Utilization of p53 and p16 Immunohistochemistry in the Classification of Human Papillomavirus-Associated, p53 Wild-Type, and p53 Abnormal Oral Epithelial Dysplasia.

p53 immunohistochemistry (IHC) has recently been shown to be a clinically useful marker for predicting risk of progression to invasive squamous cell carcinoma in oral epithelial dysplasia (OED). The literature supports the use of p53 IHC as a marker to identify TP53 mutation in in situ and invasive vulvar lesions and as a surrogate marker for high-risk human papillomavirus (HPV) infection, but there is little documentation for similar use in OED. The purpose of this study was to determine whether p53 IHC is a reliable surrogate marker for detecting both TP53 mutation and high-risk HPV infection in OED. We studied 57 cases of OED (11 mild, 18 moderate, and 28 severe), and all were stained for p16 and p53 IHC. High-risk HPV RNA in situ hybridization (ISH) was performed in selected cases (all p16-positive cases and all OED showing abundant apoptotic cells and karyorrhectic cells; N = 27). Targeted next-generation sequencing (NGS) was performed in 33 p16-negative cases and all high-risk HPV RNA ISH-negative cases (N = 36). We identified 21 cases with p53 basal sparing patterns (mid-epithelial and markedly reduced [null-like]), 14 cases with p53 wild-type patterns (scattered basal and patchy basal/parabasal), and 22 cases with p53 abnormal patterns (18 overexpression, 3 null, and 1 novel cytoplasmic pattern). Among cases with p53 basal sparing patterns, 20 were positive for p16 (20/21, 95%), and all were positive for high-risk HPV RNA ISH (21/21, 100%). The 36 sequenced cases had IHC patterns concordant with TP53 mutation status in 92% (33/36) of lesions. This study demonstrates that p53 IHC expression patterns are sensitive and specific for detection of both high-risk HPV infection and TP53 mutation. Coupled with selective p16 IHC testing, this IHC panel can accurately subclassify OED into HPV-associated, p53 wild-type (conventional), and p53 abnormal OED.

Combination immunotherapy including OncoVEXmGMCSF creates a favorable tumor immune micro-environment in transgenic BRAF murine melanoma.

Talimogene Laherparepvec (OncoVEXmGMCSF), an oncolytic virus, immune checkpoint inhibitor anti-programmed cell death protein 1 (anti-PD1), and BRAF inhibition (BRAFi), are all clinically approved for treatment of melanoma patients and are effective through diverse mechanisms of action. Individually, these therapies also have an effect on the tumor immune microenvironment (TIME). Evaluating the combination effect of these three therapies on the TIME can help determine when combination therapy is most appropriate for further study. In this study, we use a transgenic murine melanoma model (Tyr::CreER; BRAFCA/+; PTENflox/flox), to evaluate the TIME in response to combinations of BRAFi, anti-PD1, and OncoVEXmGMCSF. We find that mice treated with the triple combination BRAFi + anti-PD1 + OncoVEXmGMCSF have decreased tumor growth compared to BRAFi alone and prolonged survival compared to control. Flow cytometry shows an increase in percent CD8 + /CD3 + cytotoxic T Lymphocytes (CTLs) and a decrease in percent FOXP3 + /CD4 + T regulatory cells (Tregs) in tumors treated with OncoVEXmGMCSF compared to mice not treated with OncoVEXmGMCSF. Immunogenomic analysis at 30d post-treatment shows an increase in Th1 and interferon-related genes in mice receiving OncoVEXmGMCSF + BRAFi. In summary, treatment with combination BRAFi + anti-PD1 + OncoVEXmGMCSF is more effective than any single treatment in controlling tumor growth, and groups receiving OncoVEXmGMCSF had more tumoral infiltration of CTLs and less intratumoral Tregs in the TIME. This study provides rational basis to combine targeted agents, oncolytic viral therapy, and checkpoint inhibitors in the treatment of melanoma.

