classifier Identifies Men With Adverse Pathology After Radical Prostatectomy Who Benefit From Adjuvant Radiation Therapy”, Journal of
Clinical Oncology, Vol. 33, No. 8, pages 944-951, 2015, starts out from the premise that the optimal timing of postoperative radiotherapy (RT) after radical prostatectomy (RP) is unclear. It finds that a genomic classifier (GC) is prognostic for the development of clinical metastasis beyond routine clinical and pathologic features.
Kratochwil C. et al., “Patients Resistant Against PSMA-Targeting a-Radiation Therapy Often Harbor Mutations in DNA Damage-Repair-Associated Genes”, The Joural of Nuclear Medicine, Vol. 61, No. 5, pages 683-688, 2019, discloses that prostate-specific membrane antigen (PSMA)-targeting a-radiation therapy (TAT) is an emerging treatment modality for metastatic castration-resistant prostate cancer, wherein there is a subgroup of patients with poor response despite sufficient expression of PSMA in their tumors. The document aims at characterizing PSMA-TAT-nonresponding lesions by targeted next-generation sequencing.
Chen X. et al., “IL-6 signaling contributes to radioresistance of prostate cancer through key DNA repair-associated molecules ATM, ATR, and BRCA 1/2”, Journal of Cancer Research and Clinical Oncology, Journal of Cancer Research and Clinical Oncology, Vol. 145, No. 6, pages 1471-1484, 2019, studies an association between IL-6 signaling and resistance to radiotherapy of prostate cancer (PCa) and explores the molecular mechanisms involved.
It is an objective of the invention to provide a method of predicting a response of a prostate cancer subject to radiotherapy, which allows to make better treatment decisions. It is a further objective of the invention to provide a diagnostic kit, a use of the kit in a method of predicting a response of a prostate cancer subject to radiotherapy, a use of a gene expression profile for each of two or more DNA repair genes in radiotherapy prediction for a prostate cancer subject, and a corresponding computer program product.
In a first aspect of the present invention, a method of predicting a response of a prostate cancer subject to radiotherapy is presented, comprising:
determining or receiving the result of a determination of a gene expression profile for each of two or more, for example, 2, 3, 4 or all, DNA repair genes selected from the group consisting of: APTX, BRCA2, NUDT1, PMS2, and POLD2, said gene expression profiles being determined in a biological sample obtained from the subject, determining, preferably by a processor, the prediction of the radiotherapy response based on the gene expression profiles for the two or more DNA repair genes, and optionally, providing the prediction or a therapy recommendation based on the prediction to a medical caregiver or the subject.
In recent years, the importance of the immune system in cancer inhibition as well as in cancer initiation, promotion and metastasis has become very evident (see
Mantovani A. et al., “Cancer-related inflammation”, Nature, Vol. 454, No. 7203, pages 436-444, 2008, and Giraldo N.A. et al., “The clinical role of the TME in solid cancer”, Br J Cancer, Vol. 120, No. 1, pages 45-53, 2019). The immune cells and the molecules they secrete form a crucial part of the tumour microenvironment and most immune cells can infiltrate the tumour tissue. The immune system and the tumour affect and shape one another.
Thus, anti-tumour immunity can prevent tumour formation while an inflammatory tumour environment may promote cancer initiation and proliferation. At the same time, tumour cells that may have originated in an immune system-independent manner will shape the immune microenvironment by recruiting immune cells and can have a pro-inflammatory effect while also suppressing anti-cancer immunity.
Some of the immune cells in the tumour microenvironment will have either a general tumour-promoting or a general tumour-inhibiting effect, while other immune cells exhibit plasticity and show both tumour-promoting and tumour-inhibiting potential. Thus, the overall immune microenvironment of the tumour is a mixture of the various immune cells present, the cytokines they produce and their interactions with tumour cells and with other cells in the tumour microenvironment (see Giraldo N.A. et al., 2019, ibid).
The principles described above with regard to the role of the immune system in cancer in general also apply to prostate cancer. Chronic inflammation has been linked to the formation of benign as well as malignant prostate tissue (see Hall W.A. et al., 2016, ibid) and most prostate cancer tissue samples show immune cell infiltrates. The presence of specific immune cells with a pro-tumour effect has been correlated with worse prognosis, while tumours in which natural killer cells were more activated showed better response to therapy and longer recurrence-free periods (see Shiao S.L. et al., “Regulation of prostate cancer progression by tumor microenvironment”, Cancer Lett, Vol. 380, No. 1, pages 340-348, 2016).
