Canine malignant melanoma, a significant cause of mortality in domestic dogs, is a powerful comparative model for human melanoma, but little is known about its genetic etiology. We mapped the genomic landscape of canine melanoma through multi-platform analysis of 37 tumors (31 mucosal, 3 acral, 2 cutaneous, and 1 uveal) and 17 matching constitutional samples including long- and short-insert whole genome sequencing, RNA sequencing, array comparative genomic hybridization, single nucleotide polymorphism array, and targeted Sanger sequencing analyses. We identified novel predominantly truncating mutations in the putative tumor suppressor gene PTPRJ in 19% of cases. No BRAF mutations were detected, but activating RAS mutations (24% of cases) occurred in conserved hotspots in all cutaneous and acral and 13% of mucosal subtypes. MDM2 amplifications (24%) and TP53 mutations (19%) were mutually exclusive. Additional low-frequency recurrent alterations were observed amidst low point mutation rates, an absence of ultraviolet light mutational signatures, and an abundance of copy number and structural alterations. Mutations that modulate cell proliferation and cell cycle control were common and highlight therapeutic axes such as MEK and MDM2 inhibition. This mutational landscape resembles that seen in BRAF wild-type and sun-shielded human melanoma subtypes. Overall, these data inform biological comparisons between canine and human melanoma while suggesting actionable targets in both species.

Melanocytic lesions originating from the oral mucosa or cutaneous epithelium are common in the general dog population, with up to 100,000 diagnoses each year in the USA. Oral melanoma is the most frequent canine neoplasm of the oral cavity, exhibiting a highly aggressive course. Cutaneous melanocytomas occur frequently, but rarely develop into a malignant form. Despite the differential prognosis, it has been assumed that subtypes of melanocytic lesions represent the same disease. To address the relative paucity of information about their genomic status, molecular cytogenetic analysis was performed on the three recognized subtypes of canine melanocytic lesions. Using array comparative genomic hybridization (aCGH) analysis, highly aberrant distinct copy number status across the tumor genome for both of the malignant melanoma subtypes was revealed. The most frequent aberrations included gain of dog chromosome (CFA) 13 and 17 and loss of CFA 22. Melanocytomas possessed fewer genome wide aberrations, yet showed a recurrent gain of CFA 20q15.3–17. A distinctive copy number profile, evident only in oral melanomas, displayed a sigmoidal pattern of copy number loss followed immediately by a gain, around CFA 30q14. Moreover, when assessed by fluorescence in situ hybridization (FISH), copy number aberrations of targeted genes, such as gain of c-MYC (80 % of cases) and loss of CDKN2A (68 % of cases), were observed. This study suggests that in concordance with what is known for human melanomas, canine melanomas of the oral mucosa and cutaneous epithelium are discrete and initiated by different molecular pathways.

Background: Biliary tract cancers (BTCs) are a heterogeneous group of aggressive, rare malignancies with limited standard chemotherapeutic options for advanced disease. Recent studies have demonstrated potential novel biliary cancer targets and a possible role for immunotherapy in the treatment of patients with this disease. Intrahepatic cholangiocarcinoma (IHCC), extrahepatic cholangiocarcinoma (EHCC), and gallbladder carcinoma (GBC) are frequently grouped together in clinical trials despite differences in tumor biology. Methods: To further investigate tumor biology differences, we profiled 1,502 BTCs using next-generation sequencing (NGS), immunohistochemistry, in situ hybridization, and RNA sequencing. Results: IHCCs had higher rates of IDH1, BAP1, and PBRM1 mutations and FGFR2 fusions; EHCCs had higher rates of KRAS, CDKN2A, and BRCA1 mutations; and GBCs had higher rates of homologous recombination repair deficiency and Her2/neu overexpression and amplification. IHCCs and GBCs had higher rates of potential positive predictive biomarkers for immune checkpoint inhibition (PD-L1 expression, high microsatellite instability, and high tumor mutational burden) than EHCCs. Conclusions: These findings support clinical molecular profiling of BTCs to inform potential therapeutic selection and clinical trial design based on the primary tumor's site of origin within the biliary tree.

Purpose: GEP-NENs are rare malignancies with increasing incidence. Their molecular characteristics are still undefined. We explored the underlying biology of GEP-NENs and the differences between gastrointestinal (GI) and pancreatic (PNEN), high-grade (HG), and low-grade (LG) tumors. Experimental Design: GEP-NENs were analyzed using next-generation sequencing (NGS; MiSeq on 47 genes, NextSeq on 592 genes), IHC, and in situ hybridization. Tumor mutational burden (TMB) was calculated on the basis of somatic nonsynonymous missense mutations, and microsatellite instability (MSI) was evaluated by NGS of known MSI loci. Results: In total, 724 GEP-NENs were examined: GI (N = 469), PNEN (N = 255), HG (N = 135), and LG (N = 335). Forty-nine percent were female, and median age was 59. Among LG tumors, the most frequently mutated genes were ATRX (13%), ARID1A (10%), and MEN1 (10%). HG tumors showed TP53 (51%), KRAS (30%), APC (27%), and ARID1A (23%). Immune-related biomarkers yielded a lower prevalence in LG tumors compared with HG [MSI-H 0% vs. 4% (P = 0.04), PD-L1 overexpression 1% vs. 6% (P = 0.03), TMB-high 1% vs. 7% (P = 0.05)]. Compared with LG, HG NENs showed a higher mutation rate in BRAF (5.4% vs. 0%, P < 0.0001), KRAS (29.4% vs. 2.6%, P < 0.0001), and PI3KCA (7% vs. 0.3%, P < 0.0001). When compared with GI, PNEN carried higher frequency of MEN1 (25.9% vs. 0.0%, P < 0.0001), FOXO3 (8.6% vs. 0.8%, P = 0.005), ATRX (20.6% vs. 2.0%, P = 0.007), and TSC2 (6.3% vs. 0.0%, P = 0.007), but lower frequency of mutations in APC (1.0% vs. 13.8%, P < 0.0001). Conclusions: Significant molecular differences were observed in GEP-NENs by tumor location and grade, indicating differences in carcinogenic pathways and biology.