Evaluation of glucocorticoid-induced TNF receptor (GITR) expression in breast cancer and across multiple tumor types.

Glucocorticoid-induced TNF receptor (GITR) is an emerging immunotherapy target that is expressed at high levels on regulatory T cells. Agonistic anti-GITR antibodies have anti-tumor activity in cancer mouse models, and recent phase 1 trials have demonstrated their safe pharmacological profile. However, there is limited knowledge on the relationship between GITR expression and the tumor microenvironment. GITR protein expression was assayed by immunohistochemistry on 3992 breast cancer surgical excision specimens assembled into tissue microarrays and scored visually by a pathologist for GITR expression on tumor-infiltrating lymphocytes and on carcinoma cells. GITR expression by the malignant cells was further surveyed in gastrointestinal stromal tumor (N = 713), lung carcinoma (N = 705), pancreatic cancer (N = 486), ovarian cancer (N = 445), bladder cancer (N = 88), prostate cancer (N = 88), testicular cancer (N = 76), melanoma (N = 75), renal cell carcinoma (N = 68),  epithelioid sarcoma (N = 53), and neuroendocrine tumors (N = 41). In breast cancer, GITR expression on tumor-infiltrating lymphocytes (12.4%) correlated with other immune response biomarkers (PD-L1+ on tumor cells, and PD-1+, LAG-3+, TIM-3+ lymphocytes; p < 0.001), and T-cell markers (CD8+, FOXP3+; p < 0.001). GITR+ carcinoma cells were observed in 6.0% of breast cancer cases and correlated with worse relapse-free survival (p = 0.015). Among the additional tumor types examined, cancers with GITR+ malignant cells included bladder cancer (5.7%), primary (but not metastatic) melanoma (4.5%), and ovarian cancer (3.2%); no expression was identified among examined sarcomas. To our knowledge, this is the first immunohistochemistry study to report the frequency and pattern of GITR expression in a large breast cancer cohort, or to report membranous GITR expression on malignant cells. The co-infiltration of GITR with other immune biomarkers and T-cell markers supports a potential role for anti-GITR agents in combination immunotherapies. In addition, GITR expression on carcinoma cells could imply the existence of a novel cancer immune evasion strategy worthy of further investigation.

Identification of recurrent mutational events in anorectal melanoma.

Anorectal melanoma is a rare disease that carries a poor prognosis. To date, limited genetic analyses confirmed KIT mutations as a recurrent genetic event similar to other mucosal melanomas, occurring in up to 30% of anorectal melanomas. Importantly, a subset of tumors harboring activating KIT mutations have been found to respond to c-Kit inhibitor-based therapy, with improved patient survival at advanced tumor stages. We performed comprehensive targeted exon sequencing analysis of 467 cancer-related genes in a larger series of 15 anorectal melanomas, focusing on potentially actionable variants based on gain- and loss-of-function mutations. We report the identification of oncogenic driver events in the majority (93%) of anorectal melanomas. These included variants in canonical MAPK pathway effectors rarely observed in cutaneous melanomas (including an HRAS mutation, as well as a BRAF mutation resulting in duplication of threonine 599), and recurrent mutations in the tumor suppressor NF1 in 20% of cases, which represented the second-most frequently mutated gene after KIT in our series. Furthermore, we identify SF3B1 mutations as a recurrent genetic event in mucosal melanomas. Our findings provide an insight into the genetic diversity of anorectal melanomas, and suggest significant potential for alternative targeted therapeutics in addition to c-Kit inhibitors for this melanoma subtype.

Immune biomarkers are more accurate in prediction of survival in ulcerated than in non-ulcerated primary melanomas.

