Patent Application
20210341482
The present invention relates to cancer biomarkers, and in particular to cancer biomarkers that may be used to identify cancers that are expected to respond to endocrine and/or chemotherapy treatment. The invention also includes methods of treating cancers. The invention also extends to prognostic methods and kits for identifying cancers that are expected to respond to endocrine and/or chemotherapy treatment.
Of the 1.38 million newly diagnosed breast cancer (BC) cases each year, 65-70% of them are oestrogen receptor positive (ER+). Although the single-agent endocrine therapy (ET), which aims to prevent ER signaling through either selective ER modulators (SERM) (e.g., tamoxifen) or aromatase inhibitors (AIs) has significantly extended survival of ER+BC patients, resistance to ET is common (up to 50%). To further extend treatment benefit and delay the development of ET resistance, a combination of ET with chemotherapy (CT) with or without other modulators e.g., mammalian target of rapamycin (mTOR) inhibitors has been proven to be an effective strategy. Offering cytotoxic chemotherapy (CT) in addition to ET can improve the clinical outcome in up to 30% of ER patients and therefore leaving the remainder both being over-treated and experiencing the associated burdens of side-effects.
Currently, there is no proven test which can predict response to either ET or CT accurately. The current practice is largely based on assessment of the recurrence risk (RR) and overall survival (OS) of the patient, using the traditional clinico-pathological prognostic factors (e.g., lymph node status (LN)) and the multi-gene tests (e.g., Oncotype DX® (Genomic Health, CA, USA), MammaPrint® (Agendia, CA, USA) and Prosigna® (Nanostring Technologies, WA, USA). Nevertheless, these tests do not predict if a patient will respond to the cancer therapy, i.e. ET or CT. This therefore makes it difficult for clinicians and the patients to determine the risk/benefit ratio associated with treating an individual with ET or CT. The “benefit” of the “risk/benefit ratio” being treatment of the cancer, whereas the “risk” is the possibility that it will not treat the cancer and the possibility of any side effects associated with using ET or CT.
There is therefore a need for an improved means of determining if an individual is expected to respond to chemotherapy, endocrine therapy and/or other types of cancer therapy.
According to a first aspect of the invention, there is provided a method for determining if a cancer of a subject is expected to respond to chemotherapy, the method comprising:
Surprisingly, when SPAG5 protein is overexpressed in a cancer, the SPAG5 displays intracytoplasmic granular (punctate) staining in the cancer cells.
Furthermore, cancer cells that comprise granules of SPAG5 protein and/or overexpress the SPAG5 transcript are surprisingly responsive to chemotherapy and resistant or less responsive to endocrine therapy (see Examples 14 and 37). It is believed that this is because cancers that overexpress SPAG5 protein and/or SPAG5 transcript are rapidly growing and therefore respond better to non-specific therapies, such as chemotherapy which target cell division (growth). Since SPAG5 has an essential role in the progression of the cell cycle during the mitotic phase, the inventors believe that SPAG5 dysregulation could contribute to chromosome instability and aneuploidy, both of which are hallmarks of malignant cells and could confer vulnerability to chemotherapeutics drugs. SPAG5 protein is a microtubule-associated protein with dual localization to both centrosomes and kinetochores, and is required for mitotic spindle formation and chromosome segregation. SPAG5 protein functions directly or indirectly in the microtubule-based vesicle transport that is essential for spindle formation during mitotic (M) phase. SPAG5 is upregulated in M-phase cells and binds to microtubules as a regulator of the timing of spindle organization and separation of sister chromatids. Thus punctuate or granular staining pattern of SPAG5 protein may reflect over-production (overexpression or upregulation) of SPAG5-microtubule-based vesicle and excess spindle formation in M-phase leading to uncontrolled cell division.
Advantageously, therefore, the method according to the first aspect is a fast and accurate means of determining whether an individual is likely to respond to chemotherapy.
In one embodiment, the method according to the first aspect may further comprise the step of determining if a cell in a sample of a tumour overexpresses HER2 protein or HER2 gene (HER2+). If a cell in a sample of the tumour is HER2+ and the sample comprises a cell that expresses granules of SPAG5 protein and/or the sample overexpresses the SPAG5 transcript, the tumour is expected to respond to chemotherapy. Therefore, HER2 targeting agents, such as Trastuzumab, Pertuzumab and or Lapatinib may be deaccelerated or may not be needed. If the sample comprises a cell that is HER2+ and the sample does not comprise a cell that expresses granules of SPAG5 protein and/or does not overexpress the SPAG5 transcript, the tumour is expected to respond to HER2 targeting agents, and is not expected to respond to chemotherapy.
In another embodiment, the method according to the first aspect may further comprise the step of determining if a cell in a sample of a tumour expresses oestrogen receptor (ER+). If a cell in a sample of the tumour is ER+ and the sample comprises a cell that expresses granules of SPAG5 protein and/or cells in the sample overexpress the SPAG5 transcript, the tumour is expected to respond to chemotherapy. If the sample comprises a cell that is ER+ and the sample does not comprise a cell that expresses granules of SPAG5 protein and/or the cells in the sample do not overexpress the SPAG5 transcript, the tumour may be expected respond to endocrine therapy.
According to a second aspect of the invention, there is provided a method for determining if an ER+ cancer of a subject is expected to respond to endocrine therapy or chemotherapy, the method comprising:
According to a third aspect of the invention, there is provided a method for determining if an ER+ cancer of a subject is expected to respond to endocrine therapy, the method comprising:
According to a fourth aspect of the invention, there is provided a method for determining if an ER+ cancer of a subject may be expected to respond to chemotherapy, the method comprising:
If the sample referred to in the second, third or fourth aspect does not comprise a cell that expresses granules of SPAG5 protein and/or the cells in sample do not overexpress the SPAG5 transcript, the cancer may be expected to be resistant to chemotherapy. If the sample comprises a cell that expresses granules of SPAG5 protein and/or if cells in the sample overexpress the SPAG5 transcript, the cancer may be expected to be resistant to endocrine therapy.
If the sample referred to in the second, third or fourth aspect comprises a cell that is ER+ but does not comprise a cell that expresses granules of SPAG5 protein or cells in the sample do not overexpress the SPAG5 transcript, the sample may be taken from a cancer, which in combination with endocrine therapy, responds to a cancer therapy selected from the group comprising/consisting of: HER2 targeting therapy, such as Trastuzumab; taxane; and fluorouracil (5-FU). Preferably, the group does not comprise anthracycline, epirubicin or doxorubicin.
If the sample comprises a cell that is ER+ and comprises a cell that expresses granules of SPAG5 protein and/or if cells in the sample overexpress the SPAG5 transcript, the tumour may be expected to respond to anthracycline-based combination chemotherapy and/or taxane. Anthracycline-based combination chemotherapy is chemotherapy which comprises epirubicin (E) or doxorubicin (A) and one or more other chemotherapy drugs, including cyclophosphamide (C) and fluorouracil (5-FU; F). Thus, Anthracycline-based combination chemotherapy may comprise AC, FAC, EC or FEC. If the sample comprises a cell that is ER+ and comprises a cell that expresses granules of SPAG5 protein and/or overexpresses the SPAG5 transcript, the sample may be taken from a cancer, which in combination with chemotherapy, responds to a cancer therapy selected from the group consisting of: mTOR inhibitors, such as rapamycin and its analogs; AKT inhibitors, such as MK-2206 and GSK690693; PI3K inhibitors, such as LY294002; and CK4/6 inhibitors, such as LY2835219 and PD-0332991.
Surprisingly, ER+ cancers that are also devoid of cells that overexpress SPAG5 protein (i.e. granules of SPAG5 protein) or SPAG5 transcript are very responsive to endocrine therapy (and resistant or less responsive to chemotherapy). This may be because ER+ cancers that do not overexpress SPAG5 protein are slowly growing tumours and not as aggressive (as cancers that overexpress SPAG5 protein) and therefore respond better to hormonal therapies, which act via the oestrogen receptor (ER).
Advantageously, therefore, the method according to the second, third or fourth aspect is a fast and accurate means of determining whether an individual is likely to respond or be resistant to chemotherapy and/or endocrine therapy. Consequently, clinicians are able to provide a more tailored treatment regime for individuals with an ER+ cancer because it takes the risk/benefit ratio of chemotherapy and endocrine therapy into consideration.
The method according to the first, second, third or fourth aspect may not comprise the step of obtaining a sample from the subject. The method according to the first, second, third or fourth aspect may be used to determine the most appropriate type of cancer therapy for a subject that has cancer.
According to a fifth aspect, there is provided a method of treating cancer in a subject with chemotherapy or endocrine therapy, the method comprising:
Determining if the ER+ tumour is expected to respond to endocrine therapy, according to the fifth aspect, may comprise the method of the second or third aspect.
In one embodiment, the method according to the fifth aspect may further comprise repeating step (a), after step (b)(i) or (b)(ii), to determine if it is appropriate for the subject to be treated with the same therapy or if a different cancer therapy should be administered. The repetition of step (a) may occur after the subject has been treated with a cancer therapy. Therefore, the repetition may occur two or more weeks, three or more weeks, four or more weeks, six or more weeks, eight or more weeks, 10 or more weeks, or 12 or more weeks, 6 or more months, 1 year or more, 2 years or more, 4 years or more or 5 years or more after the subject has been treated with a cancer therapy. Preferably, the repetition of step (a) will occur after a relapse of the cancer or after failure of disappearance of cancer cells or failure of reducing the tumour growth or volume.
In another embodiment, the method according to the fifth aspect may further comprise repeating step (a), after step (b)(i) or (b)(ii), followed by performing step (b)(i) or step (b)(ii). The repetition of step (a) and performance of step (b) may occur after the subject has been treated with a cancer therapy. Therefore, the repetition may occur two or more weeks, three or more weeks, four or more weeks, six or more weeks, eight or more weeks, 10 or more weeks, or 12 or more weeks, 6 or more months, 1 year or more, 2 years or more, 4 years or more or 5 years or more after the subject has been treated with a cancer therapy. Preferably, the repetition of step (a) and performance of step (b) will occur after a relapse of the cancer or after failure of disappearance of cancer cells or failure of reducing the tumour growth or volume.
According to a sixth aspect, there is provided a method of treating cancer in a subject with chemotherapy or endocrine therapy, the method comprising:
If the cancer is expected to respond to chemotherapy (that is a sample of the cancer comprises a cell that expresses granules of SPAG5 protein or overexpresses SPAG5 transcript), the subject may be treated with chemotherapy in combination with a cancer therapy selected from the group consisting of: mTOR inhibitors, such as rapamycin and its analogs; AKT inhibitors, such as MK-2206 and GSK690693; PI3K inhibitors, such as LY294002; and CK4/6 inhibitors, such as LY2835219 and PD-0332991.
If the tumour is expected to respond to endocrine therapy (that is a sample of the cancer does not comprise a cell that expresses granules of SPAG5 protein and/or does not overexpress SPAG5 transcript but does comprise a cell that is ER+), the subject may be treated with endocrine therapy in combination with a cancer therapy selected from the group consisting of: SERM; an aromatase inhibitor; taxane and fluorouracil (5-FU). If the tumour is expected to respond to endocrine therapy, the subject may be treated with a combination of endocrine therapy and taxane or fluorouracil (5-FU) chemotherapy.
According to a seventh aspect, there is provided a method of determining the efficacy of treating a subject suffering from cancer with a cancer therapy, the method comprising:
The reference sample may be a sample of the same cancer from the same subject at an earlier time point.
Preferably the determining step of this aspect is performed using techniques such as flow cytometry, immunohistochemistry, Chromogenic in situ hybridization (CISH) or FISH (Fluorescence In-Situ Hybridization).
The cancer may be an ER+ cancer or a cancer that comprises cells that express the ER. The cancer may be a HER2+ cancer or a cancer that comprises cells that express the HER2.
According to an eighth aspect, there is provided a method of determining the prognosis of a subject that has a cancer, the method comprising:
The method according to the eighth aspect may further comprise determining if a cell in the sample from the tumour of the subject is ER+, wherein if a cell in the sample from the tumour is ER+ and the cell expresses granules of SPAG5 protein and/or the cells in sample from the tumour overexpress the SPAG5 transcript, the subject has a poor prognosis.
The method according to the eighth aspect may further comprise determining if a cell in the sample from the tumour of the subject is HER2+, wherein if a cell in the sample from the tumour is HER2+ and the cell expresses granules of SPAG5 protein and/or the sample from the tumour overexpresses the SPAG5 transcript, the subject has a poor prognosis.
