Flotte, Thomas J.

e15184 Background: Clinical responses to anti-PD1 immunotherapy in patients with metastatic melanoma (MM) remain challenging to predict. This clinical heterogeneity is also reflected in the tumor-immune microenvironment among patients and within a single tumor lesion. With the emergence of multiplex imaging platforms, defining complex phenotypes at single cell resolution has become possible. Here, we sought to objectively quantify paracrine tumor-immune interactions contributing to the variable clinical responses observed in patients receiving anti-PD1 therapy. Methods: Excisional lymph node (LN) biopsies were obtained from treatment-naïve patients with MM who underwent subsequent anti-PD1 therapy. A single 5µm section of LN tissue was used to assess a 42 analyte panel by multiplex immunofluorescence. From 30 LN samples, 418 fields of view (FOVs) were selected resulting in 14,360 high-resolution images of 4 anatomical subregions: tumor core, tumor-immune interface, tumor infiltrate and adjacent immune stroma. Following image processing, we developed an adaptive classification for tumor-centric cellular neighborhoods (TCCN) to identify and quantify critical paracrine interactions within the tumor-immune microenvironment. Results: Stratification based on responsiveness to anti-PD1 therapy resulted in 4 complete responders (CR) and 12 non-responders (NR) at 12-week follow-up. From 126 FOVs, we defined the cellular composition of 197,865 TCCN across patients based on clinical response and LN subregions. Overall, the percentage of TCCN devoid of any T cells, B cells or macrophages was significantly higher in NR compared to CR irrespective of subregion. However, other markers differentiated TCCN based on subregion. Specifically in CR, tumor core regions were enriched for CD8 T cells, while enrichment for B cells and endothelial cells was demonstrated at the tumor-immune interface. Strikingly, tumor infiltrate regions demonstrate robust immune reactivity with enrichment for M1 polarized macrophages, NK cells and B cells in CR compared to NR. Complete data from the 30 patient cohort across 418 FOVs will be presented. Conclusions: Taken together, this data suggests cellular composition of TCCN across subregions of the LN is dynamic within a single metastatic site. In this small cohort, we introduce a formalized stratification to quantify and classify critical paracrine interactions within the immune-tumor microenvironment with the potential to inform clinical responsiveness to therapy.

Abstract Purpose Delta-like protein 3 (DLL3) is being developed as a predictive biomarker for DLL3-targeting antibody-drug conjugate and other therapies. Given the neuroendocrine features of Merkel cell carcinoma (MCC), we sought to evaluate DLL3 expression and its role in MCC. Experimental Design Formalin-fixed and paraffin-embedded MCC cases were consecutively selected. Immunohistochemistry was performed for DLL3 (SC16.65 antibody) and polyomavirus large T-antigen (sc-136172 antibody). Slides were read out for percentage of positive tumor cells. Cox proportional hazards model was applied to assess the association between DLL3 expression and overall survival (OS). A patient with a DLL3-expressing MCC was treated with rovalpituzumab tesirine (Rova-T) in the “other tumor” cohort of NCT02709889 and assessed for response. Results The median H-score of DLL3 expression of 65 patients included was 60 (interquartile range, 30–100). Fifty-eight cases (89%) had ≥1% tumor cells positive for DLL3 expression with any intensity, of which the median DLL3 expression was 50% (interquartile range, 25%–70%). Thirty-four cases (52%) had ≥50% tumor cells positive for DLL3 expression with any intensity. Higher H-score of DLL3 expression was associated with higher polyomavirus nuclear expression (p = .003) when it was dichotomized to negative versus positive. H-score of DLL3 expression did not predict OS of patients with MCC (p = .4) after being adjusted for common clinicopathological factors. A patient treated with Rova-T for refractory metastatic MCC achieved partial response. Conclusions DLL3 overexpression is very common in MCC by immunohistochemistry. The response to treatment suggests that DLL3 expression may have predictive relevance for DLL3-targeting therapies in MCC. Implications for Practice Delta-like protein 3 (DLL3) is being developed as a predictive biomarker to identify patients for treatment with DLL3-targeting agents. Merkel cell carcinoma (MCC) is an aggressive neuroendocrine carcinoma of the skin. It was found that DLL3 overexpression is very common in MCC by immunohistochemistry and significantly associated with Merkel cell polyomavirus expression. Despite the lack of prognostic significance in this cohort, DLL3 expression may have predictive relevance for DLL3-targeting therapies in MCC. The high levels of DLL3 expression in a subset of MCC may potentially be used to select patients to receive DLL3-targeting therapies.

