Faculty Bibliography

Background: TERT promoter mutations in various reports have been associated with poor patient survival in early stage melanomas emphasizing it as a separate subset of melanoma. Thus far, no studies investigated whether the immune composition of the tumor microenvironment (TME) in TERT (HS) mutant melanomas differs from TERT WT melanomas. Furthermore, the mechanism underlying the worse outcome in early stage TPHS melanomas remains unclear. Herein, we aim to characterize the tumor microenvironment (TME) of TERT promoter hotspot (TPHS) mutant melanomas & compare them to TERT WT melanomas in a cohort of early and advanced stage melanomas. We also aim to elucidate the clinical significance of the TME composition.
Design(s): We analyzed tissue from a cohort of 93 melanoma patients. DNA and RNA were extracted from primary and metastatic tumor tissue, resected prior to treatment with immune checkpoint inhibitors. The extracted DNA was genotyped using a customized next generation sequencing high throughput panel that targets 580 cancer-related genes to determine TPHS mutation status. Gene expression analysis was performed on the RNA from 52 patients using a customized 770-gene expression panel combining markers 48 biologically significant signatures with the N-counter system. Differential gene expression (DGE) and Gene set enrichment analysis (GSEA) were performed using R package [(p<0.01; FDR<0.01; FDR<0.30 for GSEA] using TERT WT as a reference.
Result(s): Table 1 illustrates the clinicopathological characteristics of the cohort. TPHS mutant melanoma was associated with downregulation of melanoma-associated antigens (MAGES) and endothelial cells/angiogenesis signature (p<0.01). Notably, MAGEA4, MAGEA1, CTAG1B, PALMD & KDR were among the most downregulated genes in the (lg2fc= -3.2; -2.2; -2.66, -1.23; - 0.66, respectively). GSEA showed a significant enrichment for NOD-like receptor (NLR) signaling pathway including NFKB1, TNF & NLR3P in TPHS mutant melanoma (Figure 1). Within TPHS mutant melanoma, high endothelial cells/angiogenesis signature (score >3.85/median) was more prevalent in stage (I/II) melanomas (p=0.025). No significant association between TERT mutational status, outcome nor histologic subtype were noted. (Table presented)
Conclusion(s): Our findings show that TPHS mutant melanoma and TERT WT have distinct TME composition. The higher endothelial/angiogenesis signature seen in early stage TPHS mutant melanoma compared to stage III/IV TPHS mutant melanomas could contribute to the poor outcome reported in the former group

Background: Encapsulated papillary carcinomas (EPC) of the breast is a variant of papillary carcinoma that are confined to a cystic space, surrounded by a fibrous capsule and lack the myoepithelial coat. Despite the latter finding, it is recommended that EPC be staged as pTis due to its indolent course. Concurrent frank invasive carcinomas are staged commensurate with their size. We sought to investigate the molecular pathways differentially expressed in pure EPC and EPC with frank invasion at the genomic and transcriptomic level. In addition, we compared EPC with its corresponding invasive ductal carcinoma (IDC) at the transcriptomic level.
Design(s): We selected 3 cases of pure EPC (C1-C3) and 3 cases of EPC (C4e-C6e) with corresponding IDC (C4i-C6i).We performed whole transcriptome analysis on laser-capture microdissected samples from formalin-fixed, paraffin-embedded tissue. We used CloneTech Mammalian stranded pico kit for sequencing RNA. KEGG pathway analysis and Gene Ontology (GO) analysis was performed using the cluster Profiler R package (v3.0.0) and Database for Annotation, Visualization and Integrated Discovery. DNA analysis was performed using our in-house next generation sequencing hybrid capture covering 580 genes on C1-C3 and C4e-C6e.
Result(s): There were 5 female and 1 male patients. The mean age was 73 years (range 62-90). All cases were hormone receptor positive. C4e-C6e showed upregulation of NTRK2 and MAGI2 (lg2FC= 3.14 and lg2FC = 3.0 fold, respectively) and downregulation of PRKACB (lg2FC= -4.4) compared to C1-C3 on RNAseq. C4i-C6i showed upregulation of collagen-related genes (COL10A1, COL11A1, COL14A1, COL16A1, COL1A1, COL3A1, COL8A1) (lg2FC range: 6.28 fold change) and ADAM12/ADAMTS2 (lg2FC=6.2 and lg2FC= 6.9 fold change) compared to C4e-C6e (FDR =0.014). Pathway analysis showed upregulation of collagen fibril organization and extracellular matrix organization pathways in C4i-C6i compared to C4e-C6e and upregulation of kinase activity pathway (GO: 0016301) in C4e-C6e compared to C1-C3.Recurrent PIK3CA hotspot non-synonymous mutation was identified in C3, C4e, C5e and C6e (c.G1633A in C5 and c.A3140G in C3, C4 and C6).
Conclusion(s): Our findings suggest that kinase and matrix metalloproteinase pathways contribute to EPC with invasion compared to pure EPC cases. Furthermore, enrichment of collagen-related genes in IDCs compared to their corresponding EPC suggest a synergistic potential with the aforementioned pathways. Mechanistic studies are warranted to validate the findings