INTRODUCTION: Ulcerated melanomas may have a unique biology and microenvironment. We test whether markers of immune infiltration correlate with clinical outcome in ulcerated compared to non-ulcerated primary melanoma tumors. METHODS: Sixty-two stage II-III cutaneous melanomas, 32 ulcerated and 30 non-ulcerated, were analyzed for tumor-infiltrating lymphocytes (TILs). Immunohistochemistry (IHC) was performed for CD2, a marker previously shown to correlate with overall survival (OS) and recurrence-free survival (RFS) in this patient population. IHC using antibody, VE1, to BRAF V600E was also performed on a subset of 41 tumors to assess the relationship of BRAF mutation to immune markers. RESULTS: We found, using Cox regression models, that the presence of TILs was associated with improved OS (p = 0.034) and RFS (p = 0.002) in ulcerated melanoma tumors, but not in non-ulcerated melanoma (p = 0.632, 0.416). CD2 expression also was correlated with improved OS (p = 0.021) and RFS (p = 0.001) in ulcerated melanoma, but no relationship was seen in non-ulcerated melanoma (p = 0.427, 0.682). In this small population, BRAF status did not correlate with TILs or CD2+ count. CONCLUSION: Our data show that immune markers including TILs and CD2 count correlate more closely with survival in ulcerated melanomas than that in non-ulcerated melanomas. We propose that immune biomarkers may be particularly relevant to ulcerated, as compared to non-ulcerated, melanomas and that this merits study in larger populations.

Large-scale mitochondrial DNA analysis in Southeast Asia reveals evolutionary effects of cultural isolation in the multi-ethnic population of Myanmar.

BACKGROUND: Myanmar is the largest country in mainland Southeast Asia with a population of 55 million people subdivided into more than 100 ethnic groups. Ruled by changing kingdoms and dynasties and lying on the trade route between India and China, Myanmar was influenced by numerous cultures. Since its independence from British occupation, tensions between the ruling Bamar and ethnic minorities increased. RESULTS: Our aim was to search for genetic footprints of Myanmar's geographic, historic and sociocultural characteristics and to contribute to the picture of human colonization by describing and dating of new mitochondrial DNA (mtDNA) haplogroups. Therefore, we sequenced the mtDNA control region of 327 unrelated donors and the complete mitochondrial genome of 44 selected individuals according to highest quality standards. CONCLUSION: Phylogenetic analyses of the entire mtDNA genomes uncovered eight new haplogroups and three unclassified basal M-lineages. The multi-ethnic population and the complex history of Myanmar were reflected in its mtDNA heterogeneity. Population genetic analyses of Burmese control region sequences combined with population data from neighboring countries revealed that the Myanmar haplogroup distribution showed a typical Southeast Asian pattern, but also Northeast Asian and Indian influences. The population structure of the extraordinarily diverse Bamar differed from that of the Karen people who displayed signs of genetic isolation. Migration analyses indicated a considerable genetic exchange with an overall positive migration balance from Myanmar to neighboring countries. Age estimates of the newly described haplogroups point to the existence of evolutionary windows where climatic and cultural changes gave rise to mitochondrial haplogroup diversification in Asia.

A diagnostic algorithm to distinguish desmoplastic from spindle cell melanoma.

Spindle cell melanoma and desmoplastic melanoma differ clinically in prognosis and therapeutic implications; however, because of partially overlapping histopathological features, diagnostic distinction of spindle cell from desmoplastic melanoma is not always straightforward. A direct comparison of diagnostic and therapeutic biomarkers has not been performed. Meta-review of the literature discloses key clinicopathological differences between spindle cell and desmoplastic melanoma, including immunophenotypes. Using 50 biomarkers available in routine diagnostics, we examined 38 archival cases (n=16 spindle, 18 desmoplastic, 4 mixed spindle/desmoplastic melanoma). S100 remains as the most reliable routine marker to reach the diagnosis of melanoma in spindle cell and desmoplastic melanoma. We identified nine distinctly labeling markers with spindle cell melanoma showing positivity for laminin, p75, HMB45, c-kit, and MelanA, and desmoplastic melanoma preferentially labeling with collagen IV, trichrome, CD68, and MDM2. On the basis of comparisons of test performance measures, MelanA and trichrome were used to devise a 94% sensitive diagnostic algorithm for the distinction of desmoplastic from spindle cell melanoma. Gene amplification and expression status was assessed for a set of potentially drugable targets (HER2, EGFR, MET, MDM2, TP53, ALK, MYC, FLI-1, and KIT). Fluorescent in situ hybridizations did not reveal a significant number of gene aberrations/rearrangements; however, protein overexpression for at least one of these markers was identified in 35 of 38 cases (92%). In addition, we found BRAF mutations in 31% of spindle cell and 5% of desmoplastic melanoma, with an overall mutation frequency of 16% (n=6/38). We present the first comprehensive screening study of diagnostic and therapeutic biomarkers in spindle cell and desmoplastic melanoma. The devised algorithm allows diagnostic distinction of desmoplastic from spindle cell melanoma when routine histology is not decisive.