Abstract Background: Standard treatment for stage III melanoma is surgery followed by consideration of adjuvant therapy. This strategy is inadequate for high-risk stage III patients who have a substantial risk of subsequent relapse and distant metastasis. The goals of this trial are to improve oncologic outcomes, assess treatment response and spare patients with poorly responsive, rapidly metastatic disease from a futile operation. This protocol based on the hypothesis that treatment before surgery will immunize patients against his or her own unique tumor. While new drugs are now FDA-approved for adjuvant treatment of stage III melanoma, outcomes are still suboptimal in high-risk patients. The neoadjuvant approach is largely untested and preclinical data suggest neoadjuvant therapy is more effective than adjuvant immunotherapy. While targeted therapies can be rapidly effective, immunotherapy responses, when they occur, tend to be much more durable, prompting this study. Our specific strategy is based on the fact that BRAFm melanoma patients benefit both from BRAF/MEK inhibition (vemurafenib/cobimetinib) and PD-1 axis inhibition (atezolizumab) while patients with BRAFwt melanoma benefit from PD-1 axis inhibition (atezolizumab) and intermittent MEKi (cobimetinib) may increase effector function in tumor-infiltrating T cells. A second component of the study is robust evaluation of biomarkers of response, toxicity and outcome. Specific correlatives include assessment of the predictive value of Bim levels in tumor-related T cells, correlation of sPD-L1 with efficacy and toxicity, correlation of PD-L1 expression with efficacy, and determination of key relationships between tumor and immune cells via mIHC spatial analysis, gene expression profiles and microbiome associations with clinical response. Methods: The NeoACTIVATE trial enrolls melanoma patients with resectable clinically detectable nodal metastases (by physical examination or imaging), dual nodal basin disease or nodal recurrence of melanoma. Neoadjuvant systemic therapy is based on BRAF mutation status; participants receive either vemurafenib, cobimetinib and atezolizumab (vem/cobi/atezo), or cobimetinib and atezolizumab (cobi/atezo) before surgery. Following surgery, all patients receive atezo immunotherapy. The primary aim of the study is RFS estimates for each cohort. Secondary aims are safety/toxicity, pathologic complete response rate and prospective evaluation of biomarkers of treatment response and toxicity. This study is significant in that it is the first to test 1) combined of targeted and immunotherapy in the neoadjuvant setting for patients with resectable Stage III melanoma and 2) the novel combination of MEK-inhibition with immunotherapy in the neoadjuvant setting for resectable Stage III melanoma. As of January 1, 2019, 10% of planned patients have been enrolled and enrollment continues. Citation Format: Tina J. Hieken, Vera J. Suman, Shernan G. Holtan, Thomas J. Flotte, Lisa A. Kottschade, Matthew S. Block. Neoadjuvant combination targeted and immunotherapy for high-risk resectable stage III melanoma (NeoACTIVATE), NCT03554083 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT121.

