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Immuno-oncology therapy biomarkers differences …...Immuno-oncology therapy biomarkers differences...

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Immuno-oncology therapy biomarkers differences between polyoma-virus positive and negative Merkel cell carcinomas Zoran Gatalica, Joanne Xiu, Elma Contreras, Jeffrey Swensen Caris Life Sciences, Phoenix, Arizona, United States of America Abstract Background: Merkel cell carcinoma (MCC) is a rare, aggressive, cutaneous neuroendocrine tumor. Merkel cell polyomavirus (MCPyV) is detected in the majority of MCCs while MCPyV-negative cases are thought to arise through progressive accumulation of ultraviolet-light induced somatic mutations. In a non-selected cohort of stage IV MCC patients, treatment with Avelumab (an anti-PD-L1 monoclonal antibody), showed durable responses in less than a third of patients, regardless of their PD-L1 or MCV status. We hypothesized that there are significant differences in other predictors of response to I-O therapy in MCC related to their oncogenic origins. Methods: Forty-seven MCC samples were analyzed for the presence of MCPyV using immunohistochemistry for detection of the large T-antigen (CM2B4 clone). Biomarkers of I-O therapy response included: expression of PD-L1 (IHC), total mutational burden (TMB) and microsatellite instability (MSI). NGS was performed on genomic DNA isolated from FFPE tumor samples using either NextSeq (592-whole gene panel) or MiSeq (45-gene hot-spot panel). Results: Overall, MCPyV was detected in 16/46 cases using IHC (35%). TMB in MCPyV-positive cases (available for 9 cases) averaged 6/Mb (range 4- 11/Mb), while in MCPyV-negative cases (N=21) it was significantly (p<0.0001) higher and averaged 25/Mb (range 4-68/Mb). Similarly, MCPyV-negative cases exhibited significantly more pathogenic mutations than MCPyV- positive carcinomas (p<0.001). No microsatellite instability was detected in any of the cases (0/22). The most commonly mutated gene in MCPyV- negative cohort was TP53 detected in 20 cases, with co-mutation of RB1 in 11 cases. Other more prevalent mutations included PIK3CA (12%), NOTCH1 (9%), KMT2D and KMT2C (5% each). Only one pathogenic mutation (ARID1A) was detected in any of the nine MCPyV-positive cases. A single (MCPyV- positive) case exhibited PD-L1 expression in 5% of tumor cells. Conclusions: MCPyV associated MCC was present in a significantly lower proportion of cases in our data set (35%) than previously reported in the non-selected cases (70-80%), potentially due to the preselection of advanced disease cases. Successes of Avelumab therapy in MCC may be related to the high mutational load in MCPyV-negative cases and mediated through PD-L1+ immune cell infiltrate (IC) influence on effector (PD-1) lymphocytes, or some other mechanism. Further analysis of the status of these (and potentially other) biomarkers of response to immune checkpoint inhibitors is recommended to refine the subgroups of patients responding to therapy. Results Samples and Methods Forty-seven Merkel cell carcinomas (MCC) were analyzed for the presence of MCPyV using immunohistochemistry (IHC) for detection of the large T-antigen (CM2B4 clone). Biomarkers of I-O therapy response included: Expression of PD-L1 (IHC), total mutational burden (TMB) and microsatellite instability (MSI) (NGS assay, Illumina). Conclusions MCPyV associated MCC was present in a significantly lower proportion of cases (35%) than what had been previously reported (70-80%), potentially due to preselection of advanced disease cases. MCPyV-negative MCC are characterized by significantly higher number of pathogenic and targetable mutations as well as by significantly higher tumor mutational burden. PD-L1 expression was predominantly absent and all tested MCC cases were MSI-S. Figure 1. Merkel cell carcinoma positive for Polyomavirus by IHC (top images) and negative for MCPyV (bottom images). MCPyV status has a significant impact on mutational profile of MCC Polyomavirus status (by immunohistochemistry) in MCC Polyoma virus status in Merkel cell carcinomas Frequency Percent Merkel cell carcinomas negative 30 65% positive 16 35% Total 46 100% In MCPyV-positive cases (n=9 tested with 592-NGS ) average TMB was 6/Mb (range 4-11/Mb), while MCPyV-negative cases (n=22) had a significantly ( p<0.001) higher average TMB of 25/Mb (range 4-68/Mb). MCPyV/TMB relationship Merkel cell carcinomas TMB status Total Low High MCPyV status negative 8 (36%) 14 (64%) 22 (71%) positive 9 (100%) 0 (0%) 9 (29%) Total 17 (55%) 14 (45%) 31 The relationship between MCPyV status and tumor mutational burden (TMB), MSI and PD-L1 (biomarkers of I-O) in MCC Only one pathogenic mutation (ARID1A) was detected in a single case of MCPyV-positive MCC. A single (MCPyV-positive) case of Merkel cell carcinoma exhibited PD- L1 expression in 5% of cancer cells. All tested cases (n=22) were MSI stable (MSI-S). In contrast, MCPyV negative cases exhibited a plethora of pathogenic mutations.
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Page 1: Immuno-oncology therapy biomarkers differences …...Immuno-oncology therapy biomarkers differences between polyoma-virus positive and negative Merkel cell carcinomas Zoran Gatalica,