A poor prognosis may be an increase in relapse risk at 5 or 10 years after receiving surgery and/or therapy for cancer compared to the relapse risk of a reference individual. The increased risk of relapse may be a 1.5-fold increase or greater, 2-fold increase or greater, or 3-fold increase or greater.
A poor prognosis may be an increased risk of death from cancer at 5 or 10 years after receiving cancer surgery and/or cancer therapy compared to the relapse risk of a reference individual. The increased risk of death may be a 1.5-fold increase or greater, 2-fold increase or greater, or 3-fold increase or greater.
A good prognosis may be a decrease in relapse risk at 5 or 10 years after receiving surgery and/or therapy for cancer compared to the relapse risk of a reference individual. The decreased risk of relapse may be a 1.5-fold decrease or greater, 2-fold decrease or greater, or 3-fold decrease or greater.
A good prognosis may be a decreased risk of death from cancer at 5 or 10 years after receiving cancer surgery and/or cancer therapy compared to the relapse risk of a reference individual. The decreased risk of death may be a 1.5-fold decreased or greater, 2-fold decreased or greater, or 3-fold decreased or greater.
The reference individual may be one or more individuals with a cancer that does not comprise granules of SPAG5 protein or that does not overexpress SPAG5 transcript. The reference individual may be one or more individuals with a cancer that comprises granules of SPAG5 protein or that overexpress SPAG5 transcript. Preferably the one or more reference individuals have the same form of cancer as the individual of referred to in the invention, such as the eighth aspect.
According to a ninth aspect, there is provided a chemotherapy agent for use in treating cancer in a subject, wherein cells of the cancer comprise granules of SPAG5 protein or overexpress SPAG5 transcript.
The subject may have a cancer that is ER+. The subject may have a cancer that is HER2+.
According to a tenth aspect, there is provided an endocrine therapy for use in treating an ER+ cancer in a subject, wherein cells of the cancer do not comprise granules of SPAG5 protein or do not overexpress SPAG5 transcript.
According to an eleventh aspect of the invention, there is provided a method for determining if an HER2+ cancer of a subject is expected to respond to HER2 targeting therapy or to chemotherapy, the method comprising:
According to a twelfth aspect of the invention, there is provided a method for determining if an HER2+ cancer of a subject is expected to respond to chemotherapy, the method comprising:
If a sample referred to in the eleventh or twelfth aspect does not comprise a cell that expresses granules of SPAG5 protein and/or the sample does not overexpress the SPAG5 transcript, the cancer may be expected to be resistant to chemotherapy. If the sample comprises a cell that expresses granules of SPAG5 protein and/or if the sample overexpresses the SPAG5 transcript, the cancer may not be expected to respond to or benefit from adding HER2 target therapy in addition to chemotherapy.
If a sample referred to in the eleventh or twelfth aspect comprises a cell that is HER2+ but does not comprise a cell that expresses granules of SPAG5 protein or the sample does not overexpress the SPAG5 transcript, the sample may be taken from a cancer, which responds to HER2 targeting therapy, such as: Herceptin Pertuzumab and Lapatinib and/or taxane. The cancer may not respond to anthracycline, epirubicin or doxorubicin.
If the sample comprises a cell that is HER2+ and comprises a cell that expresses granules of SPAG5 protein and/or if the sample overexpresses the SPAG5 transcript, the tumour may be expected to respond to anthracycline-based combination chemotherapy. Anthracycline-based combination chemotherapy is chemotherapy which comprises epirubicin (E) or doxorubicin (A) and one or more other chemotherapy drugs, including cyclophosphamide (C) and fluorouracil (5-FU; F). Thus, anthracycline-based combination chemotherapy may comprise AC, FAC, EC or FEC. If the sample comprises a cell that is HER2+, ER+ and comprises a cell that expresses granules of SPAG5 protein and/or overexpresses the SPAG5 transcript, the sample may be taken from a cancer, which in combination with chemotherapy, responds to a cancer therapy selected from the group consisting of: mTOR inhibitors, such as rapamycin and its analogs; AKT inhibitors, such as MK-2206 and GSK690693; PI3K inhibitors, such as LY294002; and CK4/6 inhibitors, such as LY2835219 and PD-0332991.
Surprisingly, HER2+ cancers that are also devoid of cells that overexpress SPAG5 protein (i.e. granules of SPAG5 protein) or SPAG5 transcript are very responsive to HER2 target therapy (and resistant or less responsive to chemotherapy). This may be because HER2+ cancers that do not overexpress SPAG5 protein are slowly growing tumours and not as aggressive (as cancers that overexpress SPAG5 protein) and therefore respond better to HER2 target therapies, which act via the HER2 receptor.
Advantageously, therefore, the method according to the eleventh or twelfth aspect is a fast and accurate means of determining whether an individual is likely to respond or be resistant to chemotherapy and/or HER2 targeting therapy.
Consequently, clinicians are able to provide a more tailored treatment regime for individuals with an HER2+ cancer because it takes the risk/benefit ratio of chemotherapy and HER2 targeting therapy into consideration.
The method according to the any aspect may not comprise the step of obtaining a sample from the subject. The method according to the first, eleventh or twelfth aspect may be used to determine the most appropriate type of cancer therapy for a subject that has cancer.
According to a thirteenth aspect, there is provided a method of treating cancer in a subject with chemotherapy or HER2 target agent, the method comprising:
Determining if the HER2+ tumour is expected to respond to HER2 target therapy, according to the thirteenth aspect, may comprise the method of the eleventh or twelfth aspect.
In one embodiment, the method according to the thirteenth aspect may further comprise repeating step (a), after step (b)(i) or (b)(ii), to determine if it is appropriate for the subject to be treated with the same therapy or if a different cancer therapy should be administered. The repetition of step (a) may occur after the subject has been treated with a cancer therapy. Therefore, the repetition may occur two or more weeks, three or more weeks, four or more weeks, six or more weeks, eight or more weeks, 10 or more weeks, or 12 or more weeks, 6 or more months, 1 year or more, 2 years or more, 4 years or more or 5 years or more after the subject has been treated with a cancer therapy. Preferably, the repetition of step (a) will occur after a relapse of the cancer or after failure of disappearance of cancer cells or failure of reducing the tumour growth or volume.
In another embodiment, the method according to the thirteenth aspect may further comprise repeating step (a), after step (b)(i) or (b)(ii), followed by performing step (b)(i) or step (b)(ii). The repetition of step (a) and performance of step (b) may occur after the subject has been treated with a cancer therapy. Therefore, the repetition may occur two or more weeks, three or more weeks, four or more weeks, six or more weeks, eight or more weeks, 10 or more weeks, or 12 or more weeks, 6 or more months, 1 year or more, 2 years or more, 4 years or more or 5 years or more after the subject has been treated with a cancer therapy. Preferably, the repetition of step (a) and performance of step (b) will occur after a relapse of the cancer or after failure of disappearance of cancer cells or failure of reducing the tumour growth or volume.
According to a fourteenth aspect, there is provided a method of treating cancer in a subject with chemotherapy or HER2 target therapy, the method comprising:
If the cancer is expected to respond to chemotherapy (that is a sample of the cancer comprises a cell that expresses granules of SPAG5 protein or overexpresses SPAG5 transcript), the subject may be treated with chemotherapy in combination with a cancer therapy selected from the group comprising or consisting of: mTOR inhibitors, such as rapamycin and its analogs; AKT inhibitors, such as MK-2206 and GSK690693; PI3K inhibitors, such as LY294002; and CK4/6 inhibitors, such as LY2835219 and PD-0332991.
If the tumour is expected to respond to HER2 target therapy (that is a sample of the cancer does not comprise a cell that expresses granules of SPAG5 protein and/or does not overexpress SPAG5 transcript but does comprise a cell that is HER2+), the subject may be treated with HER2 target therapies such as Herceptin pertuzumab and/or Lapatinib in combination with a cancer therapy selected from the group consisting of; taxane and fluorouracil (5-FU). If the tumour is expected to respond to HER2 target therapy, the subject may be treated with a combination of HER2 target therapy and pertuzumab, Lapatinib, taxane or fluorouracil (5-FU) chemotherapy.
According to a fifteenth aspect, there is provided an HER2 target therapy for use in treating an HER2+ cancer in a subject, wherein cancer cells of the cancer do not comprise granules of SPAG5 protein or do not overexpress SPAG5 transcript.
According to another aspect of the invention, there is provided a method of determining a prognosis of a subject diagnosed with a cancer that does not overexpress HER2 protein or HER2 gene (HER2−) after receiving neoadjuvant chemotherapy, the method comprising:
According to another aspect of the invention, there is provided a method for determining a prognosis of a subject diagnosed with a cancer that does not overexpress HER2 protein or HER2 gene and does not express ER (HER2−/ER−) after receiving neoadjuvant chemotherapy and without receiving any further adjuvant therapy, the method comprising:
According to another aspect of the invention, there is provided a method for determining a prognosis of a subject diagnosed with a cancer that does not overexpress HER2 protein or HER2 gene but expresses ER (HER2−/ER+) after receiving neoadjuvant chemotherapy followed by 5 years of endocrine therapy, the method comprising:
According to a further aspect of the invention, there is provided a method for determining a prognosis of a subject diagnosed with a cancer that does overexpress HER2 protein or HER2 gene (HER2+) after receiving neoadjuvant chemotherapy with or without pre-operative HER2 target therapy and followed by one year of adjuvant single agent of HER2 targeting therapy and no need to add other HER2 target therapy (e.g., Pertuzumab), the method comprising:
According to the previous aspect of the invention, there is provided a method for determining a prognosis of a subject diagnosed with a cancer that overexpresses HER2 protein or HER2 gene (HER2+) after receiving neoadjuvant chemotherapy and if they would benefit from shorter course of adjuvant HER2 target therapy (6 months vs., one year), the method comprising:
According to another aspect of the invention, there is provided a method for determining a prognosis of a subject diagnosed with a cancer that overexpresses HER2 protein or HER2 gene but does not express ER (HER2+/ER−) after receiving neoadjuvant chemotherapy and one year of adjuvant HER2 target therapy and if they would benefit from adding other HER2 target agents or additional immunotherapy, the method comprising:
A subject with a good prognosis may not require any other forms of treatment.
The sample or cell(s) according to the invention may be taken from a (residual) tumour of a subject who has received chemotherapy.
According to another aspect of the invention, there is provided a method for determining a prognosis of a subject diagnosed with a cancer that overexpresses HER2 protein or HER2 gene and expresses ER (HER2+/ER+) after receiving neoadjuvant chemotherapy followed by 5 years of adjuvant endocrine therapy in addition to one year of adjuvant HER2 target therapy, the method comprising:
The sample or cell(s) may be taken from a (residual) tumour of a subject who has received chemotherapy.
If the subject is expected to have a poor prognosis, they may benefit from receiving other therapeutic treatments, such as CDK4/6 and or adding other HER2-targeting agents.
In another aspect of the invention, there is provided a method for stratification and predicting the risk of relapse of a cancer after receiving neoadjuvant therapy followed by (if eligible) adjuvant endocrine therapy for 5 years and or adjuvant HER2 Target therapy by re-valuating and retesting a number of radiological and histopathological parameters in a sample of the residual cancer after receiving neoadjuvant chemotherapy from the same individual including HER2, SPAG5, MRI volumetric measurements, lympho-vascular invasion, histopathological grade and chemotherapy related fibrotic reaction; the method comprising:
Wherein if after receiving neoadjuvant chemotherapy i) there is no granules of SPAG5 protein expression in a cell of a sample of residual tumour, and ii) there is no Lymphovascular invasion in the residual cancer and (iii) there is a reduction in the tumour volume measured by MRI equal to or more than 30% and (iv) the tumour is of low or intermediate histological grade and (v) there is a chemotherapy related histological fibrotic reaction in the residual tissue, the subject has a residual cancer that is expected to have a low risk of relapse, or
Preferably, the repetition of step (i) to (vi) will occur after failure of disappearance of cancer cells after receiving neoadjuvant chemotherapy.
According to a yet further aspect, the invention provides a method of calculating the 5 year predicted risk of relapse of a tumour subject who has no changes in HER2 expression status after receiving neoadjuvant chemotherapy, optionally followed by adjuvant endocrine therapy for 5 years and or adjuvant HER2 Target therapy, the method comprising:
An excellent prognosis (relapse risk <10%) is given if the sum of the relapse risk is 0 to 3; a good prognosis (relapse risk >=10% up to 25%) is given if the sum of the relapse risk is 4 to 6; an intermediate prognosis (relapse risk >=25% up to 50%) is given if the sum of the relapse risk is 7 to 9; and a poor prognosis (relapse risk >50%) is given if the sum of the relapse risk is 10 to 11.
