Mansfield, Aaron S.

The use of programmed cell death 1 ligand 1 (PD‐L1) as a predictive biomarker to select patients to receive programmed cell death 1 (PD‐1) or PD‐L1 inhibitors in non‐small cell lung cancer (NSCLC) is limited by the definitions of positivity, interassay agreement, and intra‐ and intertumoral heterogeneity of expression. Although PD‐L1 expression enriches for responses, the lack of expression does not exclude clinical benefit.

Abstract Purpose: The expression of programmed cell death ligand 1 (PD-L1) provides limited predictive value in identifying patients most likely to respond to immunotherapy. As the heterogeneity of PD-L1 expression may lead to sampling error and the misclassification of PD-L1 status, we assessed the distribution of PD-L1 expression in paired, resected multifocal lung cancers. Experimental Design: PD-L1 was assessed by IHC. Paired lesions were defined as independent primaries or related lesions using mate pair next-generation sequencing. Agreement statistics were used for analysis. Results: Sixty-seven multifocal lung cancers from 32 patients were sequenced and stained for PD-L1. There was agreement of PD-L1 expression by the tumor cells in paired lesions of 20 patients and disagreement of PD-L1 expression by the tumor cells in paired lesions of 12 patients (κ = 0.01). Sequencing identified that 23 patients had independent primary lung cancers and that 9 patients had related cancers. In paired lesions of patients with independent cancers, there was agreement of PD-L1 expression by the tumor cells in 12 patients and disagreement in 11 patients (κ = 0.31). In paired lesions of patients with related lung cancers, there was agreement of PD-L1 expression by the tumor cells in 8 patients and disagreement in 1 patient (κ = 0.73). Conclusions: The expression of PD-L1 is heterogeneous among paired independent lung cancers, but there are high levels of agreement in intrapulmonary metastasis. Clin Cancer Res; 22(9); 2177–82. ©2015 AACR.

Abstract We investigated the influence of PD-1 expression on the systemic antitumor response (abscopal effect) induced by stereotactic ablative radiotherapy (SABR) in preclinical melanoma and renal cell carcinoma models. We compared the SABR-induced antitumor response in PD-1–expressing wild-type (WT) and PD-1–deficient knockout (KO) mice and found that PD-1 expression compromises the survival of tumor-bearing mice treated with SABR. None of the PD-1 WT mice survived beyond 25 days, whereas 20% of the PD-1 KO mice survived beyond 40 days. Similarly, PD-1–blocking antibody in WT mice was able to recapitulate SABR-induced antitumor responses observed in PD-1 KO mice and led to increased survival. The combination of SABR plus PD-1 blockade induced near complete regression of the irradiated primary tumor (synergistic effect), as opposed to SABR alone or SABR plus control antibody. The combination of SABR plus PD-1 blockade therapy elicited a 66% reduction in size of nonirradiated, secondary tumors outside the SABR radiation field (abscopal effect). The observed abscopal effect was tumor specific and was not dependent on tumor histology or host genetic background. The CD11ahigh CD8+ T-cell phenotype identifies a tumor-reactive population, which was associated in frequency and function with a SABR-induced antitumor immune response in PD-1 KO mice. We conclude that SABR induces an abscopal tumor-specific immune response in both the irradiated and nonirradiated tumors, which is potentiated by PD-1 blockade. The combination of SABR and PD-1 blockade has the potential to translate into a potent immunotherapy strategy in the management of patients with metastatic cancer. Cancer Immunol Res; 3(6); 610–9. ©2015 AACR.

Abstract Background: The prognosis for stage III melanoma is variable, with the 5-year survival ranging from 23-87%. Previous studies have demonstrated both immune cell activation and dysfunction within the sentinel lymph node (SLN) of melanoma patients. We hypothesize that differences within the host response, specifically the immune cell profile of the SLN, may predict the heterogeneous behavior of stage III melanoma. Methods: A retrospective review of clinically node negative patients undergoing a SLN biopsy at our institution was undertaken. Thirty four SLN positive cases in which the patients also underwent a complete lymph node dissection were identified and the archived FFPE SLN blocks were obtained. A single tumor positive SLN was selected and immunohistochemistry was performed for dendritic cell markers (CD123, CD86, CD11c and HLA-DR), cytotoxic T cells (CD8), anergic T cells (PD-1) and regulatory T cells (Foxp3). Slides were digitized and analyzed for percent of the SLN staining positive for each respective marker using IHCscore. These findings were correlated with risk of further non-SLN disease in the same nodal basin as well as effect on disease-free and overall survival. Results: The SLN exhibited expression of both immune activation (CD8, CD86, and CD11c) and immune suppressive (CD123, PD-1 and Foxp3) markers. HLA-DR expression was significantly higher in females (52.07% vs. 42.32%, p=0.02). No other immune marker was associated with any baseline demographic or clinical variable. Median disease-free survival and overall survival was 6 months and not yet reached and 16 months and not yet reached for patients with and without non-SLN involvement, respectively; however, no difference was noted in any of the SLN immune markers between patients with and without non-SLN involvement. Increased CD8 expression in the SLN was associated with an improvement in disease-free survival (HR 0.94 p = 0.048) but not overall survival (HR 0.93 p = 0.07). In two variable multivariate analyses, CD8 expression remained independent for disease-free survival when adjusted for Breslow depth but not non-SLN involvement. The median disease-free survival for patients with low CD8 expression within the SLN was 19.5 months and median disease-free survival has not been reached in those with high CD8 expression in the SLN after a median of 59 months follow up (log-rank p = 0.05). Conclusion: Previous reports suggest T cell infiltration of melanoma tumors is an important prognostic factor. In this study we found that CD8 T cell accumulation within the SLN was associated with a disease-free survival advantage. Cytotoxic T cell accumulation within the SLN declines early in melanoma and the degree of CD8 depletion corresponds to the risk of disease recurrence. These findings support a key role for effector T cells in the control of melanoma progression. Citation Format: Travis E. Grotz, Aaron S. Mansfield, James W. Jakub, Svetomir N. Markovic, Tina J. Hieken. Immune cell profile of the SLN has prognostic value in stage III melanoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1266. doi:10.1158/1538-7445.AM2013-1266