TERT promoter mutations in atypical melanocytic lesions: A series of seven cases with adverse melanoma-specific outcome.

The majority of melanocytic proliferations can be readily categorized as benign or malignant based on histologic assessment under the microscope by a trained dermatopathologist. However, a subset of lesions, termed Atypical Melanocytic Proliferations (AMPs), are histologically ambiguous, leading to possible diagnostic error and suboptimal treatment. Mutations in the promoter region of the catalytic subunit of telomerase, telomerase reverse transcriptase (TERT), are commonly found in melanomas but are rare in melanocytic nevi. In this study, we aimed to determine the prevalence of hot spot TERT promoter (TERT-p) mutations in AMPs with adverse melanoma-specific outcome. Studies were approved by respective institutional review boards. Using a multi-center database, we identified seven cases of melanocytic proliferations with a clinical follow-up period of at least 4 years, which were initially diagnosed as AMPs, and which recurred either as melanoma at site of prior biopsy or as metastatic melanoma. Sequencing of the TERT-p region showed hotspot mutations in three cases (43 %), suggesting that TERT-p mutations are enriched and could aid in the identification of AMPs with adverse outcome. In comparison with existing ancillary techniques for prognostication of AMPs, TERT-p mutation analysis may have advantages in terms of cost effectiveness and turnaround time, and is a promising diagnostic parameter with potential widespread utility.

B7-H3 drives immunosuppression and Co-targeting with CD47 is a new therapeutic strategy in ß-catenin activated melanomas.

In melanoma, immune cell infiltration into the tumor is associated with better patient outcomes and response to immunotherapy. T-cell non-inflamed tumors (cold tumors) are associated with tumor cell-intrinsic Wnt/ß-catenin activation, and are typically resistant to anti-PD-1 alone or in combination with anti-CTLA-4 therapy. Reversal of the 'cold tumor' phenotype and identifying new effective immunotherapies are challenges. We sought to investigate the role of a newer immunotherapy agent, B7-H3, in this setting. RNA sequencing was used to identify co-targeting strategies upon B7-H3 inhibition in a well-defined preclinical melanoma model driven by ß-catenin. We found that immune checkpoint molecule B7-H3 confers a suppressive tumor microenvironment by modulating antiviral signals and innate immunity. B7-H3 inhibition led to an inflamed microenvironment, up-regulation of CD47/SIRPa signaling, and together with blockade of the macrophage checkpoint CD47 resulted in additive antitumor responses. We found that the antitumor effects of the B7-H3/CD47 antibody combination were dependent on cytokine signaling pathways (CCR5/CCL5 and IL4).

Exome sequencing-based copy-number variation and loss of heterozygosity detection: ExomeCNV.

MOTIVATION: The ability to detect copy-number variation (CNV) and loss of heterozygosity (LOH) from exome sequencing data extends the utility of this powerful approach that has mainly been used for point or small insertion/deletion detection. RESULTS: We present ExomeCNV, a statistical method to detect CNV and LOH using depth-of-coverage and B-allele frequencies, from mapped short sequence reads, and we assess both the method's power and the effects of confounding variables. We apply our method to a cancer exome resequencing dataset. As expected, accuracy and resolution are dependent on depth-of-coverage and capture probe design. AVAILABILITY: CRAN package 'ExomeCNV'. CONTACT: fsathira@fas.harvard.edu; snelson@ucla.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Exceptional response to combination ipilimumab and nivolumab in metastatic uveal melanoma: Insights from genomic analysis.