Advantageously, therefore, the method according to the previous aspects is a fast and accurate means of determining whether an individual is likely to benefit from HER2 targeting therapy and or endocrine therapy. Consequently, clinicians are able to provide a more tailored treatment regime for individuals with a cancer because it takes the risk/benefit ratio of chemotherapy, HER2 targeting therapy and endocrine therapy into consideration.
The method according to the previous aspects may not comprise the step of obtaining a sample from the subject. The method according to the previous aspects may be used to determine the most appropriate type of cancer therapy for a subject that has cancer.
According to another aspect, there is provided a method of determining the prognosis of a subject with patient, the method comprising determining if a test sample from a subject with a tumour, which has not been treated with systemic therapy, is HER2− and/or determining if a test sample of the tumour in a subject, after being treated with anthracycline based combination chemotherapy with or without Taxane, is HER2+,
According to another aspect, there is provided a method determining the prognosis of a subject with cancer, the method comprising determining if a test sample of a tumour from a subject with a tumour, which has not received any systemic therapy, is HER2+ and determining if a test sample of the tumour in a subject after being treated with anthracycline based combination chemotherapy with or without Taxane with or without HER2 targeting agents (e.g., Trastuzumab, Pertuzumab and or Lapatinib), is converted into HER2−,
According to a further aspect, the invention provides a method for determining if a HER2− cancer of a subject, prior to treatment with systemic therapy, is expected to benefit from adjuvant HER2 target therapy after having received neoadjuvant chemotherapy, the method comprising:
According to another aspect of the invention, there is provided a method for determining if a HER2− cancer of a subject is expected to have a good prognosis after receiving neoadjuvant chemotherapy, the method comprising:
According to a further aspect the invention there is provided a method of determining if a subject with cancer, prior to treatment with systemic therapy HER2−, is expected to have a good prognosis after receiving neoadjuvant chemotherapy, the method comprising:
A poor prognosis may be an increased risk of relapse at 5 or 10 years after receiving surgery and/or cancer therapy for cancer compared to the relapse risk of a reference individual, or wherein a poor prognosis may be an increased risk of death from cancer at 5 or 10 years after receiving cancer surgery and/or cancer therapy compared to the relapse risk of a reference individual.
The increased risk of relapse or the increased risk of death may be a 1.5-fold increase or greater, 2-fold increase or greater, or 3-fold increase or greater.
The reference individual may be one or more individuals with a cancer that has achieved complete disappearance of cancer cells in post chemotherapy samples after receiving neoadjuvant chemotherapy.
SPAG5 is an important oncogene that not only plays an essential role in the formation and progression of human cancers, but also determines their clinical behaviour by regulating mitosis, cell cycle checkpoint and apoptosis. In one embodiment, the sequence of the SPAG5 protein is 1193 amino acids long (Q96R06), and is referred to herein as SEQ ID NO. 1, as follows:
Therefore, the SPAG5 protein may comprise an amino acid sequence substantially as set out in SEQ ID No. 1, or a variant or a fragment thereof.
A sample as referred to in any of the aspects of the invention may comprise one or more cells from a subject with cancer, preferably cancer cells (not healthy/non-cancer cells). The one or more cells of the sample may comprise granular SPAG5 protein (i.e. granules of SPAG protein) and/or SPAG5 protein in a diffuse or non-granular form, such that it would have a diffuse staining pattern if, for example, it was stained using immunohistochemistry. The SPAG5 protein may comprise an amino acid sequence substantially as set out in SEQ ID No. 1, or a variant or a fragment thereof.
Granular SPAG5 protein refers to SPAG5 protein that displays a punctate intracytoplasmic staining pattern, such as spots or dots of SPAG5 protein. Granular SPAG5 protein may be one or more granules of SPAG5 protein. Granular SPAG5 protein does not occupy the entire cytoplasm of a cell. Granular SPAG5 protein only occupies a fraction of the cells cytoplasm (see, for example,
A cancer that is “ER+” or “ER positive” refers to a cancer that comprises cells which express the oestrogen receptor (ER). In one embodiment, the sequence of the oestrogen receptor (ER) protein is 595 amino acids long (P03372), and is referred to herein as SEQ ID NO. 2, as follows:
Therefore, the oestrogen receptor protein may comprise an amino acid sequence substantially as set out in SEQ ID No. 2, or a variant or a fragment thereof.
The cancer according to the invention may be selected from the group consisting of: breast cancer; lung cancer; ovarian cancer; gastric cancer; mesothelioma; Malignant Pleural Mesothelioma; uveal melanoma; melanoma; non-melanoma skin cancer; renal cancer; cholangiocarcinomas; cancer of the pleura; abdominal cancer; peritoneal cancer; cancer of the pericardium; head and neck cancers; brain cancer; liver cancer; biliary tract cancer; gastrointestinal cancers, including upper and lower tracts; urothelial cancer; prostate cancer; testicular cancer; cancer of the tunica vaginalis; cervical cancer, sarcoma; lymphoma; and leukaemia.
The cancer may or may not be metastatic.
The inventors have found that SPAG5 is not only a biomarker of proliferation but also an important genetic driver which is commonly amplified in highly proliferative “luminal B” and HER2+ BCs. Therefore, most preferably the cancer is breast cancer. More preferably, the breast cancer is a luminal B and/or HER2+ breast cancer.
A cancer that is “HER2+” or “HER2 positive” refers to a cancer that comprises cells that express the HER2 (Human Epidermal Growth Factor) receptor. In one embodiment, the sequence of the HER2 receptor is 1255 amino acids long (P04626), and is referred to herein as SEQ ID NO. 3, as follows:
Luminal tumour cells look like those of breast cancers that start in the inner (luminal) cells lining the mammary ducts. A “luminal B” cancer refers to a breast cancer that tends to be oestrogen-receptor positive. A “luminal B” cancer may be HER2+ or HER2−. A “luminal A” cancer refers to a breast cancer that is oestrogen-receptor positive and HER2 negative.
“LN+” or “LN positive” may mean that a cancer has spread to the lymph nodes, whereas “LN−” or “LN negative” means that a cancer has not spread to the lymph nodes.
The term “respond” or “responsive” means a cancer that can be treated by a particular cancer therapy, such as chemotherapy or endocrine therapy. A cancer may be considered treated if, for example, cancer cells have been killed, a tumour has been shrunk, replication of cancer cells has been halted, reduced or growth of a tumour has been stopped. The term “resistant” means a cancer that cannot be treated by a particular cancer therapy, such as chemotherapy or endocrine therapy.
The term “express(es)” or “expression of” can refer to the translation of a relevant DNA or RNA molecule into a polypeptide or protein sequence (such as SPAG5 protein or oestrogen receptor protein). The polypeptide or protein may be present within any compartment of the cell, may be present on a cell surface or may be secreted from a cell. This term may also refer to the transcription of DNA, which may be the production of transcripts, such as mRNA or cDNA, based on DNA. Thus, expression of a gene may be determined by measuring the amount of a transcript.
“Overexpresses”, as referred to herein, can refer to increased expression of a protein compared to the expression of the same protein (e.g. SPAG5) in normal (healthy/non-cancerous) individuals or it can refer to increased transcription of a gene into a transcript (e.g. mRNA or cDNA, such as that encoding SPAG5) compared the transcription of the same gene in normal (healthy/non-cancerous) individuals. “Overexpresses” may also refer to quantified expression, in a test sample, of the SPAG5 gene, such that it has a value of greater than 1 after being normalised (with respect to relevant databases) and median centred (such that the values fall in a range of 0 to 1 with the media at the centre).
SPAG5 gene expression is normalised and median centred by:
(Test sample SPAG5 gene expression value−the arithmetic mean of SPAG5 gene expression)/the standard deviation of SPAG5 gene expression.
[Normalized SPAG5 expression value of the test sample−minimal normalized value of SPAG5 among database samples]/[maximum normalized expression value of SPAG5 among the database samples−minimal normalized expression value of SPAG5 among the database samples]
The test sample according to the invention may be the sample from the subject referred to herein.
The relevant databases may be one or more of the following databases:
Preferably, the first 15 databases are used for determining the association between SPAG5 transcript and the distant relapse risk (DRR) after receiving adjuvant systemic therapy.
Preferably, the last 12 databases are used for determining the association between SPAG5 transcript expression and pathological complete response (pCR). More preferably, the last 12 databases are used for determining the association between SPAG5 transcript expression and pathological complete response (pCR) after receiving pre-operative cancer treatment. Most preferably, the preoperative cancer treatment is anthracycline based combination chemotherapy with or without Taxane and with or without Herceptin®.
SPAG5 gene expression data of each database were converted to a common scale (median equal to 0 and standard deviation equal to 1) in order to merge all of the study data that used the same platform and to create combined cohorts. To convert to a common scale each sample in the data was normalised to a mean of 0 and standard deviation of 1. Then the data was median centred for each gene whereby median of each gene is 0 (see steps 1 to 4 above). Databases using same platform have been merged and the median expression was calculated. The median expression of SPAG5 transcript for each platform has been calculated and values equal to or higher than the median coded as +1 (overexpression). Values of less than the median have been coded 0 or low SPAG5.
Overexpression of a gene may be an increase in expression of the gene in a sample in comparison to a “normal” or reference concentration. The increase may be an increase of 10% 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 or 100% or more.
The “normal” or reference concentration may be obtained from control samples (i.e. samples from subject who do not suffer from a cancer). The control samples may be taken from one or more relevant databases. Preferably, the number of control samples is statistically significant.
Detection of transcript overexpression may be performed using any of the following techniques, which are known in the art: polymerase chain reaction (PCR); northern blotting; hybridisation-based detection techniques; or gene expression array.
“Chemotherapy”, as referred to herein, can refer to any chemical drug that can be used to treat or prevent cancer, by, for example, killing cancer cells, shrinking a tumour, stopping or reducing replication of cancer cells or stopping or reducing growth of a tumour. Thus, chemotherapy may refer to a chemotherapeutic drug that inhibits mitosis or cellular division of cancer cells and/or induces cell death.
The drug may be non-specific. Chemotherapy as referred to herein does not include endocrine therapy or targeted therapy.
Chemotherapy agents according to the invention may therefore include a selection from the group comprising/consisting of: cisplatinum, taxane, cyclophosphamide, methotrexate, 5-fluorouracil, an anthracycline, epirubicin, doxorubicin and anthracycline based combination chemotherapy.
Anthracycline based combination chemotherapy comprises epirubicin (E) or doxorubicin (A) and one or more chemotherapy drugs, which may include cyclophosphamide (C) and fluorouracil (5-FU; F).
“Endocrine therapy” (or “hormonal therapy”), as referred to herein, can refer to any drugs that can be used to treat or prevent cancer by preventing ER signalling through either selective oestrogen receptor modulators (SERM) (e.g., tamoxifen) or aromatase inhibitors (AIs). The endocrine therapy may therefore be a hormone, a hormone receptor antagonist or an enzyme inhibitor (such as an aromatase inhibitor). The hormone or hormone receptor antagonist may, for example, act via the endocrine receptor. Endocrine therapy according to the invention may therefore include: tamoxifen and/or an aromatase inhibitor.
“Neoadjuvant chemotherapy”, as referred to herein, can refer to chemotherapy that is administered to a subject/patient before surgical removal of a tumour.
“Systemic therapy”, as referred to herein, can refer to chemotherapy, HER2 target therapy and/or endocrine therapy received either before or after surgical removal of the tumour.
“HER2 status” may refer to the expression the human epidermal growth factor receptor 2, preferably using the American Society of Clinical Oncology guide-lines.
The term “determining” can refer, but is not limited, to the use of any one of the following conventional assays for detecting the presence of one or more of the biomarkers (such as SPAG5 or the oesterogen receptor), or variants or fragments thereof, in a sample: flow cytometry; immunoassays, such as enzyme-linked immunosorbent assays (ELISAs), enzyme immunoassays (EIAs), radioimmunoassay (RIAs), Western Blots, immuo-precipitation or immunohistochemistry (IHC); immuno-electron microscopy; chromogenic (enzyme activity) assays; fluorometric imaging plate reader (FLIPR) assays; or high performance liquid chromatography (HPLC) tandem mass spectrometry (MS/MS). Preferably, immunohistochemistry or immuno-electron microscopy is used to detect granular SPAG5 protein.
