LAUSR

To the Editor Combined hepatocellular‐cholangiocarcinoma (cHCC‐ CCA) is rare, comprising 1%–5% of primary liver cancer. Previous molecular profiling efforts have shown differences among studies: some suggested that cHCC‐CCA has similarity to hepatocellular carcinoma, while others suggested that cHCC‐CCA is closely related to cholangiocarcinoma. This discrepancy can in part be attributed to the differences in the predominant tumor component sampled and to the use of different diagnostic criteria for cHCC‐CCA among the studies. Herein we report two patients with cHCC‐CCA. Tumors from both patients were analyzed in parallel with a paired germline control specimen using the Oncomine Comprehensive Assay v3 NGS‐based cancer panel (Thermo Fisher Scientific) by the Yale New Haven Hospital Tumor Profiling Laboratory. Both patients harbored pathogenic BRCA2 germline variants and both tumors showed loss of heterozygosity of BRCA2. This is the first study to implicate germline BRCA mutations in the pathogenesis of cHCC‐CCA. Patient 1, a 59‐year‐old woman, had a past medical history of right breast cancer status post (s/p) lumpectomy, radiation and tamoxifen for 5 years and thyroid cancer s/p partial thyroidectomy. A 2.5 cm mass in the periphery of segment VIII of the liver and also a 9mm lymph node adjacent to the distal esophagus were fund on abdominal magnetic resonance imaging (MRI). Tumor markers including CEA and CA19‐9 were within normal limits. The patient underwent a liver biopsy, which returned the diagnosis of poorly differentiated carcinoma with cholangiocarcinoma and hepatocellular carcinoma features at an outside institution. Pathological examination of the resection specimen revealed a single firm well delineated tumor with foci of hemorrhage and necrosis, measuring 4.7 × 4.4 × 3.6 cm (Figure S1a). Histologically, the tumor was composed of both hepatocellular and cholangiocytic components (Figure S2a,b), evidenced by expression of HepPar‐1 and CK19 (Figure S2c,d) on immunohistochemical staining. A prominent intratumoral lymphocytic infiltrate comprised of T and B cells was seen in both components, highlighted by CD3 and CD20 immunostains, respectively (Figure S2e,f). Background liver showed moderate macrovesicular steatosis without significant fibrosis. The excision of a paraesophageal mass measuring 2.5 cm revealed a similar morphology to the primary tumor in the liver, which is consistent with metastatic cHCC‐CCA (Table S1). A representative tissue block of the current resection specimen, containing both histological components of the cHCC‐CC with an estimated malignant cell content of 60%, was analyzed in parallel with a paired germline control specimen (peripheral blood) using the Oncomine Comprehensive Assay v3 NGS‐based cancer panel (Thermo Fisher Scientific). A pathogenic BRCA2 p.Q2859Ter variant was detected in both the tumor sample and in the germline specimen, with variant allele frequencies (VAF) of 63% and 39%, respectively. The VAF in the patient's germline specimen is consistent with heterozygous status genetically. The higher VAF in the tumor suggests loss of the normal BRCA2 allele (i.e., loss of heterozygosity) within the malignant cells. No somatic single nucleotide or multi‐nucleotide variants, gene amplifications (copy number ≥5) or gene fusions were detected in the tumor (Table 1). Patient 2, a 66‐year‐old man with a past medical history of treated chronic HCV (on Epclusa/ribavirin with sustained virological response), presented with an enlarging liver mass in the periphery of segment IVA. He underwent a liver biopsy, which showed a poorly differentiated adenocarcinoma with immunoprofile suggestive of intrahepatic cholangiocarcinoma. Liver biochemical tests revealed slightly elevated serum ALT 42 U/L (0–34 U/L) with normal alkaline phosphatase and AST. Tumor markers showed elevated CA19‐9 (78.5 U/mL [≤37.0 U/ml]) and normal AFP. Pathological examination of the resection specimen revealed a single firm well delineated tumor with foci of hemorrhage and necrosis, measuring 4.6 × 4.2 × 3.0 cm (Figure S1b). Histologically, the tumor was composed of both hepatocellular and cholangiocytic components (Figure S3a,b), and showed expression of HepPar‐1 and CK19 (Figure S3c,d) on immunohistochemical assessment. A prominent intratumoral lymphocytic infiltrate comprised of predominantly T cells was seen in both components, highlighted by CD3 immunostain (Figure S3e). CD20 immunostain was largely negative