Uveal melanoma is the most common intraocular malignancy and has a poor prognosis compared to other melanoma subtypes with a median overall survival of 6-10 months. With immune checkpoint inhibitor therapy, either PD-1 inhibitor alone or combination ipilimumab/nivolumab (anti-CTLA-4/anti-PD-1), responses are rare and often not durable. We present a case report of a now 66-year-old woman with diffuse metastatic uveal melanoma previously treated with a combination of ipilimumab/nivolumab, followed by maintenance nivolumab. Almost complete resolution of all sites of metastatic disease was observed except for one liver metastasis which regressed partially on immunotherapy. Notably, the patient had a significantly elevated BMI and developed widespread vitiligo on treatment. Whole-genome and transcriptome analysis was performed on the residual liver biopsy and molecular markers that may have contributed to the exceptional response were investigated. Several alterations were observed in genes involved in T-cell responses. Estimates of tumour infiltrating immune cells indicated a high level of plasma cells compared to other uveal melanoma cases, a finding previously associated with indolent disease. The patient also carried several germline SNPs that may have contributed to her treatment response as well as widespread vitiligo. Whole-genome and transcriptome sequencing have provided insight into potential molecular underpinnings of an exceptional treatment response in a tumour type typically associated with poor prognosis. Immunological findings suggest a role for plasma cells in the tumour microenvironment. Elevated BMI and the development of vitiligo may be clinically relevant factors for predicting response to immune checkpoint inhibitor therapy, warranting further studies in patients with uveal melanoma.

The impact of whole genome and transcriptome analysis (WGTA) on predictive biomarker discovery and diagnostic accuracy of advanced malignancies.

In this study, we evaluate the impact of whole genome and transcriptome analysis (WGTA) on predictive molecular profiling and histologic diagnosis in a cohort of advanced malignancies. WGTA was used to generate reports including molecular alterations and site/tissue of origin prediction. Two reviewers analyzed genomic reports, clinical history, and tumor pathology. We used National Comprehensive Cancer Network (NCCN) consensus guidelines, Food and Drug Administration (FDA) approvals, and provincially reimbursed treatments to define genomic biomarkers associated with approved targeted therapeutic options (TTOs). Tumor tissue/site of origin was reassessed for most cases using genomic analysis, including a machine learning algorithm (Supervised Cancer Origin Prediction Using Expression [SCOPE]) trained on The Cancer Genome Atlas data. WGTA was performed on 652 cases, including a range of primary tumor types/tumor sites and 15 malignant tumors of uncertain histogenesis (MTUH). At the time WGTA was performed, alterations associated with an approved TTO were identified in 39 (6%) cases; 3 of these were not identified through routine pathology workup. In seven (1%) cases, the pathology workup either failed, was not performed, or gave a different result from the WGTA. Approved TTOs identified by WGTA increased to 103 (16%) when applying 2021 guidelines. The histopathologic diagnosis was reviewed in 389 cases and agreed with the diagnostic consensus after WGTA in 94% of non-MTUH cases (n = 374). The remainder included situations where the morphologic diagnosis was changed based on WGTA and clinical data (0.5%), or where the WGTA was non-contributory (5%). The 15 MTUH were all diagnosed as specific tumor types by WGTA. Tumor board reviews including WGTA agreed with almost all initial predictive molecular profile and histopathologic diagnoses. WGTA was a powerful tool to assign site/tissue of origin in MTUH. Current efforts focus on improving therapeutic predictive power and decreasing cost to enhance use of WGTA data as a routine clinical test.

Southeast Asian diversity: first insights into the complex mtDNA structure of Laos.