Abstract Introduction: The appropriate assessment of HER2 amplification status is critical to determining which patients should receive HER2-directed therapy. An alternative to FISH is D-ISH (Ventana Medical Systems Tucson, Arizona) which utilizes chromogenic HER2 and chromosome 17 probes. Both FISH and D-ISH utilize formalin-fixed, paraffin-embedded (FFPE) human breast cancer tissue specimens. D-ISH has two principle advantages over FISH: it can be visualized using light microscopy and after analysis the specimens can be archived. Methods: We tested 250 samples with FISH and with D-ISH. Fifty specimens were controls: 25 cases with no HER2 or CEP17anomaly and 25 cases with HER2 amplification. There were also 200 test subjects of 50 cases each of chromosome 17 aneusomy, HER2 deletion (HER2/CEP17 ratio <0.8), HER2 duplication (HER2/CEP17 ratio 1.3–1.8), and equivocal results (HER2/CEP17 ratios between 1.8 and 2.2). FISH (Abbott Molecular) and D-ISH analysis were performed using methods described in the appropriate clinical kit package inserts, with the following exception: Four cases with CEP17 and HER2 co-amplification were also included and for FISH analysis these cases were reflexed to the use of chromosome 17 control probe (D17S122). Results: Four samples failed testing by D-ISH. There was a 63% (155/246) concordance between FISH and D-ISH by anomaly (aneusomy, duplication, deletion, etc.) and an 83% concordance by amplification status (non-amplified, equivocal, and amplified). D-ISH detected 18 (62%) of F-ISH-amplified cases. D-ISH resulted in lower estimates of HER2/CEP17 ratios than FISH, and many cases that were equivocal by FISH were normal by D-ISH. D-ISH did not detect HER2 amplification in any of the four HER2 and CEP17 co-amplified cases. Conclusions: We observed a lower concordance rate between FISH and D-ISH than the ∼95% rate in the D-ISH package insert. This lower concordance rate is likely a result of how this study was designed: it was biased toward common chromosome 17 anomalies and difficult specimens. Such specimens comprise about 60% of our current clinical HER2 biomarker practice. D-ISH may underestimate the HER2/CEP17 ratio, or FISH may overestimate this ratio. D-ISH also does not reliably detect HER2 amplification when the centromere of chromosome 17 is also co-amplifed. Compared to FISH, in this study cohort D-ISH had a significant false negative rate for the detection of HER2 amplification. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr PD02-05.

Abstract Background: Pseudomyxoma peritonei (PMP) is a rare abdominal malignancy. We hypothesized that next generation exomic sequencing would identify recurrent mutations that may have prognostic or therapeutic implications. Methods and Materials: Ten patients were selected based on availability of tissue and adequate follow-up. Mutations were tested for in 236 cancer-related genes in formalin-fixed paraffin-embedded (FFPE) slides using next-generation exomic sequencing (Foundation Medicine, Cambridge, MA). Immunohistochemistry (IHC) was used to detect MCL1 expression. Results: Detectable mutations were found in 80% of patients. Seventy percent harbored a KRAS mutation, most commonly involving codon 12 (G12D in four cases, and one case each of G12V, G12A and A59T). Four GNAS mutations (R201H/R201C substitutions) were also detected. MCL1 and JUN were concurrently amplified in three patients. One patient with MCL1 and JUN amplification had concurrent amplification of MYC and NFKBIA. ZNF703 was amplified in one patient. Patients with MCL1 amplification were also found to express MCL1 by IHC, but MCL1 expression was also detected in some patients without amplification. Conclusions: We are first to report MCL1 and JUN co-amplification in PMP. Expression of MCL1 may not be completely dependent upon amplification. The prognostic and therapeutic implications of these recurrent mutational events are the subject of ongoing investigation. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A135. Citation Format: Aaron S. Mansfield, Terence Sio, Travis E. Grotz, Rondell P. Graham, Julian R. Molina, Florencia G. Que, Robert C. Miller. Concurrent MCL1 and JUN amplification in pseudomyxoma peritonei. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A135.

The authors documented a low compliance with guidelines to screen for skin malignancy in a community-dwelling cohort of patients with newly diagnosed chronic lymphocytic leukemia.