Background: As hepatocellular carcinoma (HCC) develops from premalignant lesions (low and high grade dysplastic nodules; LGDN and HGDN), there is a corresponding accumulation of molecular alterations, some of which have been well described. However, the molecular features of lesions comprising the hepatocellular dysplasia-carcinoma sequence as they relate to different etiologies have not yet been explored. Herein, we characterize the molecular landscape of such lesions in cirrhotic explants with alcohol-related liver disease (ALD) and chronic hepatitis C (CHC).
Design(s): Tissue was assessed from 27 liver explants (14 CHC, 13 ALD) including 10 LGDNs (5 CHC, 5 ALD), 8 HGDNs (3 CHC, 5 ALD), 10 HCCs arising from HGDNs (5 CHC, 5 ALD), and 10 small HCCs defined as HCC < 2 cm (5 CHC, 5 ALD), as well as non-lesional cirrhotic parenchyma and matched normal non-liver tissue (e.g. porta hepatis structures). DNA was extracted from FFPE tissue for next generation sequencing (NGS) using a customized NGS580 panel targeting all exonic and select intronic areas in 580 cancer related genes. Data was analyzed using customized bioinformatics pipelines with an R package.
Result(s): TERT promoter HS C228T mutations were identified in 6 of 10 (60%) CHC related HCCs and only 1 of 9 (11%) alcohol related HCC (Figure 1). There was a significant association between TERT promoter HS C228T mutations and CHC related HCCs (p<0.02). In contrast, ALD related lesions showed deleterious events affecting tumor suppressor genes (NF1, BRCA1; CDKN2C) and histone methylation/chromatin remodeling genes (KMT2A; KMT2C; ASXL1; RAD21), which were found in 6 of 13 ALD related lesions (46.2% [3 of 5 small HCCs, 2 of 4 HGDNs, and 1 of 4 HCCs arising in HGDNs]). These events only occurred in 3 of 14 (21.4%) CHC related lesions (Figures 1 and 2). Within the CHC group, 1 HCC arising in HGDN showed copy number gains (CNG) in MARK4, ERCC2, FGFR4 and FLT4 and two HGDNs showed CNGs in NOTCH1 and TERT, respectively. No differences in the tumor mutational burden (TMB) were noted between CHC related and ALD related lesions, nor across the DN-HCC sequence. Non-lesional liver and LGDNs did not show recurrent mutations pertaining to a specific pathway. (Figure presented)
Conclusion(s): Our findings suggest that the pathways of hepatocarcinogenesis are distinct in ALD and CHC. While upregulation of telomerase activity (TA) and cancer cell immortalization play a pivotal role in CHC related HCC, defective chromatin remodeling appears to contribute to tumorigenesis in ALD related HCC

