Recurrent Rearrangements in PRKACA and PRKACB in Intraductal Oncocytic Papillary Neoplasms of the Pancreas and Bile Duct.

BACKGROUND & AIMS: Intraductal oncocytic papillary neoplasms (IOPNs) of the pancreas and bile duct contain epithelial cells with numerous, large mitochondria and are cystic precursors to pancreatic ductal adenocarcinoma (PDAC) and cholangiocarcinoma (CCA), respectively. However, IOPNs do not have the genomic alterations found in other pancreatobiliary neoplasms. In fact, no recurrent genomic alterations have been described in IOPNs. PDACs without activating mutations in KRAS contain gene rearrangements, so we investigated whether IOPNs have recurrent fusions in genes. METHODS: We analyzed 20 resected pancreatic IOPNs and 3 resected biliary IOPNs using a broad RNA-based targeted sequencing panel to detect cancer-related fusion genes. Four invasive PDACs and 2 intrahepatic CCAs from the same patients as the IOPNs, were also available for analysis. Samples of pancreatic cyst fluid (n = 5, collected before surgery) and bile duct brushings (n = 2) were analyzed for translocations. For comparison, we analyzed pancreatobiliary lesions from 126 patients without IOPN (controls). RESULTS: All IOPNs evaluated were found to have recurring fusions of ATP1B1-PRKACB (n = 13), DNAJB1-PRKACA (n = 6), or ATP1B1-PRKACA (n = 4). These fusions also were found in corresponding invasive PDACs and intrahepatic CCAs, as well as in matched pancreatic cyst fluid and bile duct brushings. These gene rearrangements were absent from all 126 control pancreatobiliary lesions. CONCLUSIONS: We identified fusions in PRKACA and PRKACB genes in pancreatic and biliary IOPNs, as well as in PDACs and pancreatic cyst fluid and bile duct cells from the same patients. We did not identify these gene fusions in 126 control pancreatobiliary lesions. These fusions might be used to identify patients at risk for IOPNs and their associated invasive carcinomas.

KRAS amplification in metastatic colon cancer is associated with a history of inflammatory bowel disease and may confer resistance to anti-EGFR therapy.

Mutations in RAS occur in 30-50% of metastatic colorectal carcinomas (mCRCs) and correlate with resistance to anti-EGFR therapy. Consequently, mCRC biomarker guidelines state RAS mutational testing should be performed when considering EGFR inhibitor treatment. However, a small subset of mCRCs are reported to harbor RAS amplification. In order to elucidate the clinicopathologic features and anti-EGFR treatment response associated with RAS amplification, we retrospectively reviewed a large cohort of mCRC patients that underwent targeted next-generation sequencing and copy number analysis for KRAS, NRAS, HRAS, BRAF, and PIK3CA. Molecular testing was performed on 1286 consecutive mCRC from 1271 patients as part of routine clinical care, and results were correlated with clinicopathologic findings, mismatch repair (MMR) status and follow-up. RAS amplification was detected in 22 (2%) mCRCs and included: KRAS, NRAS, and HRAS for 15, 5, and 2 cases, respectively (6-21 gene copies). Patients with a KRAS-amplified mCRC were more likely to report a history of inflammatory bowel disease (p < 0.001). In contrast, mutations in KRAS were associated with older patient age, right-sided colonic origin, low-grade differentiation, mucinous histology, and MMR proficiency (p ≤ 0.017). Four patients with a KRAS-amplified mCRC and no concomitant RAS/BRAF/PIK3CA mutations received EGFR inhibitor-based therapy, and none demonstrated a clinicoradiographic response. The therapeutic impact of RAS amplification was further evaluated using a separate, multi-institutional cohort of 23 patients. Eight of 23 patients with KRAS-amplified mCRC received anti-EGFR therapy and all 8 patients exhibited disease progression on treatment. Although the number of KRAS-amplified mCRCs is limited, our data suggest the clinicopathologic features associated with mCRC harboring a KRAS amplification are distinct from those associated with a KRAS mutation. However, both alterations seem to confer EGFR inhibitor resistance and, therefore, RAS testing to include copy number analyses may be of consideration in the treatment of mCRC.

Gene fusion characterisation of rare aggressive prostate cancer variants-adenosquamous carcinoma, pleomorphic giant-cell carcinoma, and sarcomatoid carcinoma: an analysis of 19 cases.

AIMS: To evaluate the molecular underpinnings of the rare aggressive prostate cancer variants adenosquamous carcinoma, pleomorphic giant-cell carcinoma, and sarcomatoid carcinoma. METHODS AND RESULTS: We retrieved 19 tumours with one or more variant(s), and performed ERG immunohistochemistry, a next-generation sequencing assay targeting recurrent gene fusions, and fluorescence in-situ hybridisation (FISH) for ERG and BRAF. Divergent differentiation included: sarcomatoid carcinoma (n = 10), adenosquamous carcinoma (n = 7), and pleomorphic giant-cell carcinoma (n = 7). Five patients had more than one variant. Four had variants only in metastases. ERG rearrangement was detected in nine (47%, seven via sequencing, showing TMPRSS2-ERG fusions and one GRHL2-ERG fusion, and two via FISH, showing rearrangement via deletion). ERG was immunohistochemically positive in the adenocarcinoma in eight of nine (89%) patients, but was immunohistochemically positive in the variant in only five of nine patients (56%, typically decreased). One patient had a false-positive ERG immunohistochemical result in the sarcomatoid component despite a negative FISH result. Two (11%) harboured BRAF fusions (FAM131A-BRAF and SND1-BRAF). CONCLUSIONS: ERG fusions are present in these rare prostate cancer variants with a frequency close to that in conventional prostate cancer (9/19, 47%). ERG immunohistochemistry usually detects rearrangement in the adenocarcinoma, but is less sensitive for the variant histology, with weak to negative staining. Adenosquamous and sarcomatoid variants can, particularly, occur together. Molecular assessment may be an additional tool in selected cases to confirm the prostatic origin of unusual tumours. The presence of two BRAF rearrangements suggests that this gene fusion may be enriched in this setting, as RAF kinase fusions have been previously reported in 1-2% of prostate cancers.