While a therapy will be influenced by the immune components of the tumour microenvironment, RT itself extensively affects the make-up of these components (see Barker H.E. et al., “The tumor microenvironment after radiotherapy: Mechanisms of resistance or recurrence”, Nat Rev Cancer, Vol. 15, No. 7, pages 409-425, 2015). Because suppressive cell types are comparably radiation-insensitive, their relative numbers will increase. Counteractively, the inflicted radiation damage activates cell survival pathways and stimulates the immune system, triggering inflammatory responses and immune cell recruitment. Whether the net effect will be tumour-promoting or tumour-suppressing is as yet uncertain, but its potential for enhancement of cancer immunotherapies is being investigated.
The present invention is based on the idea that, since the status of the immune system and of the immune microenvironment have an impact on therapy effectiveness, the ability to identify markers predictive for this effect might help to be better able to predict overall RT response.
DNA repair system has evolved to maintain the genomic integrity to defend against both endogenous and exogenous sources of DNA damage, such as endogenous factors include reactive oxygen species, replication errors or mistakes in meiosis and exogenous factors include ultraviolet (UV) radiation, ionizing radiation (IR), and some other chemicals or chemotherapeutic agents (see Kang Z. et al., “DNA repair in cancer”, J Oncol,
Vol. 2019, Article 8676947). Multiple repair pathways (direct repair, base excision repair, nucleotide excision repair, mismatch repair, nonhomologous end joining, and homologous recombination pathways) can be aroused from the diverse forms of DNA lesions including mismatch paired bases, small deletions or insertions, and DNA single or double-strand breaks. These repair pathways also exert crosstalk with others to complete the whole DNA repair process.
The deficient DNA repair causing prolonged existence of DNA damages can lead to genes mutations, chromosome rearrangements, genomic instability, and finally carcinogenesis. Indeed, defects in DNA repair pathways contribute to many heritable cancer predisposition syndromes; however, cancer-related DNA repair deficiency may also occur in sporadic cancer case. Defective DNA repair is common in carcinogenesis and plays a critical role in cancer progression. For example, genetic mutations in DNA mismatch repair genes are involved in reducing mismatch repair and increasing the risk to colon and uterine tumors; BRCA1, BRCA2, and PALB2 genes mutations result in defective homologous recombination repair and are associated with the carcinogenesis of breast and ovarian cancer. In these years, many cancer-related germline mutations in DNA repair have been reported; thus to detect these genetic variations gives us a chance to evaluate the cancer risk of the individual with these mutations.
Furthermore, these defects in DNA repair pathways may have therapeutic implications for clinical practice. Most recently, a series of therapeutic strategy have been exploited, such as platinum chemotherapies and PARP inhibitors in homologous recombination defected breast and ovarian cancers and inhibitors of immune checkpoints CTLA-4, PD1/PD-L1 in the case of mismatch repair deficiency cancers. These observations give the direction for further research to investigate the defects in DNA repair pathways that may serve as very useful biomarkers for the choice of suitable oncotherapy.
The identified DNA repair genes APTX, BRCA2, NUDT1, PMS2, and POLD2 were identified as follows: A group of 538 prostate cancer patients were treated with RP and the prostate cancer tissue was stored. A number of these patients experienced biochemical recurrence and was treated with SRT. For 151 of these patients, the RNA expressed in the originally stored prostate cancer tissue was analysed using RNA sequencing. The mRNA expression of DNA repair related genes was compared for the 26 out of 151 patients that died due to prostate cancer, versus the 125 out of 151 patients who survived. For the five molecules APTX, BRCA2, NUDT1, PMS2, and POLD2, the expression level was significantly different for the survivors, suggesting that they have value in the prediction of survival after SRT. Several of these five molecules were found to correlate with prostate cancer, be it in different settings, as described further below.
The term “APTX” refers to the Aprataxin gene (Ensembl: ENSG00000137074), for example, to the sequence as defined in NCBI Reference Sequence NM 175073 or in NCBI Reference Sequence NM 001195254 or in NCBI Reference Sequence NM_175069 or in NCBI Reference Sequence NM_001195250, specifically, to the nucleotide sequences as set forth in SEQ ID NO:1 or in SEQ ID NO:2 or in SEQ ID NO:3 or in SEQ ID NO:4, which correspond to the sequences of the above indicated NCBI Reference Sequences of the APTX transcript, and also relates to the corresponding amino acid sequences for example as set forth in SEQ ID NO:5 or in SEQ ID NO:6 or in SEQ ID NO:7 or in SEQ ID NO:8, which correspond to the protein sequences defined in NCBI Protein Accession Reference Sequence NP_778243 and in NCBI Protein Accession Reference Sequence NP_001182183 and in NCBI Protein Accession Reference Sequence NP_778239 and in NCBI Protein Accession Reference Sequence NP_001182179 encoding the APTX polypeptide.