Background: Immunotherapies (IT) targeting the tumor micro environment (TME) have shown revolutionary results as a treatment (TX) for advanced melanoma. Yet, dramatic responses to IT in melanoma patients is seen in only a small subset of cases highlighting the complexity of the TME. Several subtypes of melanoma exist and knowledge of the TME in rare subtypes is scarce. Primary acral lentiginous melanoma (PALM) arises from the acral skin, is aggressive, rare, and genetically distinct from primary cutaneous melanoma (PCM). The TME composition in ALM has not been well established. Herein, we aim to investigate the composition of the TME in PALM and seek to identify potential signatures that might lead to the development of new therapeutic targets and predictive biomarkers.
Design(s): Our cohort includes 35 tumors (18-PALM, 9-PCM, 8-NALM). Tumoral RNA was investigated via gene expression analysis, carried out with a customized 770-gene expression panel combining markers for 24 different immune cell types and 30 common cancer antigens, including key checkpoint blockade genes analyzed with the N-counter system. Differential gene expression (DGE) pathway analysis were performed using R package (p<0.01; FDR<0.01) through comparisons with PCM and non-lentiginous melanoma arising at acral sites (NALM). Molecular findings were correlated with clinicopathologic features, treatment status, and disease specific and overall survival.
Result(s): PALM and PCM TMEs showed a predominance of cytotoxic TCD8+ compared to NALM (p<0.001;p<0.01, respectively). An additional Mast cell signature was noted in PCM (p<.01). NALM TME showed a scarce TCD8+ signature. Most PALM showed relative abundance of CD8 over exhausted CD8 and Tregs (p<0.01) (Figure1) that significantly associated with stage I/II presentation and epithelioid cytology (p<0.05). PALMs with exhausted CD8 signatures significantly associated with ulceration and PNI (p=0.01). DGE identified significant upregulation of ICAM3, TYK2 and CD164 in PALM cases (P<0.01) which correlated with enrichment in the PI3K/Akt/mTOR, NF-kB, TNF, ERK, adhesion, and chemokine pathways (p <0.01) (Table1/Figure2). (Table presented)
Conclusion(s): PALM TME is distinct from NALM TME, showing a preponderance of TCD8+ cells over T-regs, suggesting a possible predictor for better response to IT. Upregulation of TYK2 represents a potential for combinational regimen with selective TYK2 inhibitors in cases where IT monotherapy fails. Validation of the findings through mechanistic studies is warranted

Background: When given a diagnosis of invasive melanoma, patients invariably ask their prognosis. Much of what is available to patients is based purely on stage. We seek to examine additional pathologic and clinical factors possibly associated with patient outcomes after diagnosis of invasive melanoma.
Design(s): This study retrospectively examined patients with invasive melanoma derived from a one year period of referrals to a tertiary academic care center for confirmation of pathology and clinical assessment (N=856). We utilized multivariate logistic regression to evaluate possible factors associated with disease progression over an 8-year follow-up period. Proposed factors included age at time of confirmed diagnosis, anatomic site, histologic type, Clark level, Breslow depth, radial and vertical growth phases, mitoses, ulceration, regression, lymphovascular invasion, perineural invasion, microsatellitosis, regression, concurrent nevus, and AJCC stage. Outcomes were defined as clinically or pathologically confirmed regional or distant sites of disease, recurrence or growth of residual disease, and death. Treatment was defined as whether the patient received chemotherapy, radiation, and/or immunotherapy treatment.
Result(s): Of the 856 patients referred with suspected primary invasive melanoma, 811 (95%) patients were confirmed to have primary invasive melanoma (57% male, average age at diagnosis 57 years old). Of these, 708 cases contained complete pathologic and clinical data for assessment. After diagnosis, 222 (27%) patients received treatment, 279 (34%) progressed, and 166 (20%) died within the eightyear period. Factors statistically associated with disease progression included microsatellitosis (OR=20.2, 95% CI 1.9-214) and AJCC stage (OR 1.37, 95% CI 1.1-1.7). Factors associated with death included disease progression (OR 7.5, 95% CI 3.9-14.3), age at confirmed diagnosis (OR 1.05, 95% CI 1.0-1.1), ulceration (OR 2.3, 95% CI 1.2-4.4), and treatment (OR 2.2, 95% CI 1.2-4.1). Of those without disease progression, 49 (6%) patients died within the same timeframe due to other causes. (Table presented)
Conclusion(s): Approximately one third of patients referred to an academic tertiary care center with confirmed primary invasive melanoma progressed, and of these, almost two-thirds passed away within eight years. While recent therapies have improved patient outcomes, much research is still needed to prevent and treat invasive melanoma