e21039 Background: Tumor-infiltrating neutrophils (TINs) and their myeloid precursors play an important role in cancer biology. While activated neutrophils have been shown to kill tumor cells, there is growing evidence that neutrophils may also support tumor progression by impairing T cell-dependent anti-tumor immunity. We hypothesized that low TINs would be associated with better responses to immunotherapy (IO). Methods: Pretreatment biopsies from 15 metastatic melanoma (MM) pts treated with anti-CTL4 and anti-PD-1 therapy from 2012-17 were collected and stained with myeloperoxidase (MPO). Slides were scanned at 40x magnification on the Aperio ScanScope AT Turbo brightfield instrument (Leica Biosystems) at a resolution of 0.25 microns per pixel. To indicate the region of analysis, the digital pen tool was used to trace a minimum 85% of tumor. Ten fixed-sized boxes were placed on the hottest staining region in a second annotation layer on the image. The boxes equaled 1mm2 in total area. If the tumor tracing had an area of 1mm2, or less, boxes were not placed on the image. The selected tissue was analyzed using an Aperio imaging quatification algorithm . The number of 3+ cells were considered positive for neutrophils and used in subsequent calculations. Quality control review was performed on a subset of cases by Anatomic Pathologists. Response to therapy was assessed using the Response Evaluation Criteria In Solid Tumors (RECIST). Overall survival (OS) was calculated using the Kaplan Meier method. Results: The median number of treatment lines before the biopsy was 1 (range 0-2). The treatment immediately following the biopsy consisted of ipilimumab (n = 10, 66.6%), pembrolizumab (n = 4, 26.6%), ipilimumab/nivolumab (n = 1, 6.6%). The median number of neutrophils in the biopsy hotspot was 92/mm2 (range 6-219) for the entire cohort. For patients achieving PR and CR after 4 cycles of therapy, the median neutrophils were 36/mm2 (IQ 14-78) while the median was 106/mm2 (IQ 60-138) for patients who had progressive disease,( p = 0.04). There was no correlation between the numbers of neutrophils in the biopsy with the absolute neutrophil count in the peripheral blood (R value: 0.012). Conclusions: Decreased TINs are significantly associated with better responses to IO in MM pts. Measurements of TINs may help to select patients who are likely to benefit from anti-CTL4 and anti-PD-1 therapy. Additionally, exploration of whether combining therapies that decrease neutrophils intratumorally should be explored.

e21057 Background: BRAF, NRAS, CDKN2A and P53 mutations are commonly observed pathogenic mutations in cutaneous melanoma. The influence of mutations such as BRAF, NRAS, CDKN2A and P53 on treatment response in the PD1 inhibitor era has not been well defined. Methods: This study included patients with melanoma with a cutaneous or unknown primary. All patients had genomic profiling with the Mayo Clinic 50-gene solid tumor mutation panel. Treatment response to first line immunotherapy with PD1, CTLA-4, or combination PD1/CTLA-4 inhibitors were assessed by response rate and time-to-progression (TTP) rate at 12 months. Each patient outcome was stratified by non-mutually exclusive gene mutation status into BRAF, NRAS, P53, CDKN2A, or quadruple negative (N4) groups. Results: Genomic profiling was performed on 208 patients, of whom 102 received first line immunotherapy. Most patients received a PD1 inhibitor (n = 62), followed by CTLA-4 inhibitor (n = 34), and combination therapy (n = 6). A trend towards improved outcomes was observed in CDKN2A and BRAF mutated patients with worse outcomes noted in P53, N4, and NRAS patients. The percent without progression at 12 months were BRAF 52.2%, CDKN2A 58.2%, NRAS 30.5%, P53 37.7%, and N4 26.7% (see table below). Response rates were BRAF 46.4%, CDKN2A 38.5%, NRAS 22.2%, P53 40.0%, and N4 33.3%. Conclusions: There is a trend towards improved outcomes with immunotherapy in BRAF and CDKN2A mutated patients while P53, NRAS and N4 patients have inferior outcomes. [Table: see text]