The term “APTX” also comprises nucleotide sequences showing a high degree of homology to APTX, e.g., nucleic acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in
SEQ ID NO:1 or in SEQ ID NO:2 or in SEQ ID NO:3 or in SEQ ID NO:4 or amino acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:5 or in SEQ ID NO:6 or in SEQ ID NO:7 or in SEQ ID NO:8 or nucleic acid sequences encoding amino acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:5 or in SEQ ID NO:6 or in SEQ ID NO:7 or in SEQ ID NO:8 or amino acid sequences being encoded by nucleic acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:1 or in SEQ ID NO:2 or in SEQ ID NO:3 or in SEQ ID NO:4.
The term “BRAC2” refers to the BRAC2 DNA Repair Associated gene (Ensembl: ENSG00000139618), for example, to the sequence as defined in NCBI Reference Sequence NM_000059, specifically, to the nucleotide sequence as set forth in SEQ ID NO:9, which corresponds to the sequence of the above indicated NCBI Reference Sequence of the BRAC2 transcript, and also relates to the corresponding amino acid sequence for example as set forth in SEQ ID NO:10, which corresponds to the protein sequence defined in NCBI Protein Accession Reference Sequence NP_000050 encoding the BRAC2 polypeptide.
The term “BRAC2” also comprises nucleotide sequences showing a high degree of homology to BRAC2, e.g., nucleic acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:9 or amino acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:10 or nucleic acid sequences encoding amino acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:10 or amino acid sequences being encoded by nucleic acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:9.
The term “NUDT1” refers to the Nudix Hydrolase 1 gene (Ensembl: ENSG00000106268), for example, to the sequence as defined in NCBI Reference Sequence
NM_198954 or in NCBI Reference Sequence NM_198953, specifically, to the nucleotide sequences as set forth in SEQ ID NO:11 or in SEQ ID NO:12, which correspond to the sequences of the above indicated NCBI Reference Sequences of the NUDT1 transcript, and also relates to the corresponding amino acid sequences for example as set forth in SEQ ID NO:13 or in SEQ ID NO:14, which correspond to the protein sequences defined in NCBI
Protein Accession Reference Sequence NP_945192 and in NCBI Protein Accession Reference Sequence NP_945191 encoding the NUDT1 polypeptide.
The term “NUDT1” also comprises nucleotide sequences showing a high degree of homology to NUDT1, e.g., nucleic acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:11 or in SEQ ID NO:12 or amino acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:13 or in SEQ ID NO:14 or nucleic acid sequences encoding amino acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:13 or in
SEQ ID NO:14 or amino acid sequences being encoded by nucleic acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:11 or in SEQ ID NO:12.
The term “PMS2” refers to the PMS1 Homolog 2, Mismatch Repair System Component gene (Ensembl: ENSG00000122512), for example, to the sequence as defined in
NCBI Reference Sequence NM_000535 or in NCBI Reference Sequence NM_001322005 or in NCBI Reference Sequence NM_001322008, specifically, to the nucleotide sequences as set forth in SEQ ID NO:15 or in SEQ ID NO:16 or in SEQ ID NO:17, which correspond to the sequences of the above indicated NCBI Reference Sequences of the PMS2 transcript, and also relates to the corresponding amino acid sequences for example as set forth in SEQ ID NO:18 or in SEQ ID NO:19 or in SEQ ID NO:20, which correspond to the protein sequences defined in NCBI Protein Accession Reference Sequence NP_000526 and in NCBI Protein Accession Reference Sequence NP_001308934 and in NCBI Protein Accession Reference Sequence NP_001308937 encoding the PMS2 polypeptide.
The term “PMS2” also comprises nucleotide sequences showing a high degree of homology to PMS2, e.g., nucleic acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:15 or in SEQ ID NO:16 or in SEQ ID NO:17 or amino acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:18 or in SEQ ID NO:19 or in SEQ ID NO:20 or nucleic acid sequences encoding amino acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:18 or in SEQ ID NO:19 or in SEQ ID NO:20 or amino acid sequences being encoded by nucleic acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:15 or in SEQ ID NO:16 or in SEQ ID NO:17.