BACKGROUND: Vast migrations and subsequent assimilation processes have shaped the genetic composition of Southeast Asia, an area of close contact between several major ethnic groups. To better characterize the genetic variation of this region, we analyzed the entire mtDNA control region of 214 unrelated donors from Laos according to highest forensic quality standards. To detail the phylogeny, we inspected selected SNPs from the mtDNA coding region. For a posteriori data quality control, quasi-median network constructions and autosomal STR typing were performed. In order to describe the mtDNA setup of Laos more thoroughly, the data were subjected to population genetic comparisons with 16 East Asian groups. RESULTS: The Laos sample exhibited ample mtDNA diversity, reflecting the huge number of ethnic groups listed. We found several new, so far undescribed mtDNA lineages in this dataset and surrounding populations. The Laos population was characteristic in terms of haplotype composition and genetic structure, however, genetic comparisons with other Southeast Asian populations revealed limited, but significant genetic differentiation. Notable differences in the maternal relationship to the major indigenous Southeast Asian ethnolinguistic groups were detected. CONCLUSIONS: In this study, we portray the great mtDNA variety of Laos for the first time. Our findings will contribute to clarify the migration history of the region. They encourage setting up regional and subpopulation databases, especially for forensic applications. The Laotian sequences will be incorporated into the collaborative EMPOP mtDNA database http://www.empop.org upon publication and will be available as the first mtDNA reference data for this country.

An in-depth analysis of the mitochondrial phylogenetic landscape of Cambodia.

Cambodia harbours a variety of human aboriginal populations that have scarcely been studied in terms of genetic diversity of entire mitochondrial genomes. Here we present the matrilineal gene pool of 299 Cambodian refugees from three different ethnic groups (Cham, Khmer, and Khmer Loeu) deriving from 16 Cambodian districts. After establishing a DNA-saving high-throughput strategy for mitochondrial whole-genome Sanger sequencing, a HaploGrep based workflow was used for quality control, haplogroup classification and phylogenetic reconstruction. The application of diverse phylogenetic algorithms revealed an exciting picture of the genetic diversity of Cambodia, especially in relation to populations from Southeast Asia and from the whole world. A total of 224 unique haplotypes were identified, which were mostly classified under haplogroups B5a1, F1a1, or categorized as newly defined basal haplogroups or basal sub-branches of R, N and M clades. The presence of autochthonous maternal lineages could be confirmed as reported in previous studies. The exceptional homogeneity observed between and within the three investigated Cambodian ethnic groups indicates genetic isolation of the whole population. Between ethnicities, genetic barriers were not detected. The mtDNA data presented here increases the phylogenetic resolution in Cambodia significantly, thereby highlighting the need for an update of the current human mtDNA phylogeny.

Automated digital TIL analysis (ADTA) adds prognostic value to standard assessment of depth and ulceration in primary melanoma.

Accurate prognostic biomarkers in early-stage melanoma are urgently needed to stratify patients for clinical trials of adjuvant therapy. We applied a previously developed open source deep learning algorithm to detect tumor-infiltrating lymphocytes (TILs) in hematoxylin and eosin (H&E) images of early-stage melanomas. We tested whether automated digital (TIL) analysis (ADTA) improved accuracy of prediction of disease specific survival (DSS) based on current pathology standards. ADTA was applied to a training cohort (n = 80) and a cutoff value was defined based on a Receiver Operating Curve. ADTA was then applied to a validation cohort (n = 145) and the previously determined cutoff value was used to stratify high and low risk patients, as demonstrated by Kaplan-Meier analysis (p ≤ 0.001). Multivariable Cox proportional hazards analysis was performed using ADTA, depth, and ulceration as co-variables and showed that ADTA contributed to DSS prediction (HR: 4.18, CI 1.51-11.58, p = 0.006). ADTA provides an effective and attainable assessment of TILs and should be further evaluated in larger studies for inclusion in staging algorithms.

