Tyrosine kinase alterations in colorectal cancer with emphasis on the distinct clinicopathological characteristics.

AIMS: Tyrosine kinase (TK) alterations, such as anaplastic lymphoma kinase (ALK) fusion, neurotrophic tyrosine receptor kinase (NTRK) fusion, c-ros oncogene 1 (ROS1) fusion and mesenchymal-epithelial transition factor (MET) exon 14 skipping, have been reported in colorectal cancers (CRC). We have previously reported CRCs with NTRK fusion among our cohort. However, their clinicopathological features have not been fully elucidated. METHODS AND RESULTS: Tissue microarray (TMA)-based immunohistochemistry (IHC) was performed on 951 CRC lesions from 944 patients. IHC was evaluated as positive or negative for ALK and ROS1 and 0 to 3+ for c-MET. For ALK and ROS1 IHC-positive cases, RNA-based imbalanced gene expression assays, Archer FusionPlex assays and reverse transcription-polymerase chain reaction (RT-PCR) followed by Sanger sequencing were performed. For c-MET IHC 3+ cases, RT-PCR followed by Sanger sequencing were performed. ALK IHC was positive in three cases (0.2%) and all showed imbalanced ALK gene expression. The following ALK fusions were confirmed: EML4 (exon 21)::ALK (exon 20), EML4 (exon 6)::ALK (exon 19) and HMBOX1 (exon 6)::ALK (exon 20). Two showed microsatellite instability-high/mismatch repair (MMR)-deficient, and all were located in the right colon. ROS1 IHC was positive in one case; however, imbalanced expression and ROS1 fusion was negative. Forty-two cases (4.4%) showed c-MET IHC3+. MET exon 14 skipping was confirmed in nine cases. All cases were microsatellite stable/MMR-proficient, and eight were located in the left colon and rectum. CONCLUSIONS: CRCs with these TK alterations had distinct clinicopathological features. Together with our previous study, 15 cases (1.6%) harboured targetable TK alterations (three NTRK fusion, three ALK fusion, nine MET exon 14 skipping).

Molecular and clinicopathological analysis of three cases of gastric juvenile polyposis.

Background and Aim: Juvenile polyposis (JP) is a rare disease known to be associated with mutations either in SMAD4/BMPR1A. JP is known to often develop into malignant tumors, with a reported probability of 9-50%. However, the mechanisms of its carcinogenesis are not fully understood. We tried to elucidate the mechanisms of malignant transformation underlying this condition in three cases of gastric JP. Methods: We selected polyps from each patient displaying varying degrees of atypia and their nearby normal polyps and compared them using immunohistochemistry, Sanger sequencing, and loss of heterozygosity (LOH) analysis of SMAD4, BMPR1A, and TP53. Results: Two of the three cases were suspected of having germline SMAD4 mutations based on their familial medical histories; the remaining case was found to have a SMAD4 germline mutation following preoperative genetic testing. All three cases were shown to present with both SMAD4 positive and negative areas across each lesion, with the neoplastic lesions tending to show stronger nuclear SMAD4 expression. This expression was closely associated with the SMAD4 LOH status; however, we also noted paradoxical SMAD4 expression in the neoplastic lesions despite the biallelic inactivation of SMAD4 revealed in the genetic evaluation. Conclusions: These data suggest that strong nuclear expression of SMAD4, even when seemingly paradoxical, seems to be closely associated with dysplastic polyps in JP. Complete inactivation of SMAD4 was not shown to be essential for the development of dysplastic polyps in gastric JP, and other pathways seemed to be involved in the acquisition of the malignant phenotype.

Mucin phenotypes and clinicopathological features of colorectal adenocarcinomas: Correlation with colorectal adenocarcinoma with enteroblastic differentiation.

BACKGROUND: The mucin phenotypes of colorectal carcinoma (CRC) is related to the biological behavior and prognosis. But there has been no studies evaluating phenotypic characteristics in a large number of cases. Furthermore, colorectal adenocarcinoma with enteroblastic differentiation (CAED) is a rare subtype of CRC and having poor prognosis. The aims of this study were to clarify the correlation between mucin phenotypes and tumor development, including biological behavior in CRC, as well as to investigate characteristic of mucin phenotypes in CAED. METHODS AND RESULTS: 974 CRC cases and 42 CAED cases of CRCs were classified five types (large-intestinal, small-intestinal, gastric, mixed, and unclassified) of mucin phenotypes by using immunohistochemistry (IHC). IHC was performed on tissue microarrays with antibodies against followings: MUC2, MUC5AC, MUC6, and CD10. In CRCs, large-intestine type has a relatively better prognosis, small-intestinal type frequently shows venous invasions, and liver metastases, gastric type has more high-histological grades and lymphatic invasions, mixed type shows originating from the right side of the colon, larger tumor size and mucinous type, but less venous invasions and liver metastasis, whereas the unclassified type showed poorer prognosis in overall survival with statistical significance. The majority of CAED were found to be small-intestinal type or unclassified type. CONCLUSIONS: The phenotypic classification is useful for predicting the prognosis of CRCs. Small-intestinal type and unclassified type showed dismal prognosis in CRCs. We speculate that CAED having aggressive behavior and poor prognosis might reflect characteristics of small-intestinal and unclassified types.