The term “POLD2” refers to the DNA Polymerase Delta 2, Accessory Subunit gene (Ensembl: ENSG00000106628), for example, to the sequence as defined in NCBI Reference Sequence NM_001127218 or in NCBI Reference Sequence NM_006230, specifically, to the nucleotide sequences as set forth in SEQ ID NO:21 or in SEQ ID NO:22, which correspond to the sequences of the above indicated NCBI Reference Sequences of the POLD2 transcript, and also relates to the corresponding amino acid sequences for example as set forth in SEQ ID NO:23 or in SEQ ID NO:24, which correspond to the protein sequences defined in NCBI Protein Accession Reference Sequence NP_001120690 and in NCBI
Protein Accession Reference Sequence NP_006221 encoding the POLD2 polypeptide.
The term “POLD2” also comprises nucleotide sequences showing a high degree of homology to POLD2, e.g., nucleic acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:21 or in SEQ ID NO:22 or amino acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:23 or in SEQ ID NO:24 or nucleic acid sequences encoding amino acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:23 or in SEQ ID NO:24 or amino acid sequences being encoded by nucleic acid sequences being at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence as set forth in SEQ ID NO:21 or in SEQ ID NO:22.
The term “biological sample” or “sample obtained from a subject” refers to any biological material obtained via suitable methods known to the person skilled in the art from a subject, e.g., a prostate cancer patient. The biological sample used may be collected in a clinically acceptable manner, e.g., in a way that nucleic acids (in particular RNA) or proteins are preserved.
The biological sample(s) may include body tissue and/or a fluid, such as, but not limited to, blood, sweat, saliva, and urine. Furthermore, the biological sample may contain a cell extract derived from or a cell population including an epithelial cell, such as a cancerous epithelial cell or an epithelial cell derived from tissue suspected to be cancerous. The biological sample may contain a cell population derived from a glandular tissue, e.g., the sample may be derived from the prostate of a male subject. Additionally, cells may be purified from obtained body tissues and fluids if necessary, and then used as the biological sample. In some realizations, the sample may be a tissue sample, a urine sample, a urine sediment sample, a blood sample, a saliva sample, a semen sample, a sample including circulating tumour cells, extracellular vesicles, a sample containing prostate secreted exosomes, or cell lines or cancer cell line.
In one particular realization, biopsy or resections samples may be obtained and/or used. Such samples may include cells or cell lysates.
It is also conceivable that the content of a biological sample is submitted to an enrichment step. For instance, a sample may be contacted with ligands specific for the cell membrane or organelles of certain cell types, e.g., prostate cells, functionalized for example with magnetic particles. The material concentrated by the magnetic particles may subsequently be used for detection and analysis steps as described herein above or below.
Furthermore, cells, e.g., tumour cells, may be enriched via filtration processes of fluid or liquid samples, e.g., blood, urine, etc. Such filtration processes may also be combined with enrichment steps based on ligand specific interactions as described herein above.
The term “prostate cancer” refers to a cancer of the prostate gland in the male reproductive system, which occurs when cells of the prostate mutate and begin to multiply out of control. Typically, prostate cancer is linked to an elevated level of prostate-specific antigen (PSA). In one embodiment of the present invention the term “prostate cancer” relates to a cancer showing PSA levels above 3.0. In another embodiment the term relates to cancer showing PSA levels above 2.0. The term “PSA level” refers to the concentration of PSA in the blood in ng/ml.
The term “non-progressive prostate cancer state” means that a sample of an individual does not show parameter values indicating “biochemical recurrence” and/or “clinical recurrence” and/or “metastases” and/or “castration-resistant disease” and/or “prostate cancer or disease specific death”.
The term “progressive prostate cancer state” means that a sample of an individual shows parameter values indicating “biochemical recurrence” and/or “clinical recurrence” and/or “metastases” and/or “castration-resistant disease” and/or “prostate cancer or disease specific death”.
The term “biochemical recurrence” generally refers to recurrent biological values of increased PSA indicating the presence of prostate cancer cells in a sample. However, it is also possible to use other markers that can be used in the detection of the presence or that rise suspicion of such presence.
The term “clinical recurrence” refers to the presence of clinical signs indicating the presence of tumour cells as measured, for example using in vivo imaging.