Gab2-mediated signaling promotes melanoma metastasis.

Metastatic melanoma is a disease with a poor prognosis that currently lacks effective treatments. Critical biological features of metastasis include acquisition of migratory competence, growth factor independence, and invasive potential. In an attempt to identify genes that contribute to melanoma pathogenesis, a genome-wide search using bacterial artificial chromosome array comparative genomic hybridization and single nucleotide polymorphism arrays in a series of 64 metastatic melanoma samples and 20 melanoma cell lines identified increased copy numbers of Gab2 located on 11q14.1. Gab2 is an adaptor protein that potentiates the activation of the Ras-Erk and PI3K-Akt pathways and has recently been implicated in human cancer; however, its role in melanoma has not been explored. In this study, we found that Gab2 was either amplified (approximately 11%) and/or overexpressed (approximately 50%) in melanoma. Gab2 protein expression correlated with clinical melanoma progression, and higher levels of expression were seen in metastatic melanomas compared with primary melanoma and melanocytic nevi. We found that overexpression of Gab2 potentiates, whereas silencing of Gab2 reduces, migration and invasion of melanoma cells. Gab2 mediated the hyperactivation of Akt signaling in the absence of growth factors, whereas inhibition of the PI3K-Akt pathway decreased Gab2-mediated tumor cell migration and invasive potential. Gab2 overexpression resulted in enhanced tumor growth and metastatic potential in vivo. These studies demonstrate a previously undefined role for Gab2 in melanoma tumor progression and metastasis.

Lack of distinct molecular profile of Primary Dermal Melanoma.

Primary dermal melanoma (PDM) is a rare variant of melanoma which simulates metastatic melanoma to the skin. Diagnosis of PDM cannot be established on histologic grounds alone but requires absence of evidence of melanoma elsewhere by clinical history and/or imaging studies. Despite this entity being clinically well documented, limited data on molecular characterization are available. We performed comprehensive mutation and copy number variation analysis in a series of PDMs in search for distinctive molecular features.Studies were approved by respective institutional review boards. Six cases fulfilling strict histologic criteria of PDM were identified in patients with absent history of melanoma elsewhere, negative sentinel lymph node biopsies and imaging studies. DNA was extracted from formalin-fixed, paraffin-embedded (FFPE) tissue, and five cases passed quality control measures and were subjected to targeted exon sequencing using the Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets 410 panel. Two of five PDM carried NRAS hotspot mutations characteristic of cutaneous melanoma (CM) genomic subtypes. One case showed a probable low-activating NRAS mutation in combination with additional aberrations in other mitogen-activated protein kinase (MAPK) pathway effectors. Hotspot mutations were also identified in the TERT promoter region, and one tumor showed a mutation in the SWI/SNF remodeling complex. No oncogenic mutations were identified in one case. Furthermore, none of the tumors analyzed showed activating mutations in Gα subunits, including GNAQ and GNA11. Copy number alterations included deletions of Chr 9p, characteristic of CM.Despite PDM showing mutational heterogeneity, our findings suggest predominance of MAPK pathway aberrations in agreement with the mutational profile of CMs in general. Given the absence of genetic overlap with other distinct primary dermal melanocytic proliferations, mutational profiling will unlikely aid in the difficult differential diagnosis of PDM versus melanoma metastasis. Thorough metastatic workup remains crucial in establishing this rare diagnosis.

Topography, morphology, and etiology of lymphocytic gastritis: a single institution experience.

