Faculty Bibliography

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

Soft tissue tumors can be categorized molecularly into two categories: tumors which are known to have recurrent molecular alterations and tumors which do not have consistent recurrent molecular alterations or translocations. These "non-translocation" associated sarcomas are clinically more aggressive than their more stable counterparts. However, recent advances in RNA sequencing have discovered recurrent novel fusions within the latter group, namely TERT-TRIO fusions. Furthermore, a recent report discovered this fusion in a spindle cell liposarcoma. Our case describes a novel fusion of CTNND2, a neighbor gene of TRIO, and TERT in a spindle cell liposarcoma, and provides further evidence that spindle cell liposarcoma should be a distinct entity from spindle cell dedifferentiated liposarcoma. This article is protected by copyright. All rights reserved.

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.

Mycobacterium abscessus and M. chelonae belong to the rapid-growing nontuberculous mycobacteria (NTM) group, which are defined by their ability to form visible colonies on agar within 7 days of subculture. Cutaneous infections by this complex show a heterogeneous clinical presentation with varied histopathologic findings. However, the presence of vacuoles in many specimens has been reported as a specific histologic finding. Herein, we correlate the histopathology of patients with tissue-culture positive M. abscessus/M. chelonae complex in order to identify features that may prompt a rapid categorization of the infectious etiology. The cohort includes 33 skin punch biopsy specimens from 28 patients who had associated positive tissue cultures. The most frequent clinical presentation was a single or multiple nodule. Twenty-seven specimens (81.81%) were found to have vacuoles. The observation of certain histologic features (ie, polymorphonuclear microabscesses and epithelioid granuloma formation) should raise the possibility of infection by NTM. In addition to these findings, we believe the presence of vacuoles in the dermal and subcutaneous inflammation should raise suspicion for NTM.

Salivary gland secretory carcinoma, also termed mammary analogue secretory carcinoma (MASC), is a recently described salivary gland neoplasm with characteristic histomorphologic findings similar to those of secretory carcinoma of the breast and harboring recurrent ETV6-NTRK3 fusions. Recent findings have expanded the molecular profile of salivary gland secretory carcinoma to include multiple novel ETV6 fusion partners, including RET, MET, and MAML3. Here, we report a case of cystic MASC with cribriform and papillary histology harboring two gene fusions, ETV6-RET and EGFR-SEPT14, identified by targeted RNA sequencing. The presence of the rearrangements was confirmed by FISH, RT-PCR, and Sanger sequencing. This is the first EGFR-SEPT14 fusion reported in secretory carcinoma as a single event or in association with an ETV6 rearrangement. This finding adds to the expanding molecular profile of this tumor entity, and may translate into novel treatment strategies.