The term “metastases” refers to the presence of metastatic disease in organs other than the prostate.
The term “castration-resistant disease” refers to the presence of hormone-insensitive prostate cancer; i.e., a cancer in the prostate that does not any longer respond to androgen deprivation therapy (ADT).
The term “prostate cancer specific death or disease specific death” refers to death of a patient from his prostate cancer.
It is preferred that the two or more DNA repair genes comprise three or more, preferably, all of the DNA repair genes.
It is preferred that the determining of the prediction of the radiotherapy response comprises combining the gene expression profiles for two or more, for example, 2, 3, 4 or all, of the DNA repair genes with a regression function that had been derived from a population of prostate cancer subjects.
Cox proportional-hazards regression allows analyzing the effect of several risk factors on time to a tested event like survival. Thereby, the risk factors maybe dichotomous or discrete variables like a risk score or a clinical stage but may also be a continuous variable like a biomarker measurement or gene expression values. The probability of the endpoint (e.g., death or disease recurrence) is called the hazard. Next to the information on whether or not the tested endpoint was reached by e.g. subject in a patient cohort (e.g., patient did die or not) also the time to the endpoint is considered in the regression analysis. The hazard is modeled as: H(t)=Ho(t) exp(wiVi+w2 V2+W3 V3+), where Vi, V2, V3 . . . are predictor variables and Ho(t) is the baseline hazard while H(t) is the hazard at any time t. The hazard ratio (or the risk to reach the event) is represented by Ln[H(t)/Ho(t)]=wi.Vi+w2. V2 +W3 V3+ . . . , where the coefficients or weights w1, w2, w3 . . . are estimated by the Cox regression analysis and can be interpreted in a similar manner as for logistic regression analysis.
In one particular realization, the prediction of the radiotherapy response is determined as follows:
(w1·APTX)+(w2·BRCA2)+(w3NUDT1)+(w4PMS2)+(w5POLD2) (1)
where w1 to w5 are weights and APTX, BRCA2, NUDT1, PMS2, and POLD2 are the expression levels of the DNA repair genes.
In one example, w1 may be about −1.0 to 0.0, such as −0.4783, w2 may be about 0.5 to 1.5, such as 1.0455, w3 may be about 0.5 to 1.5, such as 1.1879, w4 may be about −2.0 to −1.0, such as −1.5361, and w5 may be about −2.0 to −1.0, such as −1.7216. The prediction of the radiotherapy response may also be classified or categorized into one of at least two risk groups, based on the value of the prediction of the radiotherapy response. For example, there may be two risk groups, or three risk groups, or four risk groups, or more than four predefined risk groups. Each risk group covers a respective range of (non-overlapping) values of the prediction of the radiotherapy response. For example, a risk group may indicate a probability of occurrence of a specific clinical event from 0 to <0.1 or from 0.1 to <0.25 or from 0.25 to <0.5 or from 0.5 to 1.0 or the like.
It is further preferred that the determining of the prediction of the radiotherapy response is further based on one or more clinical parameters obtained from the subject.
As mentioned above, various measures based on clinical parameters have been investigated. By further basing the prediction of the radiotherapy response on such clinical parameter(s), it can be possible to further improve the prediction.
It is preferred that the one or more clinical parameters comprise one or more of: (i) a prostate-specific antigen (PSA) level; (ii) a pathologic Gleason score (pGS); iii) a clinical tumour stage; iv) a pathological Gleason grade group (pGGG); v) a pathological stage; vi) one or more pathological variables, for example, a status of surgical margins and/or a lymph node invasion and/or an extra-prostatic growth and/or a seminal vesicle invasion; vii) CAPRA-S; and viii) another clinical risk score.
It is further preferred that the determining of the prediction of the radiotherapy response comprises combining the gene expression profiles for the two or more DNA repair genes and the one or more clinical parameters obtained from the subject with a regression function that had been derived from a population of prostate cancer subjects.
In one particular realization, the prediction of the radiotherapy response is determined as follows:
(w6·DNArep_model)+(w7·pGGG) (2)
where w6 and w7 are weights, DNArep_model is the above-described regression model based on the expression profiles for the two or more, for example, 2, 3, 4 or all, of the DNA repair genes, and pGGG is the pathological Gleason grade group.
In one example, w6 may be about 0.5 to 1.5, such as 0.9015, and w7 may be about 0.5 to 1.5, such as 0.7818.