Advantageously, IHC and immuno-electron microscopy allow stained cancer tissues that comprise cells containing granules of SPAG5 protein to be identified easily, i.e. by simply looking at the cells under a microscope.
The term “sample” refers to a specimen taken from the body of a subject. The sample may comprise blood, plasma, serum, spinal fluid, urine, sweat, saliva, tears, breast aspirate, prostate fluid, seminal fluid, vaginal fluid, stool, cervical scraping, cytes, intraocular fluid, animal tissue, cell lysates, tumour tissue, skin, bone marrow, cartilage, or combinations thereof. In another embodiment, the sample may be contained within the subject, which maybe an experimental animal (e.g. a mouse or rat) or a human, wherein the method is an in vivo based test. Alternatively, the sample may be an ex vivo sample or an in vitro sample. Therefore, the cells being tested may be in a tissue sample (for ex vivo based tests) or the cells maybe grown in culture (an in vitro sample). Preferably, the biological sample is an ex vivo sample. The sample may be a (biological) tissue. Preferably the tissue is a solid tissue, such as breast tissue, bone tissue, lung tissue or liver tissue. Most preferably, the sample comprises tumour tissue. The sample may be any sample that comprises one or more cancer cells.
The “subject” may be a vertebrate, mammal or domestic mammal. Hence, the methods according to the invention may be used to treat any animal, for example, pigs, cats, dogs, horses, sheep or cows. Preferably, the subject is a human being.
“A cell” may be one or more cells. Preferably, it means two or more cells, or three or more cells.
It will be appreciated that the invention relates to or extends to any nucleic acid or peptide or variant, derivative or analogue thereof, which comprises substantially the amino acid or nucleic acid sequences of any of the sequences referred to herein, including variants or fragments thereof. The terms “substantially the amino acid/nucleotide/peptide sequence”, “variant” and “fragment”, can be a sequence that has at least 40% sequence identity with the amino acid/nucleotide/peptide sequences of any one of the sequences referred to herein, for example 40% identity with the polypeptide identified as SEQ ID No. 1, and so on.
Amino acid/polynucleotide/polypeptide sequences with a sequence identity which is greater than 50%, more preferably greater than 65%, 70%, 75%, and still more preferably greater than 80% sequence identity to any of the sequences referred to are also envisaged. Preferably, the amino acid/polynucleotide/polypeptide sequence has at least 85% identity with any of the sequences referred to, more preferably at least 90%, 92%, 95%, 97%, 98%, and most preferably at least 99% identity with any of the sequences referred to herein.
The skilled technician will appreciate how to calculate the percentage identity between two amino acid/polynucleotide/polypeptide sequences. In order to calculate the percentage identity between two amino acid/polynucleotide/polypeptide sequences, an alignment of the two sequences must first be prepared, followed by calculation of the sequence identity value. The percentage identity for two sequences may take different values depending on:—(i) the method used to align the sequences, for example, ClustalW, BLAST, FASTA, Smith-Waterman (implemented in different programs), or structural alignment from 3D comparison; and (ii) the parameters used by the alignment method, for example, local vs global alignment, the pair-score matrix used (e.g. BLOSUM62, PAM250, Gonnet etc.), and gap-penalty, e.g. functional form and constants. Having made the alignment, there are many different ways of calculating percentage identity between the two sequences. For example, one may divide the number of identities by: (i) the length of shortest sequence; (ii) the length of alignment; (iii) the mean length of sequence; (iv) the number of non-gap positions; or (iv) the number of equivalenced positions excluding overhangs. Furthermore, it will be appreciated that percentage identity is also strongly length dependent. Therefore, the shorter a pair of sequences is, the higher the sequence identity one may expect to occur by chance.
Hence, it will be appreciated that the accurate alignment of protein or DNA sequences is a complex process. The popular multiple alignment program ClustalW (Thompson et al, 1994, Nucleic Acids Research, 22, 4673-4680; Thompson et al, 1997, Nucleic Acids Research, 24, 4876-4882) is a preferred way for generating multiple alignments of proteins or DNA in accordance with the invention. Suitable parameters for ClustalW may be as follows: For DNA alignments: Gap Open Penalty=15.0, Gap Extension Penalty=6.66, and Matrix=Identity. For protein alignments: Gap Open Penalty=10.0, Gap Extension Penalty=0.2, and Matrix=Gonnet. For DNA and Protein alignments: ENDGAP=−1, and GAPDIST=4. Those skilled in the art will be aware that it may be necessary to vary these and other parameters for optimal sequence alignment. Preferably, calculation of percentage identities between two amino acid/polynucleotide/polypeptide sequences may then be calculated from such an alignment as (N/T)*100, where N is the number of positions at which the sequences share an identical residue, and T is the total number of positions compared including gaps but excluding overhangs. Hence, a most preferred method for calculating percentage identity between two sequences comprises (i) preparing a sequence alignment using the ClustalW program using a suitable set of parameters, for example, as set out above; and (ii) inserting the values of N and T into the following formula:—Sequence Identity=(N/T)*100. Alternative methods for identifying similar sequences will be known to those skilled in the art. For example, a substantially similar nucleotide sequence will be encoded by a sequence which hybridizes to any sequences referred to herein or their complements under stringent conditions. By stringent conditions, it is meant that the nucleotide hybridises to filter-bound DNA or RNA in 3× sodium chloride/sodium citrate (SSC) at approximately 45° C. followed by at least one wash in 0.2×SSC/0.1% SDS at approximately 20-65° C. Alternatively, a substantially similar polypeptide may differ by at least 1, but less than 5, 10, 20, 50 or 100 amino acids from the sequences shown in SEQ ID No. 1.
Due to the degeneracy of the genetic code, it is clear that any nucleic acid sequence described herein could be varied or changed without substantially affecting the sequence of the protein encoded thereby, to provide a variant thereof. Suitable nucleotide variants are those having a sequence altered by the substitution of different codons that encode the same amino acid within the sequence, thus producing a silent change. Other suitable variants are those having homologous nucleotide sequences but comprising all, or portions of, sequence, which are altered by the substitution of different codons that encode an amino acid with a side chain of similar biophysical properties to the amino acid it substitutes, to produce a conservative change. For example small non-polar, hydrophobic amino acids include glycine, alanine, leucine, isoleucine, valine, proline, and methionine. Large non-polar, hydrophobic amino acids include phenylalanine, tryptophan and tyrosine. The polar neutral amino acids include serine, threonine, cysteine, asparagine and glutamine. The positively charged (basic) amino acids include lysine, arginine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. It will therefore be appreciated which amino acids may be replaced with an amino acid having similar biophysical properties, and the skilled technician will know the nucleotide sequences encoding these amino acids.
All of the features described herein (including the accompanying claims, abstract and drawings) and/or all of the steps of any method or process so disclosed, may be combined with any of the above aspects in any combinations, except combinations where at least some of such features and/or steps are mutually exclusive.
For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the accompanying Figures, in which:—
Materials and Methods
SPAG5 Protein Expression (i.e. Examples 1 to 25, 46 to 48, 51, 52, 55, 56, 58 and 59)
Immunohistochemistry (IHC) and tissue microarray (TMA) analysis of SPAG5 protein expression in breast cancer was investigated by IHC analysis at two independent centres:
The IHC of SPAG5 protein has been externally validated at University of Queensland centre using Queensland breast cancer follow-up (QFU) cohort, which comprises TMAs of 547 invasive breast carcinomas (sampled in duplicate at a minimum) with associated clinical data, including breast cancer-specific survival up to 35 years post-diagnosis (median follow-up 14.1 years, range 0.03-41.75 years). The samples were collected from the Royal Brisbane and Women's Hospital (RBWH) between 1987 and 1994. Pathological and clinical data for these patients were obtained from a combination of clinical diagnostic pathology reports (Pathology Queensland), the Queensland Cancer Registry and internal histopathology review (SRL). The use of samples and clinical data for this study were approved by human research ethics committees of the University of Queensland and RBWH.
Four pm TMA sections were processed in a decloaker for antigen retrieval in sodium citrate buffer (pH 6.0) for 20 mins, and then IHC was performed using an anti-SPAG5 antibody (Sigma HPA022479; 1:50), and the Mach 1 Universal HRP-Polymer Detection kit (Biocare Medical). Haematoxylin-counterstained, mounted sections were then scanned at 40× magnification on an Aperio AT Turbo slide scanner (Leica Biosystems). Digital images of individual tissue cores were scored by three experienced molecular pathologists according to the intensity of tumour cell staining: punctate cytoplasmic staining was recorded as 1+ (moderate) or 2+ (strong), and multiplied by the percentage of tumour cells stained to derive a final score ranged between 0 to 200. Faint, diffuse staining was recorded as 0. Using the maximum score of duplicate tissue cores for each case, associations between SPAG5 expression and clinicopathologic variables were investigated.
Statistical analysis: Statistical analyses were performed using STATISTICA (Stat Soft Ltd, Tulsa, USA) and SPSS (version 17, Chicago, USA) by the authors who were blinded to the clinical data. Where appropriate, Pearson's chi-squared; student's t-test and ANOVA tests were used. Positivity for SPAG5 protein both pre- and post-¬chemotherapy was calculated and compared using McNemar's test. Cumulative survival probabilities and 10-year BCSS and DRFS were estimated using the univariate Cox proportional hazards models and the Kaplan-Meier plot method where appropriate, and differences between survival rates were tested for significance using the log-rank test. Multivariable analysis for survival was performed using the Cox proportional hazard model. The proportional hazards assumption was tested using standard log-log plots. Hazard ratios (HR) and 95% confidence intervals (95% CI) were estimated for each variable. All tests were two-sided with a 95% CI and a p value <0.05 was considered to be indicative of statistical significance. The interaction between SPAG5 and chemotherapy was tested in Cox proportional hazard model. For multiple comparisons, p values were adjusted according to Benjamini-Hochberg method. Ethical approval was obtained from the Institutes Research Ethics Committees.
Also Immunohistochemistry (IHC) and tissue microarray (TMA) analysis of SPAG5 protein expression in other tumours types was investigated by IHC analysis at Nottingham City Hospital including:
SPAG5 Transcript Expression
A) Breast Cancer—Assessment Death and Distant Relapse Risks after Surgery and Receiving Adjuvant Systemic Therapy if Eligible in Early Stage Breast Cancer (i.e. Examples 26 to 37, 45, 49, 50, 53, 54 and 57)
The inventors evaluated the prognostic utility of SPAG5-mRNA expression in a large combined breast cancer (BC) cohort which was sourced from 15 publically-available, global datasets (n=3538) using the following access numbers.
All the databases had been downloaded and processed. The gene expression data were converted to a common scale (median equal to 0 and standard deviation equal to 1) in order to merge all of the studies data and create combined cohorts. Each sample in the data was standardised to a mean of 0 and standard deviation of 1. Then the data was median centred for each gene whereby median of each gene is 0.
In addition the SPAG5-transcript analysis was validated using the Molecular Taxonomy of BC International Consortium [METABRIC (n=1980; median follow-up time in years (MFUT) (inter-quantile range (IQR)): 9.1(5.2-12.9)) and The Cancer Genome Atlas-Breast Cancer project 13 (TCGA-BRCA; n=709; MFUT (IQR): 1.9 (1.7-3.6).
In these cohorts, oestrogen receptor positive (ER+) and/or lymph-node negative (LN−) patients did not receive adjuvant chemotherapy, whereas ER negative (ER−) and/or lymph-node positive (LN+) patients received adjuvant chemotherapy. Additionally, none of the human epidermal growth factor receptor-2 (HER2) overexpression (+) patients received trastuzumab.
Cut-Off Determination:
SPAG5 gene expression data of each database were converted to a common scale (median equal to 0 and standard deviation equal to 1) in order to merge all of the studies data that used the same platform and create combined cohorts. To convert to a common scale each sample in the data was standardised to a mean of 0 and standard deviation of 1. Then the data was median centred for each gene whereby median of each gene is 0. Databases using same platform have been merged and the median expression was calculated. The median expression of SPAG5 transcript for each platform has been calculated and values equal to or higher than the median coded as +1 (overexpression). Values of less than the median have been coded 0 or low SPAG5.
For HG-U133A-Affymetrix; GPL96 platform: SPAG5 median was 0.3858.