Abstract In patients with metastatic melanoma, high blood levels of galectin‐9 are correlated with worse overall survival and a bias towards a Th2 inflammatory state supportive of tumor growth. Although galectin‐9 signaling through TIM3 on T cells has been described, less is known about the interaction of galectin‐9 with macrophages. We aimed to determine whether galectin‐9 is a binding partner of CD206 on macrophages and whether the result of this interaction is tumor‐supportive. It was determined that incubation of CD68 + macrophages with galectin‐9 or anti‐CD206 blocked target binding and that both CD206 and galectin‐9 were detected by immunoprecipitation of cell lysates. CD206 and galectin‐9 had a binding affinity of 2.8 × 10 −7 m . Galectin‐9 causes CD206 + macrophages to make significantly more FGF2 and monocyte chemoattractant protein (MCP‐1), but less macrophage‐derived chemokine (MDC). Galectin‐9 had no effect on classical monocyte subsets, but caused expansion of the non‐classical populations. Lastly, there was a positive correlation between increasing numbers of CD206 macrophages and galectin‐9 expression in tumors, and high levels of CD206 macrophages correlated negatively with melanoma survival. These results indicate that galectin‐9 binds to CD206 on M2 macrophages, which appear to drive angiogenesis and the production of chemokines that support tumor growth and poor patient prognoses. Targeting this interaction systemically through circulating monocytes may therefore be a novel way to improve local anti‐tumor effects by macrophages. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The presence of enlarged epithelioid/spindled nests located deep in the reticular dermis of a biphasic melanocytic neoplasm can mimic melanoma arising in a pre‐existing nevus, causing over‐interpretation of malignancy. We aimed to define the clinicopathologic significance of epithelioid/spindled nests in melanocytic nevi. Retrospectively using clinical and histologic information, we characterized 121 patients with a single lesion showing epithelioid/spindled melanocytes in the reticular dermis or subcutaneous fat, surrounded by melanophages, sometimes blending in with the adnexa. The majority of nevi occurred in women in the ages of 10 to 39 years, where the most frequent presentation was a changing mole. While 78% of the lesions displayed an anatomic (Clark’s) level of IV‐V, there was no ulceration, significant regression or inflammation. Up to 2 mitoses were found in only 12% of the cases, not correlating with the severity of cytological atypia. No recurrence or metastasis occurred during 45.5 months (mean) of clinical follow up in 26 patients. Notwithstanding the deep dermal extension, these findings suggest a benign histopathology and clinical outcome. Having compared the overlapping histopathology and clinical features between deep penetrating/clonal nevus and combined nevus, we posit that “inverted type‐A nevus” might be considered a variant of the two.

BACKGROUNDThe amount of time available to pathologists with which to perform research is becoming limited due to an increasing manpower shortage in pathology, decreased reimbursement, and increased workload. This is occurring at the same time as demands escalate for pathologists to develop new companion tests, correlate the molecular findings with traditional methods, and assist in the development of individualized medicine. This study examined whether cytotechnologists may be integrated into a research team that uses their expertise in understanding pathology and clinical disease to provide interpretations of experiments that traditionally were performed by pathologists.METHODSCytotechnologists worked with pathologists to choose blocks for tissue microarrays (TMAs) and to interpret immunohistochemically stained TMA slides. The pathologist met with the cytotechnologist to review the study design. The cytotechnologists reviewed the slides and blocks and chose the most appropriate blocks for the TMA. Either 10% or all of the slides/blocks selected for TMA construction were reviewed by the supervising pathologist. The final selections were given to the TMA technologist to make the TMA. A minimum of 10% of the immunohistochemically stained TMA slides were reviewed by the supervising pathologist.RESULTSA total of 32 TMAs were created with 6 cytotechnologists collaborating with 6 pathologists. Immunohistochemical stains of 190 TMAs were interpreted by 4 cytotechnologists collaborating with 3 pathologists. All the TMAs and TMA interpretation data were used successfully for the research for which they were designed.CONCLUSIONSThe collaboration of cytotechnologists and pathologists in research can improve the quality of effort and increase satisfaction and productivity. Cancer Cytopathol 2018;126:232‐5. © 2018 American Cancer Society.