It is preferred that the biological sample is obtained from the subject before the start of the radiotherapy. The gene expression profiles may be determined in the form of mRNA or protein in tissue of prostate cancer. Alternatively, if the DNA repair genes are present in a soluble form, the gene expression profiles may be determined in blood.
It is further preferred that the radiotherapy is radical radiotherapy or salvage radiotherapy.
It is preferred that the prediction of the radiotherapy response is negative or positive for the effectiveness of the radiotherapy, wherein a therapy is recommended based on the prediction and, if the prediction is negative, the recommended therapy comprises one or more of: (i) radiotherapy provided earlier than is the standard; (ii) radiotherapy with an increased radiation dose; (iii) an adjuvant therapy, such as androgen deprivation therapy; and iv) an alternative therapy that is not a radiation therapy. The degree to which the prediction is negative may determine the degree to which the recommended therapy deviates from the standard form of radiotherapy.
In a further aspect of the present invention, an apparatus for predicting a response of a prostate cancer subject to radiotherapy is presented, comprising:
In a further aspect of the present invention, a computer program product is presented comprising instructions which, when the program is executed by a computer, cause the computer to carry out a method comprising:
BRCA2, NUDT1, PMS2, and POLD2, said gene expression profiles being determined in a biological sample obtained from a prostate cancer subject,
In a further aspect of the present invention, a diagnostic kit is presented, comprising:
In a further aspect of the present invention, a use of the kit as defined in claim 12 is presented.
It is preferred that the use as defined in claim 13 is in a method of predicting a response of a prostate cancer subject to radiotherapy.
In a further aspect of the present invention, a method is presented, comprising:
In a further aspect of the present invention, a use of a gene expression profile for each of two or more, for example, 2, 3, 4 or all, DNA repair genes selected from the group consisting of: APTX, BRCA2, NUDT1, PMS2, and POLD2, in a method of predicting a response of a prostate cancer subject to radiotherapy is presented, comprising:
determining, preferably by a processor, the prediction of the radiotherapy response based on the gene expression profiles for the two or more DNA repair genes, and optionally, providing the prediction or a therapy recommendation based on the prediction to a medical caregiver or the subject.
It shall be understood that the method of claim 1, the apparatus of claim 10, the computer program product of claim 11, the diagnostic kit of claim 12, the use of the diagnostic kit of claim 13, the method of claim 15, and the use of the gene expression profiles of claim 16 have similar and/or identical preferred embodiments, in particular, as defined in the dependent claims.
It shall be understood that a preferred embodiment of the present invention can also be any combination of the dependent claims or above embodiments with the respective independent claim.
These and other aspects of the invention will be apparent from and elucidated with reference to the embodiments described hereinafter.
In the following drawings:
(DNArep 2.4 model). The clinical endpoint that was tested was the time to prostate cancer specific death (PCa Death) after the start of salvage radiation therapy (SRT) due to post-surgical disease recurrence.
(DNArep 3.2 model). The clinical endpoint that was tested was the time to prostate cancer specific death (PCa Death) after the start of salvage radiation therapy (SRT) due to post-surgical disease recurrence.
Overview Of Radiotherapy Response Prediction
The method begins at step S100.
At step S102, a biological sample is obtained from each of a first set of patients (subjects) diagnosed with prostate cancer. Preferably, monitoring prostate cancer has been performed for these prostate cancer patients over a period of time, such as at least one year, or at least two years, or about five years, after obtaining the biological sample.
At step S104, a gene expression profile for each of two or more, for example, 2, 3, 4 or all, DNA repair genes selected from the group consisting of: APTX, BRCA2, NUDT1, PMS2, and POLD2, is obtained for each of the biological samples obtained from the first set of patients, e.g., by performing RT-qPCR (real-time quantitative PCR) on RNA extracted from each biological sample. The exemplary gene expression profiles include an expression level (e.g., value) for each of the two or more DNA repair genes.
At step S106, a regression function for assigning a prediction of the radiotherapy response is determined based on the gene expression profiles for the two or more DNA repair genes, APTX, BRCA2, NUDT1, PMS2, and/or POLD2, obtained for at least some of the biological samples obtained for the first set of patients and respective results obtained from the monitoring. In one particular realization, the regression function is determined as specified in Eq. (1) above.
At step S108, a biological sample is obtained from a patient (subject or individual). The patient can be a new patient or one of the first set.
At step S110, a gene expression profile is obtained for each of the two or more, for example, 2, 3, 4 or all, DNA repair genes, e.g., by performing PCR on the biological sample.