For HG-U133_Plus_2-Affymetrix; GPL570 SPAG5 median was 0.4205
For [HG_U95Av2] AFFYMETRIX GPL8300 platform: SPAG5 median was 0.1259
For H_v2.1.1 55K SWEGENE GPL5345 platform: SPAG5 median was 0.4163
For MLRG Human 21K V-Human QiagenGPL5049 platform: SPAG5 median was 0.4012
For OLIGO-ARRAY-35K-DKFZ GPL6486 platform: SPAG5 median was 0.6963
For Expression BeadC-ILLUMINA GPL6098 platform: SPAG5 median was 0.3535
B) Breast Cancer—Assessment of the Clinical Dynamic Response after Receiving Preoperative Endocrine Therapy (Aromatase Inhibitor) (i.e. Example 38)
The association between SPAG5 mRNA expression and clinical response after receiving preoperative therapy has been analysed in a total of 101 patients from a consecutive series of 255 postmenopausal women who presented to Western General Hospital in Edinburgh with large primary histologically confirmed estrogen receptor (ER)-rich (Allred scores 6 to 8) invasive breast cancer and who were recruited between 2003 and 2011 fulfilled the requirements to be included in this. All patients gave informed consent and the study was approved by the local regional ethics committee (2001/8/80 and 2001/8/81). Patients were treated with letrozole (Femara, 2.5 mg; Novartis Pharma AG, Basel, Switzerland) daily for at least 3 months. The clinical response was assessed by three-dimensional ultrasound measurements using dynamic (serial) changes in tumor volumes. Tumours with <50% reduction in volume were classified as non-responders. SPAG5 mRNA expression data were retrieved from the publically available gene expression microarray datasets at the National Center for Biotechnology Information Gene Expression Omnibus under access numbers (GSE59515, GSE55374, and GSE20181).
Gene Expression Analysis and Data Processing
For “GSE59515” and “GSE55374” cases, RNA was extracted, labelled, and hybridized to HumanHT-12 v4 Illumina BeadChips (GPL10558) according to the standard protocol for NuGEN amplified samples. For GSE20181 cases, samples were processed on Affymetrix U133A gene chip (GPL96). There were no significant differences in clinical or pathologic features of patients or tumours from the three datasets. Illumina and Affymetrix data were independently normalized (quantile using the lumi Bioconductor package2l and the log scale robust multi-array analysis expression measure was implemented with the affy packages) and mapped to Ensembl gene identifiers using re-annotation and mapping for oligonucleotide array technologies and custom chip definition file, respectively, before detection filtering and batch correction with cross-platform normalization. Biopsies were taken from the same subjects at three time-points: pre-treatment, after 10-14 days Letrozol (2.5 mg/day, oral), and after 90 days Letrozol (2.5 mg/day, oral).
C) Assessment of Complete Pathological Response (pCR; Complete Disappearance of Tumour Cells in Breast and Lymph Node) after Receiving Pre-Operative Anthracycline Based Combination Chemotherapy Therapy for ER+ Locally Advanced Breast Cancer Patients (i.e. Examples 39 to 44)
The association between SPAG5 mRNA expression and pCR was evaluated in 2208 patients with locally advanced BC who received anthracycline based combination chemotherapy (E, AC, or FEC) with (+) or without (−) taxane (T). SPAG5 mRNA expression data has been retrieved from 11 publically available gene expression microarray datasets at the National Centre for Biotechnology Information Gene Expression Omnibus using the following access numbers.
The patients had received the following pre-operative chemotherapy.
Cut-Off Determination:
SPAG5 gene expression data of each database were converted to a common scale (median equal to 0 and standard deviation equal to 1) in order to merge all of the studies data that used the same platform and create combined cohorts. To convert to a common scale each sample in the data was standardised to a mean of 0 and standard deviation of 1. Then the data was median centred for each gene whereby median of each gene is 0. Databases using same platform have been merged and the median expression was calculated. The median expression of SPAG5 transcript for each platform has been calculated and values equal to or higher than the median coded as +1 (overexpression). Values of less than the median have been coded 0 or low SPAG5.
For HumanWG-6 v3.0-ILLUMINA; GPL6884 platform: SPAG5 median was 0.356
For HG-U133A_2-Affymetrix; GPL571 platform: SPAG5 median was 0.5505
For HG-U133_X3P-Affymetrix; GPL1352 platform: SPAG5 median was 0.3102
For Agilent-014850-Agilent; GPL1708 platform: SPAG5 median was 0.3373
For HG-U133A-Affymetrix; GPL96 platform: SPAG5 median was 0.5075
For HG-U133_Plus_2—Affymetrix; GPL570 SPAG5 median was 0.4368
Ovarian Cancer: Assessment death and distant relapse risks after surgery and receiving adjuvant systemic therapy if eligible in ovarian epithelial cancer. The inventors evaluated the prognostic utility of SPAG5-mRNA expression in a large combined ovarian (OVC) cohort which was sourced from 14 publically-available, global datasets (n=1434) using the following access numbers: GSE14764, GSE15622, GSE18520, GSE19829, GSE23554, GSE26193, GSE26712, GSE27651, GSE30161, GSE3149, GSE51373, GSE63885, GSE9891 and TCGA. Data download and processed as mentioned before in breast cancer. Median cut off was 0.4
Lung cancer: Assessment death and distant relapse risks after surgery and receiving adjuvant systemic therapy if eligible in lung cancer. The inventors evaluated the prognostic utility of SPAG5-mRNA expression in a large combined lung cohort which was sourced from 14 publically-available, global datasets (n=1926) using the following access numbers: CAARRAY, GSE8894, GSE50081, GSE4573, GSE43580, GSE37745, GSE31908, GSE3141, GSE231210, GSE30219, GSE29013, GSE519188, GSE14814, and TCGA. Data download and processed similar to breast cancer.
Gastric cancer: Assessment death and distant relapse risks after surgery and receiving adjuvant systemic therapy if eligible in lung cancer. The inventors evaluated the prognostic utility of SPAG5-mRNA expression in a large combined lung cohort which was sourced from 6 publically-available, global datasets (n=876) using the following access numbers: GSE14216, GSE15459, GSE22377, GSE29272, GSE51105 and GSE62254. Data download and processed similar to breast cancer.
Liver cancer: The relationship between SPAG5 mRNA using SPAG5 RNA sequence ID 10615 and survival was analysed in 364 patients.
Immunohistochemistry (IHC) and tissue microarray (TMA) analysis of SPAG5 protein expression in HER2+ breast cancer was investigated by IHC analysis as four independent cohorts:
A) Nottingham University Hospital Early Stage HER2+ Breast Cancer (NUH-ES-HER2+BC) Cohort (Example 59 and
This cohort of HER2+BC include patients (age>71 years) who were diagnosed and treated uniformly between 1986 and 2006 at the Nottingham City Hospital (NCH), Nottingham, UK. Patients within the good prognosis group (Nottingham Prognostic Index (NPI)<3.4) did not receive systemic adjuvant therapy. Pre-menopausal patients within the moderate and poor prognosis groups were candidates for CMF chemotherapy. Conversely, postmenopausal ER-positive patients with moderate or poor NPI were offered hormonal therapy, whereas ER-negative patients received CMF chemotherapy. None of the patients received trastuzumab. Clinical data were maintained on a prospective basis with a median follow-up of 143 months (Inter-quartile range (IQR)=114-174). Survival data were maintained on a prospective basis. Breast cancer specific survival (BCSS) was defined as the number of months from diagnosis to the occurrence of breast cancer related death. Survival was censored if the patient was still alive, lost to follow-up, or died from other causes. Relapse free survival (RFS) was defined as the number of months from diagnosis to relapse either locally, regionally or distantly. Distant relapse free survival (DRFS) was defined as the number of months from diagnosis to distant metastases relapse.
B) Nottingham University Hospital Early Stage HER2+ Anthracycline Based Combination Chemotherapy Treated Breast Cancer Cohort (NUH− ES− HER2+ ACT-BC; n=275; Example 60 and
To evaluate the survival benefit of SPAG5-protein expression, its expression was analysed in a consecutive series of 275 early stage HER2+BC's who had been diagnosed and managed at NCH between 2000 and 2006 who received adjuvant anthracycline-based combination chemotherapy. None of the patients received trastuzumab.
C) Nottingham University Hospital Early Stage HER2+ Trastuzumab Treated Breast Cancer Cohort (NUH− ES-HER2+ HERCEPTIN-BC; n=235; Example 61 and
To evaluate the survival benefit of SPAG5-protein expression, its expression was analysed in a consecutive series of 235 early stages HER2+BC's who had been diagnosed and managed at NCH between 2006 and 2010 who received adjuvant Trastuzumab with and without anthracycline-based combination chemotherapy and or Taxane.
D) Nottingham University Hospital HER2+ Locally Advanced BC (NUH− HER2+ LABC; n=162); Example 62
The relationship between SPAG5-protein expression and response to chemotherapy was evaluated by investigating its expression in pair-matched pre-chemotherapy core biopsies and post-chemotherapy surgical specimens, from 162 female patients with HER2+ locally-advanced primary BC (HER2+ LAP-BC) (stage IIIA-C) that had been treated with anthracycline-based Neo-ACT (AC-Neo-ACT) with or without Taxane and with or without Trastuzumab at NCH between 1996 and 2015. Twenty-eight percent of patients received six cycles of an anthracycline-based therapy (FEC: 5-fluorouracil (5-FU) 500 md mg m−2, epirubicin 75-100 md mg m−2, cyclophosphamide 500 mg m−2, on day 1 of a 21 day cycle), whereas 27% of patients received FEC plus Taxane. Forty five percent of patients have received Trastuzumab in addition to FEC plus Taxane. All patients underwent mastectomy or breast-conserving surgery and axillary dissection, followed by adjuvant radiation therapy. All patients offered one years of adjuvant Trastuzumab. Patients with HER2+ ER+ BCs were offered additionally 5 years of adjuvant endocrine therapy. The median follow-up time was 67 months (IRQ 27-81).
SPAG5 Transcript Expression
A) Adjuvant HER2+ Breast Cancer Transcript Cohort—Assessment Death and Distant Relapse Risks after Surgery and Receiving Adjuvant Systemic Therapy if Eligible in Early Stage HER+ Breast Cancer (i.e. Examples 63, 64 and 65)
The inventors evaluated the prognostic utility of SPAG5-mRNA expression in a combined HER2+ breast cancer (BC) cohort which was sourced from 8 publically-available, global datasets (n=446) using the following access numbers.
All the databases had been downloaded and processed. The gene expression data were converted to a common scale (median equal to 0 and standard deviation equal to 1) in order to merge all of the studies data and create combined cohorts. Each sample in the data was standardised to a mean of 0 and standard deviation of 1. Then the data was median centred for each gene whereby median of each gene is 0. No systemic therapy has been prescribed for 132/436 of patients (30%) while 35% (155/436) of patients have received FEC plus Trastuzumab. FEC alone has been offered to 142/436 (33%) while CMF was given to 7 patients. Endocrine therapy has been offered to ER+ HER2+ patient (144/444).
B) Assessment of Complete Pathological Response (pCR; Complete Disappearance of Tumour Cells in Breast and Lymph Node) after Receiving Pre-Operative Anthracycline Based Combination Chemotherapy Therapy for HER2+ Locally Advanced Breast Cancer Patients (i.e. Example 66)
The association between SPAG5 mRNA expression and pCR was evaluated in 476 patients with locally advanced BC who received anthracycline based combination chemotherapy (E, AC, or FEC) with (+) or without (−) taxane (T). Fifty one percent of patients (244/476) have offered FEC plus Taxane plus Trastuzumab while 43% (205/476) has received FEC plus Taxane. Only 6% (27/476) has been prescribed FEC.
SPAG5 mRNA expression data has been retrieved from 10 publically available gene expression microarray datasets at the National Centre for Biotechnology Information Gene Expression Omnibus using the following access numbers.
Advanced Breast Cancer
SPAG5 Immunohistochemistry (IHC) and HER2 protein and gene expression in locally advanced breast cancer was investigated by IHC analysis in three independent cohorts (examples 67-74).