Renal cell tumors with clear cell histology and intact VHL and chromosome 3p: a histological review of tumors from the Cancer Genome Atlas database.

Clear cell renal cell carcinoma is by far the most common form of kidney cancer; however, a number of histologically similar tumors are now recognized and considered distinct entities. The Cancer Genome Atlas published data set was queried (http://cbioportal.org) for clear cell renal cell carcinoma tumors lacking VHL gene mutation and chromosome 3p loss, for which whole-slide images were reviewed. Of the 418 tumors in the published Cancer Genome Atlas clear cell renal cell carcinoma database, 387 had VHL mutation, copy number loss for chromosome 3p, or both (93%). Of the remaining, 27/31 had whole-slide images for review. One had 3p loss based on karyotype but not sequencing, and three demonstrated VHL promoter hypermethylation. Nine could be reclassified as distinct or emerging entities: translocation renal cell carcinoma (n=3), TCEB1 mutant renal cell carcinoma (n=3), papillary renal cell carcinoma (n=2), and clear cell papillary renal cell carcinoma (n=1). Of the remaining, 6 had other clear cell renal cell carcinoma-associated gene alterations (PBRM1, SMARCA4, BAP1, SETD2), leaving 11 specimens, including 2 high-grade or sarcomatoid renal cell carcinomas and 2 with prominent fibromuscular stroma (not TCEB1 mutant). One of the remaining tumors exhibited gain of chromosome 7 but lacked histological features of papillary renal cell carcinoma. Two tumors previously reported to harbor TFE3 gene fusions also exhibited VHL mutation, chromosome 3p loss, and morphology indistinguishable from clear cell renal cell carcinoma, the significance of which is uncertain. In summary, almost all clear cell renal cell carcinomas harbor VHL mutation, 3p copy number loss, or both. Of tumors with clear cell histology that lack these alterations, a subset can now be reclassified as other entities. Further study will determine whether additional entities exist, based on distinct genetic pathways that may have implications for treatment.

Acquired cystic disease associated renal cell carcinoma: A clinicopathologic and molecular study of 31 tumors.

Acquired cystic disease associated renal cell carcinomas (ACD-RCC) are rare and their molecular and histopathological characteristics are still being explored. We therefore investigated the clinicopathologic and molecular characteristics of 31 tumors. The patients were predominantly male (n = 30), with tumors mainly left-sided (n = 17), unifocal (n = 19), and unilateral (n = 29) and a mean tumor size of 25 mm (range, 3-65 mm). Microscopically, several histologic patterns were present, including pure classic sieve-like (n = 4), and varied proportions of mixed classic sieve-like with papillary (n = 23), tubulocystic (n = 9), compact tubular (n = 4) and solid (n = 1) patterns. Calcium-oxalate crystals were seen in all tumors. Molecular analysis of 9 tumors using next generation sequencing showed alterations in SMARCB1 in 3 tumors (1 with frameshift deletion and 2 with copy number loss in chromosome 22 involving SMARCB1 region), however, INI1 stain was retained in all. Nonrecurrent genetic alterations in SETD2, NF1, NOTCH4, BRCA2 and CANT1 genes were also seen. Additionally, MTOR p.Pro351Ser was identified in one tumor. Copy number analysis showed gains in chromosome 16 (n = 5), 17 (n = 2) and 8 (n = 2) as well as loss in chromosome 22 (n = 2). In summary, ACD-RCC is a recognized subtype of kidney tumors, with several histological architectural patterns. Our molecular data identifies genetic alterations in chromatin modifying genes (SMARCB1 and SETD2), which may suggest a role of such genes in ACD-RCC development.

Renal Cell Carcinoma With Chromosome 6p Amplification Including the TFEB Gene: A Novel Mechanism of Tumor Pathogenesis?

Amplification of chromosome 6p has been implicated in aggressive behavior in several cancers, but has not been characterized in renal cell carcinoma (RCC). We identified 9 renal tumors with amplification of chromosome 6p including the TFEB gene, 3 by fluorescence in situ hybridization, and 6 from the Cancer Genome Atlas (TCGA) databases. Patients' ages were 28 to 78 years (median, 61 y). Most tumors were high stage (7/9 pT3a, 2/9 pN1). Using immunohistochemistry, 2/4 were positive for melanocytic markers and cathepsin K. Novel TFEB fusions were reported by TCGA in 2; however, due to a small composition of fusion transcripts compared with full-length transcripts (0.5/174 and 3.3/132 FPKM), we hypothesize that these represent secondary fusions due to amplification. Five specimens (4 TCGA, 1 fluorescence in situ hybridization) had concurrent chromosome 3p copy number loss or VHL deletion. However, these did not resemble clear cell RCC, had negative carbonic anhydrase IX labeling, lacked VHL mutation, and had papillary or unclassified histology (2/4 had gain of chromosome 7 or 17). One tumor each had somatic FH mutation and SMARCB1 mutation. Chromosome 6p amplification including TFEB is a previously unrecognized cytogenetic alteration in RCC, associated with heterogenous tubulopapillary eosinophilic and clear cell histology. The combined constellation of features does not fit cleanly into an existing tumor category (unclassified), most closely resembling papillary or translocation RCC. The tendency for high tumor stage, varied tubulopapillary morphology, and a subset with melanocytic marker positivity suggests the possibility of a unique tumor type, despite some variation in appearance and genetics.