At step S112, a prediction of the radiotherapy response based on the gene expression profiles for the two or more DNA repair genes is determined for the patient using the regression function. This will be described in more detail later in the description.
At S114, a therapy recommendation may be provided, e.g., to the patient or his or her guardian, to a doctor, or to another healthcare worker, based on the prediction. To this end, the prediction may be categorized into one of a predefined set of risk groups, based on the value of the prediction. In one particular realization, the prediction of the radiotherapy response may be negative or positive for the effectiveness of the radiotherapy. If the prediction is negative, the recommended therapy may comprise one or more of: (i) radiotherapy provided earlier than is the standard; (ii) radiotherapy with an increased radiation dose; (iii) an adjuvant therapy, such as androgen deprivation therapy; and iv) an alternative therapy that is not a radiation therapy.
The method ends at S116.
In one embodiment, the gene expression profiles at steps S104 and S110 are determined by detecting mRNA expression using two or more primers and/or probes and/or two or more sets thereof.
In one embodiment, steps S104 and S110 further comprise obtaining one or more clinical parameters from the first set of patients and the patient, respectively. The one or more clinical parameters may comprise one or more of: (i) a prostate-specific antigen (PSA) level; (ii) a pathologic Gleason score (pGS); iii) a clinical tumour stage; iv) a pathological Gleason grade group (pGGG); v) a pathological stage; vi) one or more pathological variables, for example, a status of surgical margins and/or a lymph node invasion and/or an extra-prostatic growth and/or a seminal vesicle invasion; vii) CAPRA-S; and viii) another clinical risk score. The regression function for assigning the prediction of the radiotherapy response that is determined in step S106 is then further based on the one or more clinical parameters obtained from at least some of the first set of patients. In step S112, the prediction of the radiotherapy response is then further based on the one or more clinical parameters, e.g., the pathological Gleason grade group (pGGG), obtained from the patient and is determined for the patient using the regression function.
The immune system interacts in a strong manner with prostate cancer, both on a systemic level and in the tumour microenvironment. DNA repair genes play a central role in the regulation of immune activity. DNA repair genes may therefore provide information on the effectiveness of RT. However, which DNA repair genes may have predictive value in this application is extremely difficult to deduce from existing literature due to the many factors that influence the exact function of DNA repair genes.
We investigated the extent to which the expression of DNA repair genes in prostate cancer tissue correlates with the recurrence of disease after radical RT or SRT.
We have identified five members of the family of DNA repair genes for which the degree of expression in prostate cancer tissue significantly correlates with mortality after SRT, in a cohort of 151 prostate cancer patients (TABLE 1).
Based on the significant correlation with outcome after RT, we expect that the identified molecules will provide predictive value with regard to the effectiveness of radical RT and/or SRT.
We then set out to test whether the combination of these five DNA repair genes will exhibit more prognostic value. With Cox regression we modelled the expression levels of the five DNA repair genes to prostate cancer specific death after post-surgical salvage RT either with (DNArep&pGGG model) or without (DNArep_model) the presence of the variable pathological Gleason grade group (pGGG). We tested the two models in ROC curve analysis as well as in Kaplan-Meier survival analysis.
The Cox regression functions were derived as follows:
DNArep_model:
(w1 ·APTX)+(w2 ·BRCA2)+(w3 ·NUDT1)+(w4 ·PMS2)+(w5 ·POLD2)
DNArep&pGGG model:
(w6·DNArep_model)+(w7·pGGG)
The details for the weights wi to w7 are shown in the following TABLE 3.
Next, we tested the combined Cox regression model as outlined above its power to predict 10-year prostate cancer specific death after start of salvage radiation due to post-surgical disease recurrence. The performance of the model was compared to the clinical risk score CAPRA-S(see Cooperberg M.R. et al., “The CAPRA-S score: A straightforward tool for improved prediction of outcomes after radical prostatectomy”, Cancer, Vol. 117, No. 22, pages 5039-5046, 2011).
For Kaplan-Meier curve analysis, the Cox regression function of the two risk models (DNArep_model and DNArep&pGGG model) was categorized into two sub-cohorts based on a cut-off (see description of figures below). The goal was to create patient classes by separating the classes with the mean risk score as calculated from the DNArep_model and DNArep&pGGG model, respectively.