The histopathological and radiological alterations of tumor characteristics after receiving neoadjuvant chemotherapy (NACT) was evaluated and the clinical significance of the changes of adjuvant therapy based on these findings was determined. Methods: A pathological assessment of tumor features including ER, PR, HER2 and proliferation markers (Ki67 and SPAG5) status in pre and post neoadjuvant tumors tissues was centrally evaluated in Nottingham University Hospital (NUH; n=465). Since 2013 any change in the ER and HER2 status from negative (−) [in the pre NACT biopsies] to positive (+) [in the post neoadjuvant surgical specimens] received additional adjuvant therapy (Endocrine therapy (ET) for ER+ and Trastuzumab for HER2+ cases). The primary end point was disease free survival (DFS). The relationship between SPAG5-protein expression and response to chemotherapy was evaluated by investigating its expression in pair-matched pre-chemotherapy core biopsies and post-chemotherapy surgical specimens. Twenty-eight percent of patients received six cycles of an anthracycline-based therapy (FEC: 5-fluorouracil (5-FU) 500 mg m−2, epirubicin 75-100 mg m−2, cyclophosphamide 500 mg m−2, on day 1 of a 21 day cycle), whereas 27% of patients received FEC plus Taxane. Forty five percent of patients received Trastuzumab in addition to FEC plus Taxane. All patients underwent mastectomy or breast-conserving surgery and axillary dissection, followed by adjuvant radiation therapy. All patients offered one years of adjuvant Trastuzumab if HER2+. Patients with ER+ BCs were offered additionally 5 years of adjuvant endocrine therapy. The median follow-up time was 67 months (IRQ 27-81).
Granules of SPAG5 protein have been detected in the cytoplasm of cells of breast cancer tissue stained using immunohistochemistry (see
The absence of staining or presence of faint or diffuse intracytoplasmic SPAG5 staining have been detected in the cytoplasm of breast cancer tissues stained using immunohistochemistry (see
Early stage breast cancer patients with SPAG5 overexpression in their cancerous tissue at the time of surgery had 2-fold increase in the risk of death from BC at 10 year of follow up after the time of surgery compared to those with no SPAG5 protein overexpression (i.e. no expression of granular SPAG5 protein) (see
ER+ Early stage breast cancer patients with SPAG5 overexpression in their cancerous tissue at the time of surgery had 2-fold increase in the risk of death from BC at 10 year of follow up after the time of surgery compared to those with low SPAG5 protein overexpression (i.e. no staining or faint or diffuse staining (no intracytoplasmic punctate expression of SPAG5 protein) (see
In early stage BC, the interaction analysis (using multivariate Cox regression model) of SPAG5 protein with other validated prognostic factors confirmed that SPAG5+ protein overexpression was independently associated with 2 fold increase in the risk of death from breast cancer at 10 year of follow up after the time of surgery compared to those with low SPAG5 protein expression (i.e. no staining or faint or diffuse staining (no intracytoplasmic punctate expression of SPAG5 protein) (see Table 1).
ER+ Early stage breast cancer patients with SPAG5 overexpression in their cancerous tissue at the time of surgery had 1.5 fold increases in the risk of distant relapse at 10 year of follow up after the time of surgery compared to those with low SPAG5 protein expression (i.e. no staining or faint or diffuse staining (no intracytoplasmic punctate expression of SPAG5 protein) (see
In ER+LN− early stage BC patients (i.e. patients that have a breast cancer that has not spread to the lymph nodes (LN−) but contains cells that express the oestroegen receptor (ER+)) who did not receive any systemic therapy, SPAG5 protein overexpression in the cancerous tissue at the time of surgery was associated with a three-fold increase of distant relapse risk at 5 years follow up compared to low SPAG5 protein expression (i.e. no staining or faint or diffuse staining (no intracytoplasmic punctate expression of SPAG5 protein) (see
In ER+LN− early stage BC patients who received 5-year adjuvant endocrine therapy (Tamoxifen) alone, SPAG5 protein overexpression in the cancerous tissue at the time of surgery was associated with 2.5-fold increase of distant relapse risk at 5 years follow up compared to low SPAG5− protein expression (i.e. no staining or faint or diffuse staining (no intracytoplasmic punctate expression of SPAG5 protein) (see
In ER+LN− early stage BC patients who received adjuvant anthracycline based combination (AC) therapy with or without taxane chemotherapy in addition to adjuvant 5-year endocrine therapy (Tamoxifen), SPAG5 protein overexpression in the cancerous tissue at the time of surgery had a similar distant relapse risk at 5 years follow up after surgery to low SPAG5 protein expression (i.e. no staining or faint or diffuse staining (no intracytoplasmic punctate expression of SPAG5 protein) (see
In ER+LN+ early stage BC patients who received 5-year adjuvant endocrine therapy (Tamoxifen) alone, SPAG5 protein overexpression in the cancerous tissue at the time of surgery was associated with 2-fold increase of distant relapse risk at 5 years follow up after surgery compared to low SPAG5 protein expression (i.e. no staining or faint or diffuse staining (no intracytoplasmic punctate expression of SPAG5 protein) (see
In ER+LN+ early stage BC patients who received adjuvant CMF (cyclophosphamide, methotrexate and 5-fluorouracil) chemotherapy in addition to adjuvant 5-year endocrine therapy (Tamoxifen), SPAG5 protein overexpression in the cancerous tissue at the time of surgery was associated with 3-fold increase of distant relapse risk at 5 years follow up after surgery compared to low SPAG5 protein expression (i.e. no staining or faint or diffuse staining)(see
In ER+LN+ early stage BC patients who received adjuvant anthracycline based combination with or without taxane chemotherapy in addition to adjuvant 5-year endocrine therapy (Tamoxifen), SPAG5 protein overexpression in the cancerous tissue at the time of surgery had similar distant relapse risk at 5 years of follow up after surgery to low SPAG5 protein expression (i.e. no staining or faint or diffuse staining (no intracytoplasmic punctate expression of SPAG5 protein) (see
In early stage BC, the interaction analysis (using multivariate Cox regression model) of SPAG5 protein expression with systemic therapy and other validated prognostic factors confirmed that SPAG5+ protein overexpression was independently associated with 2 fold increase in the risk of distant relapse at 5 year of follow up after the time of surgery compared to those with low SPAG5 protein expression (i.e. no staining or faint or diffuse staining (no intracytoplasmic punctate expression of SPAG5 protein) (see Table 2 below).
In locally advanced breast cancer, compared to low SPAG5 protein expression (no intracytoplasmic punctate expression of SPAG5 protein) in the tumour biopsy that had been taken from patients at the time of the diagnosis and before receiving any treatment, SPAG5 protein overexpression (i.e. The intracytoplasmic granular (punctate) staining pattern of SPAG5 protein) predicted 2-fold higher rate of complete disappearance of the cancer cells in the breast and lymph node (i.e., pathological complete response) in the surgically removed specimen after receiving preoperative anthracycline based combination chemotherapy (AC) with or without taxane (36% (45/126) vs., 21% (16/77); odd ratio (OR) (CI 95%)=5.78 (3.47-9.63); p<0.0001) (see
In ER negative locally advanced breast cancer, compared to low SPAG5 protein expression (no intracytoplasmic punctate expression of SPAG5 protein) in the tumour biopsy that had been taken from patients at the time of the diagnosis and before receiving any treatment, SPAG5 protein overexpression (i.e. The intracytoplasmic granular (punctate) staining pattern of SPAG5 protein) predicted 6-fold higher rate of complete disappearance of the cancer cells in the breast and lymph node (i.e., pathological complete response) in the surgically removed specimen after receiving preoperative anthracycline based combination chemotherapy (AC) with or without taxane (33% (73/219) vs., 8% (23/289); odd ratio (OR) (CI 95%)=2.12 (1.10-4.10); p=0.024) (see
In ER positive locally advanced breast cancer, compared to low SPAG5 protein expression (no intracytoplasmic punctate expression of SPAG5 protein) in the tumour biopsy that had been taken from patients at the time of the diagnosis and before receiving any treatment, SPAG5 protein overexpression (i.e. The intracytoplasmic granular (punctate) staining pattern of SPAG5 protein) predicted 13-fold higher rate of complete disappearance of the cancer cells in the breast and lymph node (i.e., pathological complete response) in the surgically removed specimen after receiving preoperative anthracycline based combination chemotherapy (AC) with or without taxane (30% (28/93) vs., 3% (7/212); odd ratio (OR) (CI 95%)=12.62 (5.26-30.23); p<0.0001)(see
In HER2 negative locally advanced breast cancer, compared to low (no intracytoplasmic punctate expression of SPAG5 protein) SPAG5 protein expression in the tumour biopsy that had been taken from patients at the time of the diagnosis and before receiving any treatment, SPAG5 protein overexpression (i.e. The intracytoplasmic granular (punctate) staining pattern of SPAG5 protein) predicted 6-fold higher rate of complete disappearance of the cancer cells in the breast and lymph node (i.e., pathological complete response) in the surgically removed specimen after receiving preoperative anthracycline based combination chemotherapy (AC) with or without taxane (27% (40/149) vs., 6% (12/199); odd ratio (OR) (CI 95%)=5.72 (2.88-11.37); p<0.0001) (see
In HER2 positive locally advanced breast cancer, compared to low SPAG5 protein expression (no intracytoplasmic punctate expression of SPAG5 protein) in the tumour biopsy that had been taken from patients at the time of the diagnosis and before receiving any treatment, SPAG5 protein overexpression (i.e. The intracytoplasmic granular (punctate) staining pattern of SPAG5 protein) predicted 7-fold higher rate of complete disappearance of the cancer cells in the breast and lymph node (i.e., pathological complete response) in the surgically removed specimen after receiving preoperative anthracycline based combination chemotherapy (AC) with or without taxane (50% (30/60) vs., 13% (10/80); odd ratio (OR) (CI 95%)=7.00 (3.04-16.11); p<0.0001) (see
In locally advanced breast cancer, compared to low SPAG5 protein expression (no intracytoplasmic punctate expression of SPAG5 protein) in the tumour biopsy that had been taken from patients at the time of the diagnosis and before receiving any treatment, SPAG5 protein overexpression (i.e. The intracytoplasmic granular (punctate) staining pattern of SPAG5 protein) predicted 9-fold higher rate of complete disappearance of the cancer cells in the breast and lymph node (i.e., pathological complete response) in the surgically removed specimen after receiving preoperative anthracycline based combination chemotherapy (AC) alone (28% (25/89) vs., 4% (4/93); odd ratio (OR) (CI 95%)=8.70 (2.88-26.20); p<0.0001) (see
In locally advanced breast cancer, compared to low SPAG5 protein expression in the tumour biopsy that had been taken from patients at the time of the diagnosis and before receiving any treatment, SPAG5 protein overexpression predicted 7-fold higher rate of complete disappearance of the cancer cells in the breast and lymph node (i.e., pathological complete response) in the surgically removed specimen after receiving preoperative taxane in addition to anthracycline based combination chemotherapy (33% (34/103) vs., 7% (11/160); odd ratio (OR) (CI 95%)=6.70 (3.19-13.95); p<0.0001) (see
In HER2 positive locally advanced breast cancer, compared to low SPAG5 protein expression (no intracytoplasmic punctate expression of SPAG5 protein) in the tumour biopsy that had been taken from patients at the time of the diagnosis and before receiving any treatment, SPAG5 protein overexpression (i.e. The intracytoplasmic granular (punctate) staining pattern of SPAG5 protein) predicted 4-fold higher rate of complete disappearance of the cancer cells in the breast and lymph node (i.e., pathological complete response) in the surgically removed specimen after receiving preoperative trastuzumab and taxane in addition to anthracycline based combination chemotherapy (52% (14/27) vs., 22% (8/36); odd ratio (OR) (CI 95%)=3.80 (1.27-11.21); p=0.015) (see
In ER positive locally advanced breast cancer, compared to low SPAG5 protein expression (no intracytoplasmic punctate expression of SPAG5 protein) in the tumour biopsy that had been taken from patients at the time of the diagnosis and before receiving any treatment, SPAG5 protein overexpression (i.e. The intracytoplasmic granular (punctate) staining pattern of SPAG5 protein) predicted 16 fold higher rate of complete disappearance of the cancer cells in the breast and lymph node (i.e., pathological complete response) in the surgically removed specimen after receiving preoperative anthracycline based combination chemotherapy (AC) alone (16% (6/38) vs., 0% (0/62); p=0.001) (see
In ER positive locally advanced breast cancer, compared to low SPAG5 protein expression (no intracytoplasmic punctate expression of SPAG5 protein) in the tumour biopsy that had been taken from patients at the time of the diagnosis and before receiving any treatment, SPAG5 protein overexpression (i.e. The intracytoplasmic granular (punctate) staining pattern of SPAG5 protein) predicted 15-fold higher rate of complete disappearance of the cancer cells in the breast and lymph node (i.e., pathological complete response) in the surgically removed specimen after receiving preoperative taxane in addition to anthracycline based combination chemotherapy (37% (16/43) vs., 4% (5/128); odd ratio (OR) (CI 95%)=14.60 (4.92-43.24); p<0.0001) (see
In ER positive/Her2 positive locally advanced breast cancer, compared to low SPAG5 protein expression (no intracytoplasmic punctate expression of SPAG5 protein) in the tumour biopsy that had been taken from patients at the time of the diagnosis and before receiving any treatment, SPAG5 protein overexpression (i.e. The intracytoplasmic granular (punctate) staining pattern of SPAG5 protein) predicted 10-fold higher rate of complete disappearance of the cancer cells in the breast and lymph node (i.e., pathological complete response) in the surgically removed specimen after receiving preoperative taxane in addition to anthracycline based combination chemotherapy (37% (16/43) vs., 4% (5/128); odd ratio (OR) (CI 95%)=10.00 (1.60-63.10); p=0.0007) (see
In summary SPAG5 protein overexpression is associated with higher pCR regardless the molecular subclass of breast cancer after receiving either Anthracycline based combination chemotherapy alone or with Taxane or with Taxane and Herceptin®.