Background: Molecular screening for therapeutically targetable alterations is considered standard of care in the management of non-small cell lung cancer. However, most molecular assays utilize tumor tissue, which may not always be available. This has led to the development of "liquid biopsies": Plasma-based Next Generation Sequencing (NGS) tests that use circulating tumor DNA as a substrate to identify relevant targets. In this study, we sought to determine the level of agreement between the two tests as they are used in clinical practice and to investigate the utility of concurrent plasma/tissue testing.
Design(s): We identified 47 cases of lung adenocarcinoma diagnosed over the past 2 years, who received concurrent testing (within 24 weeks) with both our institution's tissue (DNA and RNA based) NGS assay and a commercial plasma-based NGS assay. The results were reviewed to establish concordance in the identification of mutations or fusions deemed clinically relevant or for which a targeted therapy was available.
Result(s): Patients in our cohort represented both new diagnoses (31 cases, 66%) and disease progression on treatment (16 cases, 34%). The majority (83%) had stage 4 disease. Tissue NGS identified clinically relevant mutations in 39 cases (83%), including in 14 (88%) of the previously treated cases. By comparison, plasma NGS identified clinically relevant mutations in 20 cases (43%, p<0.001), including 6 treated cases (38%, p=0.01). Tissue NGS identified therapeutic targets in 55% of cases and 75% of previously treated cases; while plasma NGS identified targets in 28% and 25% respectively (p<0.001 and p=0.01 respectively). All clinically relevant mutations identified by plasma NGS were also detected by tissue NGS, while plasma NGS detected only 51% those identified by tissue NGS. Discrepant cases involved hotspot mutations and actionable fusions including those in EGFR, KRAS, and ROS1 (Table 1).(Table presented)
Conclusion(s): Tissue NGS detects more clinically relevant alterations and therapeutic targets compared to plasma NGS, especially in the post-treatment setting, suggesting that tissue NGS should be the preferred method for molecular testing of lung adenocarcinoma. Additionally, all clinically relevant mutations identified by plasma NGS were also detected by tissue NGS, suggesting that tissue/plasma cotesting provides little additional benefit over tissue NGS alone. Plasma NGS can detect clinically relevant targets, and still plays an important role when tissue testing is impractical or not possible

Background: Melanoma is the most common among the fatal forms of skin cancer. Our institution routinely performs a second-opinion review of pertinent previous pathologic material on patients referred for further care. In this current study, we evaluated the extent of discordance between primary histopathologic diagnosis and secondary review of benign and malignant melanocytic lesions; parameters of melanoma, and the subsequent impact on clinical management and follow-up were reviewed.
Design(s): In a retrospective review of 1718 referral cases of melanocytic lesions from 1/2010 to 1/2011, initial diagnoses from the outside institution were compared to second opinion reports. Consultation cases were excluded from the study. If the diagnosis was that of "invasive melanoma", the following parameters were collected: Histologic type, Clark level, Breslow thickness, mitotic count, ulceration status, regression, lymphovascular invasion, perineural invasion, microsatellitosis, tumor infiltrating lymphocytes, associated nevus, and outcome. Discordance categories were classified as major or minor. Major discordance was defined as a change in the stage or diagnosis that would directly change the management. Minor discordance category included discrepancies that would not alter the stage or management.
Result(s): The final diagnoses were metastatic melanoma - 517 cases (30%), invasive melanoma - 808 cases (47%), melanoma in-situ (MIS) - 298 cases (17.3%), dysplastic nevus (DN) - 73 cases (4.2%), nevus - 19 cases (1.1%), and no melanocytic lesion seen - 3 cases (0.2%). The concordance rates were as follows: For metastatic melanoma - 514 cases (99.4%), invasive melanoma - 775 cases (95.9%), M

Gene fusions are caused by chromosomal rearrangements and encode fusion proteins that can act as oncogenic drivers in cancers. Traditional methods for detecting oncogenic fusion transcripts include fluorescence in situ hybridization (FISH), reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC). However, these methods are limited in scalability and pose significant technical and interpretational challenges. Next-generation sequencing (NGS) is a high-throughput method for detecting genetic abnormalities and providing prognostic and therapeutic information for cancer patients. We present our experience with the validation of a custom-designed Archer Anchored Multiplex PCR (AMP™) technology-based NGS technology, "NYU FUSION-SEQer" using RNA sequencing. We examine both analytical performance and clinical utility of the panel using 75 retrospective validation samples and 84 prospective clinical samples of solid tumors. Our panel showed robust sequencing performance with strong enrichment for target regions. The lower limit of detection was 12.5% tumor fraction at 125 ng of RNA input. The panel demonstrated excellent analytic accuracy, with 100% sensitivity, 100% specificity and 100% reproducibility on validation samples. Finally, in the prospective cohort, the panel detected fusions in 61% cases (n = 51), out of which 41% (n = 21) enabling diagnosis and 59% (n = 30) enabling treatment and prognosis. We demonstrate that the fusion panel can accurately, efficiently and cost-effectively detect the majority of known fusion genes, novel clinically relevant fusions and provides an excellent tool for discovery of new fusion genes in solid tumors.