The patient classes represent an increasing risk to experience the tested clinical endpoints of time to development of metastases (
For example, the Kaplan-Meier analysis as shown in
This section shows additional results for Cox regression models based on only two and three of the identified DNA repair genes, respectively. In total, eight different 2 gene models and four different 3 gene models were tested. The details for the weights are shown in the following TABLES 4 and 5, respectively.
For Kaplan-Meier curve analysis, the Cox regression function of the twelve risk models (DNArep_2.1 model to DNArep_2.8 model and DNArep_3.1 model to DNArep_3.4 model) was categorized into two sub-cohorts based on a cut-off (see description of figures below), as described above.
The patient classes represent an increasing risk to experience the tested clinical endpoint of time to prostate cancer specific death since the start of salvage RT for the created risk models.
The Kaplan-Meier analysis as shown in
The effectiveness of both radical RT and SRT for localized prostate cancer is limited, resulting in disease progression and ultimately death of patients, especially for those at high risk of recurrence. The prediction of the therapy outcome is very complicated as many factors play a role in therapy effectiveness and disease recurrence. It is likely that important factors have not yet been identified, while the effect of others cannot be determined precisely. Multiple clinico-pathological measures are currently investigated and applied in a clinical setting to improve response prediction and therapy selection, providing some degree of improvement. Nevertheless, a strong need remains for better prediction of the response to radical RT and to SRT, in order to increase the success rate of these therapies.
We have identified molecules of which expression shows a significant relation to mortality after radical RT and SRT and therefore are expected to improve the prediction of the effectiveness of these treatments. An improved prediction of effectiveness of RT for each patient be it in the radical or the salvage setting, will improve therapy selection and potentially survival. This can be achieved by 1) optimizing RT for those patients where RT is predicted to be effective (e.g. by dose escalation or a different starting time) and 2) guiding patients where RT is predicted not to be effective to an alternative, potentially more effective form of treatment. Further, this would reduce suffering for those patients who would be spared ineffective therapy and would reduce cost spent on ineffective therapies.
Other variations to the disclosed realizations can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims.
In the claims, the word “comprising” does not exclude other elements or steps, and the indefinite article “a” or “an” does not exclude a plurality.
One or more steps of the method illustrated in
Alternatively, the one or more steps of the method may be implemented in transitory media, such as a transmittable carrier wave in which the control program is embodied as a data signal using transmission media, such as acoustic or light waves, such as those generated during radio wave and infrared data communications, and the like.
The exemplary method may be implemented on one or more general purpose computers, special purpose computer(s), a programmed microprocessor or microcontroller and peripheral integrated circuit elements, an ASIC or other integrated circuit, a digital signal processor, a hardwired electronic or logic circuit such as a discrete element circuit, a programmable logic device such as a PLD, PLA, FPGA, Graphical card CPU (GPU), or PAL, or the like. In general, any device, capable of implementing a finite state machine that is in turn capable of implementing the flowchart shown in
The computer program instructions may also be loaded onto a computer, other programmable data processing apparatus, or other devices to cause a series of operational steps to be performed on the computer, other programmable apparatus or other devices to produce a computer implemented process such that the instructions which execute on the computer or other programmable apparatus provide processes for implementing the functions/acts specified herein.
Any reference signs in the claims should not be construed as limiting the scope.
The invention relates to a method of predicting a response of a prostate cancer subject to radiotherapy, comprising determining or receiving the result of a determination of a gene expression profile for each of two or more, for example, 2, 3, 4 or all, DNA repair genes selected from the group consisting of: APTX, BRCA2, NUDT1, PMS2, and POLD2, said gene expression profiles being determined in a biological sample obtained from the subject, determining, preferably by a processor, the prediction of the radiotherapy response based on the gene expression profiles for the two or more DNA repair genes, and optionally, providing the prediction or a therapy recommendation based on the prediction to a medical caregiver or the subject. Since the status of the immune system and of the immune microenvironment have an impact on therapy effectiveness, the ability to identify markers predictive for this effect might help to be better able to predict overall RT response. DNA repair genes play a central role in the regulation of immune activity. The identified DNA repair genes were found to exhibit a significant correlation with outcome after RT, wherefore we expect that they will provide predictive value with regard to the effectiveness of radical RT and/or SRT.
The attached Sequence Listing, entitled 2020PF00060_Sequence Listing_ST25 is incorporated herein by reference, in its entirety.
| Number | Date | Country | Kind |
|---|---|---|---|
| 20164829.2 | Mar 2020 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2021/056833 | 3/17/2021 | WO |