NTRK fusion in Japanese colorectal adenocarcinomas.

NTRK fusion-positive tumors are known to be highly sensitive to TRK inhibitors, such as larotrectinib and entrectinib. Therefore, identification of patients who can potentially benefit from these inhibitors is important; however, the frequency of NTRK fusions in Japanese patients with colorectal cancer (CRC) is unknown. We performed pan-TRK staining using TMA-based immunohistochemistry (IHC) on samples from 971 consecutive Japanese CRC cases from a single institution. Positive cases were further analyzed using NanoString and subsequent targeted RNA sequencing. We found three positive cases using TRK-IHC. Furthermore, the Nanostring assay supported the presence of NTRK fusion in these cases. Subsequent targeted RNA-sequencing and RT-PCR revealed two cases with TPM3-NTRK1 and one with TPR-NTRK1. The TNM stages of these cases were stage I, stage IIA, and stage IIIB, and two showed microsatellite instability-high status. Next-generation sequencing analysis using Cancer hotspot panel revealed TP53 and SMAD4 mutations in separate cases. IHC of ß-catenin did not show nuclear accumulation. We found three cases (0.31%) of CRC with NTRK1 fusion among 971 consecutive Japanese CRC cases. No potential driver alterations other than NTRK fusion were identified in these three patients.

Molecular and clinicopathological features of colorectal adenocarcinoma with enteroblastic differentiation.

BACKGROUND: Colorectal adenocarcinoma with enteroblastic differentiation (CAED) is a rare subtype of colorectal malignancy with expression of enteroblastic markers (glypican 3, SALL4, AFP); however, the clinicopathological and epidemiological features are not fully elucidated. AIMS: The aims of this study were to elucidate and establish the molecular and clinicopathological characteristics of CAED. MATERIALS AND METHODS: In addition to three cases recently diagnosed as CAED, colorectal carcinoma (CRC) with expression of enteroblastic markers were selected by using immunohistochemistry (IHC) on tissue microarrays of 988 advanced CRC. We employed next-generation sequencing (NGS) and Sanger sequencing for the detection of genetic alterations. IHC for p53 and HER2, HER2-FISH and MSI status was also investigated. Survival analyses for clinicopathological parameters were performed using Kaplan-Meier methods. RESULTS: Thirty-nine cases (4.0%) were positive for at least one enteroblastic marker. Histological evaluation of the total of 42 cases revealed that 10 contained tumour cells with clear cytoplasm. Enteroblastic marker-positive cases had aggressive behaviour and poor prognosis. NGS revealed TP53 as the most frequently mutated gene. The rate of HER2-positive cases and MSI-H cases was 9.5% (four of 42) and 12.2% (five of 41), respectively. Among these 42 cases, there were no molecular and clinicopathological differences according to the presence of tumour cells with clear cytoplasm. CONCLUSIONS: Enteroblastic marker-positive CRC could be grouped together as CAED regardless of clear cell cytoplasm. Using this definition, the frequency of CAED is 4.0% and has a poorer prognosis than that for conventional CRCs. HER2 targeted therapy would be a meaningful treatment for CAED, and CAEDs contain both MSI-H and MSI-stable CRCs, although the MSS phenotype is dominant.

Colorectal adenocarcinoma with enteroblastic differentiation: a clinicopathological study of five cases.

AIMS: Colorectal adenocarcinoma with enteroblastic differentiation (CAED) is a rare malignancy, and its clinicopathological characteristics have not yet been fully elucidated. This study aimed to elucidate the clinicopathological features of CAED through immunostaining of enteroblastic lineage markers alpha-fetoprotein (AFP), glypican-3 (GPC3), and spalt-like transcription factor 4 (SALL4). METHODS AND RESULTS: We identified five CAED cases (0.3%) from 1666 colorectal carcinomas, analysed the clinicopathological characteristics and performed immunostaining for AFP, GPC3 and SALL4. Three patients were male and two were female. All cases were located in the sigmoid colon or rectum. Histologically, all cases showed adenocarcinoma composed of cuboidal or columnar cells, with clear cytoplasm resembling the primitive gut; one case exhibited a partial hepatoid pattern. The depth of invasion was T2 and T3 in two and three cases, respectively. Lymphatic/venous invasion was found in all cases (100%), lymph node metastases in four of five cases (80%) and distant metastases in three of five cases (60%) (liver, two cases; lung, one case). Two patients died as a result of their disease during follow-up. Immunohistochemically, SALL4 and GPC3 were each positive in four of five cases, whereas one case with a hepatoid component was positive for AFP. All three CAED cases with distant metastases were GPC3-positive. CONCLUSIONS: CAED was frequently located in the sigmoid colon or rectum, showed aggressive behaviour, such as lymph node metastasis and distant metastasis, and had a dismal prognosis. In addition, CAED was immunoreactive to AFP, GPC3 or SALL4, indicating that these markers may be characteristic of CAED.