SPAG5+ mRNA expression was observed in 43% of ER+ early stage BC tissue at the time of surgery. SPAG5 transcript overexpression is more common in PAM-50 luminal B, PAM-50 HER2+ breast cancer than in luminal A, TP53 mutation, BRCA2 mutation, highly genomic instable integrative clusters (1, 2, 5, 6, 9 and 10), Luminal genomic complex and 17q12 genomic patterns and 8qcis-acting/20q amplification.
In ER+ early stage breast cancer, at 10 years of follow up after surgically removing the primary breast cancer regardless of the other risk factors and did not receive adjuvant systemic therapy, SPAG5 overexpression in breast cancer tissue at the time of surgery was associated with 2-fold increase in the distant relapse risk compare to low SPAG5 mRNA expression (see
In ER+ early stage BC patients with lymph node negative, after receiving 5-year of adjuvant endocrine therapy (Tamoxifen), breast cancers that exhibited overexpression of SPAG5+ mRNA at the time of surgery had 3-fold increase of distant relapse risk compared to those with low SPAG5 (see
In ER+ early stage BC patients with lymph node negative, after receiving adjuvant anthracycline combination chemotherapy followed by 5-year-adjuvant endocrine therapy (Tamoxifen), breast cancers that exhibited overexpression of SPAG5+ mRNA at the time of surgery had similar distant relapse risk compared to those who did not receive any systemic therapy (see
In ER+ early stage BC patients with lymph node negative and low SPAG5 mRNA expression at the time of surgery, receiving 5-year-adjuvant endocrine therapy (Tamoxifen) alone had the similar distant relapse risk of receiving adjuvant anthracycline combination chemotherapy in addition to the 5-year-adjuvant endocrine therapy (Tamoxifen) (13% vs 15%)(see
In ER+ early stage BC patients with lymph node negative and SPAG5+ mRNA overexpression at the time of surgery, receiving adjuvant anthracycline combination chemotherapy in addition to the 5-year-adjuvant endocrine therapy (Tamoxifen), had reduced the distant relapse risk by 3-fold (11%) compared to receiving the 5-year-adjuvant endocrine therapy (Tamoxifen) alone (33%)(see
In ER+ early stage BC patients with lymph node negative and SPAG5+ mRNA overexpression at the time of surgery, receiving adjuvant anthracycline combination chemotherapy in addition to 5-year-adjuvant endocrine therapy (Tamoxifen), had reduced the distant relapse risk (11%) by 4-fold compared to receiving no systemic therapy (39%) (see
In ER+ early stage BC patients with lymph node positive (LN+), after receiving 5-year-Adjuvant endocrine therapy (Tamoxifen) alone, breast cancers that exhibited overexpression of SPAG5+ mRNA at the time of surgery had a two-fold higher distant relapse risk compared to those with low SPAG5 (see
In ER+ early stage BC patients with lymph node positive (LN+) after receiving adjuvant anthracycline combination chemotherapy in addition to 5-year-adjuvant endocrine therapy (Tamoxifen), tumours that exhibited overexpression of SPAG5+ mRNA had a similar distant relapse risk compared to those with low SPAG5 mRNA expressing (see
In ER+ early stage BC patients with lymph node positive (LN+) and SPAG5+ mRNA overexpression at the time of surgery, receiving adjuvant anthracycline combination chemotherapy in addition to the 5-year-adjuvant endocrine therapy (Tamoxifen), had reduced the distant relapse risk by 2 fold (24%) compared to receiving the 5-year-adjuvant endocrine therapy (Tamoxifen) alone (46%) (see
In ER+ early stage BC patients with lymph node positive (LN+) and low SPAG5+ mRNA at the time of surgery, receiving 5-year-adjuvant endocrine therapy (Tamoxifen) alone had similar distant relapse risk of receiving adjuvant anthracycline combination chemotherapy in addition to the 5-year-adjuvant endocrine therapy (Tamoxifen), (22% vs 20%) (see
In ER+ early stage BC, the interaction analysis (using multivariate Cox regression model) of SPAG5 mRNA with systemic therapy and other validated prognostic factors confirmed that SPAG5+ mRNA expression was a better independent biomarker for predicting poor 5-year distant relapse risk.
In ER+ early stage BC, the interaction analysis (using multivariate Cox regression model) between SPAG5 mRNA with anthracycline based combination chemotherapy therapy has confirmed that SPAG5 mRNA is both a prognostic and predictive biomarker 5-year distant relapse risk.
In ER+ early stage BC, the interaction analysis (using multivariable cox regression) of SPAG5 mRNA with Nottingham Prognostic Index (NPI) has revealed that SPAG5+ mRNA was a better independent biomarker for predicting 5-year distant relapse risk.
In ER+ early stage BC, the interaction analysis (using multivariable cox regression) of SPAG5 mRNA with 72-proliferation-gene-signature has revealed that SPAG5+ mRNA was a better independent biomarker for predicting 5-year distant relapse risk.
In ER+ early stage BC, the interaction analysis (using multivariable cox regression) of SPAG mRNA with 76-gene prognostic signature (Veridex) has revealed that SPAG5+ mRNA was a better independent biomarker for predicting 5-year distant relapse risk.
In ER+ early stage BC, the interaction analysis (using multivariable cox regression) of SPAG mRNA with PAM-50-molecular subgroups has revealed that SPAG5+ transcript was a better independent biomarker for predicting 5-year distant relapse risk (Table 3).
The expression level of SPAG5 mRNA in a tumour biopsy that has been taken from BC patients with an ER+ tumour after 2 weeks of receiving pre-operative endocrine therapy was significantly downregulated in 72% (68/92) of cases compared to the expression level in the biopsy that has been taken before the starting of the treatment I (p<0.00001, Wilcoxon-test).
There was a significant further reduction in the level of SPAG5 mRNA expression in the tumour biopsy that has been taken from patients after 12 weeks of receiving pre-operative endocrine therapy compared to the expression level in the biopsy that has been taken after 2 weeks of receiving pre-operative endocrine therapy (p=0.023, Wilcoxon).
In 76% of tumours that have shown reduction in the tumour volume by 50% after receiving preoperative endocrine therapy for 2 weeks (i.e. responding tumours), a significant down-regulation of SPAG5 mRNA expression occurred in the tumour biopsy that has been taken from patients after 2 weeks of receiving pre-operative endocrine therapy (p<0.00001, Wilcoxon-test).
In tumours that have not shown reduction in the tumour volume by 50% after receiving preoperative endocrine therapy for either 2 or 12 weeks (i.e. non-responding tumours), there was no significant change in SPAG5 mRNA expression level in the tumour biopsy that has been taken from patients either after 2 or 12 weeks of receiving pre-operative endocrine therapy compared to the expression level in the biopsy that has been taken before the starting of the treatment (p=0.265 and 0.563; respectively, Wilcoxon-test).
After 12 weeks of receiving pre-operative endocrine therapy, tumours that did not show a reduction in the tumour volume by 50% (i.e. non-responding tumours) expressed a higher level of SPAG5 mRNA level compared to those that showed reduction in the tumour volume by 50% (i.e. responding tumours) (p=0.014, Mann-Whitney);
In ER+ locally advanced breast cancer, compared to low SPAG5 mRNA expression in the tumour biopsy that had been taken from patients at the time of the diagnosis and before receiving any treatment, SPAG5 mRNA overexpression predicted 2-fold higher rate of complete disappearance of the cancer cells in the breast and lymph node (i.e., pathological complete response) in the surgically removed specimen after receiving preoperative anthracycline based combination chemotherapy alone (25% vs 12%)); p<0.0001; see
In ER+ locally advanced breast cancer, compared to low SPAG5 mRNA expression in the tumour biopsy that had been taken from patients at the time of the diagnosis and before receiving any treatment, SPAG5 mRNA overexpression predicted 2-fold higher rate of complete disappearance of the cancer cells in the breast and lymph node (i.e., pathological complete response) in the surgically removed specimen after receiving preoperative taxane in addition to anthracycline based combination chemotherapy (17% vs 8%); p<0.0001.
In ER+ HER2− locally advanced breast cancer, compared to low SPAG5 mRNA expression in the tumour biopsy that had been taken from patients at the time of the diagnosis and before receiving any treatment, SPAG5 mRNA overexpression predicted 2.5 fold higher rate of complete disappearance of the cancer cells in the breast and lymph node (i.e., pathological complete response) in the surgically removed specimen after receiving preoperative taxane in addition to anthracycline based combination chemotherapy [17% (57/328) vs., 8% (43/552); OR (CI 95%): 2.49 (1.63-3.80); p=0.00001].
In ER+ HER2+ locally advanced breast cancer, compared to low SPAG5 mRNA expression in the tumour biopsy that had been taken from patients at the time of the diagnosis and before receiving any treatment, SPAG5 mRNA overexpression predicted a trend of higher rate of complete disappearance of the cancer cells in the breast and lymph node (i.e., pathological complete response) in the surgically removed specimen after receiving preoperative Trastuzumab and taxane in addition to anthracycline based combination chemotherapy (33% vs 22%; p=0.15).
Overall, in ER+ locally advanced breast cancer, compared to low SPAG5 mRNA expression in the tumour biopsy that had been taken from patients at the time of the diagnosis and before receiving any treatment, SPAG5 mRNA overexpression predicted 2.5-fold higher rate of complete disappearance of the cancer cells in the breast and lymph node (i.e., pathological complete response) in the surgically removed specimen after receiving preoperative anthracycline based combination chemotherapy with or without taxane (20% (86/430) vs., 9% (58/627); odd ratio (OR) (CI 95%)=2.5 (1.71-3.51); p<0.0001).
The interaction analysis of SPAG5 mRNA with systemic therapy and other validated prognostic factors (using multivariate logistic regression model) confirmed that SPAG5 mRNA overexpression in the tumour biopsy that had been taken from patients at the time of the diagnosis and before receiving any treatment, predicted 2-fold higher rate of complete disappearance of the cancer cells in the breast and lymph node (i.e., pathological complete response) in the surgically removed specimen after receiving anthracycline based combination preoperative chemotherapy regardless HER2 status or receiving additional taxane or Trastuzumab (OR (CI 95%): 1.92 (1.01-3.64); p=0.047; Table 4A; MC-Neo-Adj-CT cohort).