Immuno-oncology therapy biomarkers differences between polyoma-virus positive and negative Merkel cell carcinomasZoran Gatalica, Joanne Xiu, Elma Contreras, Jeffrey SwensenCaris Life Sciences, Phoenix, Arizona, United States of America

Abstract

Background: Merkel cell carcinoma (MCC) is a rare, aggressive, cutaneous neuroendocrine tumor. Merkel cell polyomavirus (MCPyV) is detected in the majority of MCCs while MCPyV-negative cases are thought to arise through progressive accumulation of ultraviolet-light induced somatic mutations. In a non-selected cohort of stage IV MCC patients, treatment with Avelumab (an anti-PD-L1 monoclonal antibody), showed durable responses in less than a third of patients, regardless of their PD-L1 or MCV status. We hypothesized that there are significant differences in other predictors of response to I-O therapy in MCC related to their oncogenic origins.Methods: Forty-seven MCC samples were analyzed for the presence of MCPyV using immunohistochemistry for detection of the large T-antigen (CM2B4 clone). Biomarkers of I-O therapy response included: expression of PD-L1 (IHC), total mutational burden (TMB) and microsatellite instability (MSI). NGS was performed on genomic DNA isolated from FFPE tumor samples using either NextSeq (592-whole gene panel) or MiSeq (45-gene hot-spot panel). Results: Overall, MCPyV was detected in 16/46 cases using IHC (35%). TMB in MCPyV-positive cases (available for 9 cases) averaged 6/Mb (range 4-11/Mb), while in MCPyV-negative cases (N=21) it was significantly (p<0.0001) higher and averaged 25/Mb (range 4-68/Mb). Similarly, MCPyV-negative cases exhibited significantly more pathogenic mutations than MCPyV-positive carcinomas (p<0.001). No microsatellite instability was detected in any of the cases (0/22). The most commonly mutated gene in MCPyV-negative cohort was TP53 detected in 20 cases, with co-mutation of RB1 in 11 cases. Other more prevalent mutations included PIK3CA (12%), NOTCH1(9%), KMT2D and KMT2C (5% each). Only one pathogenic mutation (ARID1A) was detected in any of the nine MCPyV-positive cases. A single (MCPyV-positive) case exhibited PD-L1 expression in 5% of tumor cells.Conclusions: MCPyV associated MCC was present in a significantly lower proportion of cases in our data set (35%) than previously reported in the non-selected cases (70-80%), potentially due to the preselection of advanced disease cases. Successes of Avelumab therapy in MCC may be related to the high mutational load in MCPyV-negative cases and mediated through PD-L1+ immune cell infiltrate (IC) influence on effector (PD-1) lymphocytes, or some other mechanism. Further analysis of the status of these (and potentially other) biomarkers of response to immune checkpoint inhibitors is recommended to refine the subgroups of patients responding to therapy.

Results

Samples and MethodsForty-seven Merkel cell carcinomas (MCC) were analyzed for the presence of MCPyV using immunohistochemistry (IHC) for detection of the large T-antigen (CM2B4 clone). Biomarkers of I-O therapy response included: Expression of PD-L1 (IHC), total mutational burden (TMB) and microsatellite instability (MSI) (NGS assay, Illumina).

Conclusions• MCPyV associated MCC was present in a significantly

lower proportion of cases (35%) than what had been previously reported (70-80%), potentially due to preselection of advanced disease cases.

• MCPyV-negative MCC are characterized by significantly higher number of pathogenic and targetable mutations as well as by significantly higher tumor mutational burden.

• PD-L1 expression was predominantly absent and all tested MCC cases were MSI-S.

Figure 1. Merkel cell carcinoma positive for Polyomavirus by IHC (top images) and negative

for MCPyV (bottom images).

MCPyV status has a significant impact on mutational profile of MCC

Polyomavirus status (by immunohistochemistry) in MCC

Polyoma virus status in Merkel cell carcinomas

Frequency Percent

Merkel cell carcinomas

negative 30 65%

positive 16 35%

Total 46 100%

In MCPyV-positive cases (n=9 tested with 592-NGS ) average TMB was

6/Mb (range 4-11/Mb), while MCPyV-negative cases (n=22) had a

significantly (p<0.001) higher average TMB of 25/Mb (range 4-68/Mb).

MCPyV/TMB relationship

Merkel cell carcinomas

TMB status

TotalLow High

MCPyV

status

negative 8 (36%) 14 (64%) 22 (71%)

positive 9 (100%) 0 (0%) 9 (29%)

Total 17 (55%) 14 (45%) 31

The relationship between MCPyV status and tumor mutational burden (TMB), MSI and PD-L1 (biomarkers of I-O) in MCC

Only one pathogenic mutation (ARID1A) was detected in a

single case of MCPyV-positive

MCC.

A single (MCPyV-positive) case of Merkel cell carcinoma exhibited PD-L1 expression in 5% of cancer cells.All tested cases (n=22) were MSI stable (MSI-S).

In contrast, MCPyVnegative cases

exhibited a plethora of pathogenic

mutations.

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