Lymphocytic gastritis (LG) is an uncommon reaction pattern of gastric injury characterized by intraepithelial lymphocytosis of the surface foveolar epithelium and chronic inflammation in the lamina propria. It most commonly occurs in association with gluten-sensitive enteropathy, Helicobacter pylori gastritis, non-steroidal anti-inflammatory drugs, and microscopic colitis. While the topography of LG has been described in gluten-sensitive enteropathy and H. pylori infection, no definite morphologic features have been used to further subcategorize LG based on possible etiologies. Furthermore, new immunotherapy agents have been associated with lymphocytic infiltrate in the gastrointestinal tract, but their association with LG has not been reported. Cases of LG were collected from our institution in the period between August 2011 and September 2017. The topography of LG and morphologic features such as glandular microabscesses, intestinal metaplasia, lymphoid aggregates, surface vs pit distribution of lymphocytes, and number of intraepithelial lymphocytes per 100 epithelial cells were assessed for each case using the updated Sydney System where applicable. Twenty-seven cases of LG were identified in the recent 6-year period at our institution. Gluten-sensitive enteropathy is the main reported cause of LG followed by NSAID injury. Cases of LG associated with gluten-sensitive enteropathy are antral predominant, those associated with H. pylori are body predominant, and those occurring in the setting of NSAID injury show pangastritis. Glandular microabscesses are observed in all cases of LG associated with H. pylori, and not in the setting of GSE or NSAID injury. In addition, a case of LG associated with melanoma immunotherapy has been identified. Topography and morphology of lymphocytic gastritis may point to the cause of injury, allowing for proper treatment of the underlying disease.

Linking Transcriptomic and Imaging Data Defines Features of a Favorable Tumor Immune Microenvironment and Identifies a Combination Biomarker for Primary Melanoma.

Patients with resected stage II-III melanoma have approximately a 35% chance of death from their disease. A deeper understanding of the tumor immune microenvironment (TIME) is required to stratify patients and identify factors leading to therapy resistance. We previously identified that the melanoma immune profile (MIP), an IFN-based gene signature, and the ratio of CD8+ cytotoxic T lymphocytes (CTL) to CD68+ macrophages both predict disease-specific survival (DSS). Here, we compared primary with metastatic tumors and found that the nuclei of tumor cells were significantly larger in metastases. The CTL/macrophage ratio was significantly different between primary tumors without distant metastatic recurrence (DMR) and metastases. Patients without DMR had higher degrees of clustering between tumor cells and CTLs, and between tumor cells and HLA-DR+ macrophages, but not HLA-DR- macrophages. The HLA-DR- subset coexpressed CD163+CSF1R+ at higher levels than CD68+HLA-DR+ macrophages, consistent with an M2 phenotype. Finally, combined transcriptomic and multiplex data revealed that densities of CD8 and M1 macrophages correlated with their respective cell phenotype signatures. Combination of the MIP signature with the CTL/macrophage ratio stratified patients into three risk groups that were predictive of DSS, highlighting the potential use of combination biomarkers for adjuvant therapy. SIGNIFICANCE: These findings provide a deeper understanding of the tumor immune microenvironment by combining multiple modalities to stratify patients into risk groups, a critical step to improving the management of patients with melanoma.

Deep Learning Based on Standard H&E Images of Primary Melanoma Tumors Identifies Patients at Risk for Visceral Recurrence and Death.

PURPOSE: Biomarkers for disease-specific survival (DSS) in early-stage melanoma are needed to select patients for adjuvant immunotherapy and accelerate clinical trial design. We present a pathology-based computational method using a deep neural network architecture for DSS prediction. EXPERIMENTAL DESIGN: The model was trained on 108 patients from four institutions and tested on 104 patients from Yale School of Medicine (YSM, New Haven, CT). A receiver operating characteristic (ROC) curve was generated on the basis of vote aggregation of individual image sequences, an optimized cutoff was selected, and the computational model was tested on a third independent population of 51 patients from Geisinger Health Systems (GHS). RESULTS: Area under the curve (AUC) in the YSM patients was 0.905 (P < 0.0001). AUC in the GHS patients was 0.880 (P < 0.0001). Using the cutoff selected in the YSM cohort, the computational model predicted DSS in the GHS cohort based on Kaplan-Meier (KM) analysis (P < 0.0001). CONCLUSIONS: The novel method presented is applicable to digital images, obviating the need for sample shipment and manipulation and representing a practical advance over current genetic and IHC-based methods.