Table 4: Multivariable logistic regression models analysis for pathological complete response (pCR) after neo-adjuvant chemotherapy
In ovarian cancer, SPAG5 mRNA overexpression in ovarian cancer tissue at the time of surgery was associated with 20% reduction in the risk of relapse after receiving cisplatinum and taxane at 5 years of follow up after surgically removing the primary ovarian cancer compare to low SPAG5 mRNA expression (HR (95% CI)=0.80 (0.65-0.98); p=0.035 (see
Granular SPAG5 protein is expressed in ovarian (see
In ovarian cancer, SPAG5 protein overexpression in ovarian cancer tissue at the time of surgery was associated with 60% reduction in the risk of death after receiving cisplatinum and taxane at 10 years of follow up after surgically removing the primary ovarian cancer compare to low SPAG5 protein expression (HR (95% CI)=0.40 (0.26-0.62); p<0.0001 (see
In ovarian cancer, SPAG5 protein overexpression in ovarian cancer tissue at the time of surgery was associated with 52% reduction in the risk of relapse after receiving cisplatinum and taxane at 10 years of follow up after surgically removing the primary ovarian cancer compare to low SPAG5 protein expression (HR (95% CI)=0.48 (0.33-0.71); p<0.0001 (see
In gastric cancer, SPAG5 mRNA in gastric cancer tissue at the time of surgery was associated with 25% reduction in the risk of relapse after receiving preoperative cisplatinum followed by surgery compare to low SPAG5 mRNA expression at 5 years of follow up after surgically removing the primary gastric cancer, (HR (95% CI=0.75 (0.60-0.93); p=0.0088) (n=499) (see
In gastric cancer, SPAG5 mRNA overexpression in gastric cancer tissue at the time of surgery was associated with three times higher risk of relapse after receiving 5-flu adjuvant therapy at 5 years of follow up after surgically removing the primary gastric cancer compared to low SPAG5 mRNA expression (HR (95% CI)=2.82 (1.81-4.39); p<0.0001) (n=499) (see
Granular SPAG5 protein is expressed in gastric cancer (see
In gastric cancer, SPAG5 protein overexpression in gastric cancer tissue at the time of surgery was associated with 40% reduction in the risk of death (see
In lung cancer, SPAG5 mRNA overexpression in lung cancer tissue at the time of surgery was associated with 2-fold increase in the risk of death at 10 years of follow up after surgically removing the primary lung cancer compare to low SPAG5 mRNA expression (HR (95% CI): 1.95 (1.66-2.29), p<0.0001; n=1926, expression level of SPAG5 probe=26-5014, median cut-off expression level=254) (see
In lung cancer, SPAG5 mRNA overexpression in lung cancer tissue at the time of surgery was associated with 2-fold increase in the risk of relapse at 10 years of follow up after surgically removing the primary lung cancer compare to low SPAG5 mRNA expression (HR (95% CI): 1.94 (1.58-2.38), p<0.0001; n=1926, expression level of SPAG5 probe=26-5014, median cut-off expression level=254) (see
Granular SPAG5 protein is expressed in lung cancer (see
In lung cancer, SPAG5 protein overexpression in lung cancer tissue at the time of surgery was associated with higher risk of death at 10 years of follow up after surgically removing the primary lung cancer compare to low SPAG5 protein expression (HR (95% CI): 1.17 (1.04-1.30), p=0.007) (see
In liver cancer, SPAG5 mRNA overexpression in liver cancer tissue at the time of surgery was associated with 2-fold increase in the risk of relapse at 5 years of follow up after surgically removing the primary liver cancer compare to low SPAG5 mRNA expression (HR (95% CI): 1.61(1.14-2.28), p=0.0069; n=364) (see
In colon cancer, SPAG5 protein overexpression in colon cancer tissue at the time of surgery was associated with 20% reduction in the risk of death after receiving taxane and cisplatin at 5 years of follow up after surgically removing the primary colon cancer compare to low SPAG5 protein expression (p=0.016) (see
In rectal cancer, SPAG5 protein overexpression in rectal cancer tissue at the time of surgery was associated with 2 fold increase in the risk of death after receiving radiotherapy at 5 years of follow up after surgically removing the primary rectal cancer compare to low SPAG5 protein expression (p=0.005) (see
In HER2+ early stage BC patients (i.e. patients that have a breast cancer that express the human epidermal growth factor receptor 2 (HER+2) who did not receive any systemic chemotherapy and HER2 target therapy, SPAG5 protein overexpression in the cancerous tissue at the time of surgery was associated with increase of distant relapse risk at 5 years follow up compared to low SPAG5 protein expression (i.e. no staining or faint or diffuse staining (no intracytoplasmic punctate expression of SPAG5 protein) (see
In HER2+ early stage BC patients (i.e. patients that have a breast cancer that express the human epidermal growth factor receptor 2 (HER+2) who received anthracycline based chemotherapy but no Trastuzumab, SPAG5 protein overexpression in the cancerous tissue at the time of surgery was associated with increase of distant relapse risk at 5 years follow up compared to low SPAG5 protein expression (i.e. no staining or faint or diffuse staining (no intracytoplasmic punctate expression of SPAG5 protein) (see
In HER2+ early stage BC patients (i.e. patients that have a breast cancer that express the human epidermal growth factor receptor 2 (HER+2) who received anthracycline based chemotherapy plus Trastuzumab, SPAG5 protein overexpression in the cancerous tissue at the time of surgery was associated with reduction of distant relapse risk at 5 years follow up compared to low SPAG5 protein expression (i.e. no staining or faint or diffuse staining (no intracytoplasmic punctate expression of SPAG5 protein) (see
In HER2 positive locally advanced breast cancer with low SPAG5 protein expression (no intracytoplasmic punctate expression of SPAG5 protein) in the tumour biopsy that had been taken from patients at the time of the diagnosis and before receiving any treatment, receiving anthracycline based chemotherapy plus Taxane plus HER2 targeting Neo-adjuvant therapy was associated with higher pCR compared to anthracycline based chemotherapy with or without taxane (21% vs., 4%; OR (95% CI): 6.6 (1.4-32.6), p=0.01). Whereas receiving anthracycline based chemotherapy with or without taxane was associated with similar pCR to anthracycline based chemotherapy plus Taxane plus HER2 targeting Neo-adjuvant therapy in patients with HER2+ SPAG5+ protein (49% vs., 53%; OR (95% CI): 1.2 (0.5-3.2), p=0.702). Receiving anthracycline based chemotherapy plus Taxane plus HER2 targeting Neo-adjuvant therapy was associated with lower DRR compared to anthracycline based chemotherapy plus Taxane Neo-adjuvant therapy [HR (95% CI): 0.82 (0.08-0.97); p=0.045] in patients with SPAG5− protein expression but not in those with SPAG5+ protein [HR (95% CI): 1.08 (0.34-3.36); p=0.901].
In HER2+ early stage BC patients (i.e. patients that have a breast cancer that express the human epidermal growth factor receptor 2 (HER+2) who did not receive any systemic chemotherapy and HER2 target therapy, SPAG5 transcript overexpression in the cancerous tissue at the time of surgery was associated with increase of distant relapse risk at 5 years follow up compared to low SPAG5 transcript expression (see
In HER2+ early stage BC patients (i.e. patients that have a breast cancer that express the human epidermal growth factor receptor 2 (HER+2) who treated with anthracycline based chemotherapy but no Trastuzumab, SPAG5 transcript overexpression in the cancerous tissue at the time of surgery was associated with reduced distant relapse risk at 5 years follow up compared to low SPAG5 transcript expression (see
In HER2+ early stage BC patients (i.e. patients that have a breast cancer that express the human epidermal growth factor receptor 2 (HER+2) who treated with anthracycline based chemotherapy plus Trastuzumab, SPAG5 transcript overexpression in the cancerous tissue at the time of surgery was associated with similar distant relapse risk at 5 years follow up compared to low SPAG5 transcript expression (see
In HER2 positive locally advanced breast cancer, patients with SPAG5 mRNA overexpression (+;> median), those who had received anthracycline based chemotherapy with or without taxane Neo-adjuvant therapy alone achieved similar pCR to those who had received anthracycline based chemotherapy plus Taxane plus HER2 targeting Neo-adjuvant therapy (38% vs., 37%; OR (95% CI): 1.0 (0.6-1.6), p=0.923) in either ER− (46% vs., 52%; OR (95% CI): 1.3 (0.5-3.0), p=0.58) or ER+ subgroups (25% vs., 26%; OR (95% CI): 1.1 (0.4-3.4), p=0.88). Whereas in patients with low SPAG5 mRNA (−), those who had received anthracycline based chemotherapy plus Taxane plus HER2 targeting Neo-adjuvant therapy had achieved 2.5 fold increase in pCR compared to those who received anthracycline based chemotherapy with or without taxane alone (47% vs., 26%; OR (95% CI): 2.5 (1.4-4.4), p=0.001) in either ER− (60% vs., 31%; OR (95% CI): 3.4 (1.7-6.7), p<0.001) or ER+ subgroups (42% vs., 16%; OR (95% CI): 4.0 (1.2-12.9), p=0.018).
Eight percent (8%; 30/361) of pre neoadjuvant chemotherapy HER2− samples had been converted into HER2+ in post neoadjuvant chemotherapy samples had achieved 92% 5-year relapse free survival compared to those who remained HER− in post neoadjuvant chemotherapy (58% 5-year DFS); (HR (95% CI)=0.18 (0.05-0.74); p=0.017). Noteworthy 19 out of 30 HER2 converted cases (63%) have subsequently received adjuvant Trastuzumab while 11 out of the 30 converted cases did not receive either adjuvant chemotherapy or HER2 target therapy (see
Eight percent (8%; 30/361) of pre neoadjuvant chemotherapy HER2− samples had been converted into HER2+ in post neoadjuvant chemotherapy samples. The 19 out of 30 HER2 converted cases who have subsequently received adjuvant Trastuzumab had achieved 100% 5-year relapse free survival compared to those who did not receive either adjuvant chemotherapy or HER2 target therapy (58% 5-year DFS); p=0.040) (see
In pre systemic therapy chemotherapy locally advanced BC patients that were HER2− (i.e. patients that have a breast cancer that doesn't express the human epidermal growth factor receptor 2 (HER+2) in both pre and post neoadjuvant chemotherapy samples, the presence of granular SPAG5 protein expression was reported in 26% of the post neoadjuvant chemotherapy residual cancerous tissue and was associated with 3 fold in increase in the risk of relapse at 5 years follow up compared to the absence of granular SPAG5 protein expression (65% vs., 35% 5-year DFS); (HR (95% CI)=2.96 (2.06-4.26); p<0.0001) (see
In pre systemic therapy chemotherapy locally advanced BC patients that were HER2−/ER− (i.e. patients that have a breast cancer that doesn't express the human epidermal growth factor receptor 2 (HER+2) and oestrogen receptor) in the pre neoadjuvant chemotherapy samples and remained HER2− in the post chemotherapy samples, the presence of granular SPAG5 protein expression was reported in 41% of the post neoadjuvant chemotherapy residual cancerous tissue samples and was associated with 3 fold in increase in the risk of relapse at 5 years follow up compared to the absence of granular SPAG5 protein expression (67% vs., 34% 5-year DFS); (HR (95% CI)=2.68 (1.53-4.68); p=0.001) (see
In pre systemic therapy chemotherapy locally advanced BC patients that were HER2−/ER+ (i.e. patients that have a breast cancer that doesn't express the human epidermal growth factor receptor 2 (HER+2) but expresses oestrogen receptor) in the pre neoadjuvant chemotherapy samples and remained HER2− in the post chemotherapy samples, the presence of granular SPAG5 protein expression was reported in 25% of the post neoadjuvant chemotherapy residual cancerous tissue samples and was associated with four fold in increase in the risk of relapse at 5 years follow up compared to the absence of granular SPAG5 protein expression (72% vs., 32% 5-year DFS); (HR (95% CI)=4.06 (2.53-6.51); p<0.001) (see
Twenty-one percent (21%; 19/92) of pre neoadjuvant chemotherapy HER2+ samples had been converted into HER2− in post neoadjuvant chemotherapy residual samples and had achieved 100% 5-year relapse free survival compared to those who remained HER+ in post neoadjuvant chemotherapy residual sample (66% 5-year DFS); p=0.025). Noteworthy 15 out of 19 HER2 converted cases (79%) have received HER2 target neoadjuvant therapy in addition to anthracycline based combination plus taxane chemotherapy followed by adjuvant Trastuzumab for one year (see
Seventy-nine percent (79%; 73/92) of pre neoadjuvant chemotherapy HER2+ samples had remained HER2+ in post neoadjuvant chemotherapy samples. The majority of these cases 88%; (64/73) have received adjuvant HER2 Target therapy after the neoadjuvant chemotherapy and achieved a better prognosis (75% 5-year DFS) compared to those who did not receive any HER2 Target therapy (n=9/73 (12%); 26% 5-year DFS; p<0.001 (see
In pre systemic therapy chemotherapy locally advanced BC patients that were HER2+ (i.e. patients that have a breast cancer that express the human epidermal growth factor receptor 2 (HER+2) in both pre and post neoadjuvant chemotherapy samples, the presence of granular SPAG5 protein expression was reported in 16% of the post neoadjuvant chemotherapy residual cancerous tissue. In cases that has received HER2 target therapy. The presence of the granules of SPAG5 protein was associated with increase in the risk of relapse at 5 years follow up compared to the absence of granular SPAG5 protein expression either in ER negative or ER positive breast cancer (see
| Number | Date | Country | Kind |
|---|---|---|---|
| 1807879.0 | May 2018 | GB | national |
| 1817968.9 | Nov 2018 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/GB2019/051319 | 5/14/2019 | WO | 00 |