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The increasing rate of colorectal cancers (CRC), primarily distal tumors, among young adults poses a global health issue. This study investigates the molecular differences between younger (≤45 years old) and older (≥65) adults with left-sided CRCs. Younger patients more frequently harbor mutations in genes associated with cancer-predisposing syndromes. Higher rates of microsatellite instability-high and tumor mutational burden-high tumors occur in younger patients, who could benefit from immune-checkpoint inhibitors. Finally, histone modifiers are more frequently mutated in younger patients and could serve as a new promising therapeutic target. This study provides new insights into mutations that may guide development of novel tailored therapy in younger CRC patients.

Background Primary Extra‐mammary Paget's disease (EMPD) is a very rare cutaneous adenocarcinoma affecting anogenital or axillary regions. It is characterized by a prolonged course with recurrences and eventually distant metastatic spread for which no specific therapy is known. Methods Eighteen EMPD (13 vulvar and five scrotal) and ten mammary Paget's disease (MPD) cases were comprehensively profiled for gene mutations, fusions and copy number alterations, and for therapy‐relevant protein biomarkers). Results Mutations in TP53 and PIK3CA were the most frequent in both cohorts: 7/15 and 5/15 in EMPD; 1/6 and 4/7 in MPD HER2 gene amplification was detected in 4/18 EMPD (3 vulvar and 1 scrotal case) in contrast to MPD where it was detected in the majority (7/8) of cases. TOP2A gene amplification was seen in 2/12 EMPD and 1/6 MPD, respectively. Similarly, no difference in estrogen receptor expression was seen between the EMPD (4/15) and MPD (3/10). Androgen receptor was also expressed in the majority of both cohorts (12/16 EMPD) and (7/8 MPD).Here ARv7 splice variant was detected in 1/7 EMPD and 1/4 MPD cases, respectively. PD‐L1 expression on immune cells was exclusively observed in three vulvar EMPD. In contrast to MPD, six EMPDs harbored a “high” tumor mutation burden (≥10 mutations/Mb). All tested cases from both cohorts were MSI stable. Conclusions EMPD shares some targetable biomarkers with its mammary counterpart (steroid receptors, PIK3CA signaling pathways, TOP2A amplification). HER2 positivity is notably lower in EMPD while biomarkers to immune checkpoint inhibitors (high TMB and PD‐L1) were observed in some EMPD. Given that no consistent molecular alteration characterizes EMPD, comprehensive theranostic profiling is required to identify individual patients with targetable molecular alterations.

BRAF mutations are relatively common in many cancers, particularly melanoma, colorectal cancer, and thyroid cancer and to a lesser extent in lung cancer. These mutations can be targeted by BRAF and MEK inhibitors, which exhibit good clinical activity. There are conflicting reports of the various relative rates of BRAF Class I mutations (V600 locus), defined as those that exhibit extremely strong kinase activity by stimulating monomeric activation of BRAF, Class II, define as non-V600 mutations that activate BRAF to signal as a RAS-independent dimer, and Class III mutations, defined as “kinase-dead” with low kinase activity as compared to wild type BRAF. Prospective studies have largely focused on patients with tumors harboring Class I BRAF mutations (limited to the V600 locus) where response rates up to 70% with BRAF plus MEK inhibition have been demonstrated. We report on the relative prevalence of various types of BRAF mutations across human cancers in a cohort of 114,662 patients that received comprehensive genomic profiling using next-generation sequencing. Of these patients, 4517 (3.9%) a pathogenic or presumed pathogenic BRAF mutation (3.9%). Of these, 1271 were seen in melanoma, representing 39.7% of all melanomas sequenced, representing the highest rate in all tumors. Class I (V600) mutations were seen overall in 2841 patients (62.1% of BRAF mutations, 2.4% of total cancers). Class II mutations were seen in 746 tumors (16.5% of BRAF mutant, 0.7% of total), and Class III mutations were seen in 801 tumors (17.7% of BRAF, 0.7% of total). Knowledge of the relative prevalence of these types of mutations can aid in the development of agents that might better address non-V600 mutations in cancer. Impact statement These data represent the largest aggregation of BRAF mutations within a single clinical database to our knowledge. The relative proportions of both BRAF V600 mutations and non-V600 mutations are informative in all cancers and by malignancy, and can serve as a definitive gold-standard for BRAF mutation cancer incidence by malignancy. The rate of BRAF mutation in human cancer in a real-world large database is lower than previously reported likely representing testing more broadly across tumor types. The relative percentages of Class II and Class III BRAF mutations are higher than previously reported, representing almost 35% of BRAF mutations in cancer. These findings provide support for the development of effective treatments for non-V600 BRAF mutations in cancer.