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Background & Aims Germline variants in mismatch repair genes MLH1 , MSH2 (EPCAM), MSH6 , or PMS2 cause Lynch syndrome. Patients with these variants have an increased risk of developing colorectal cancers (CRCs) that differ from sporadic CRCs in genetic and histologic features. It has been a challenge to study CRCs associated with PMS2 variants (PMS2 -associated CRCs) because these develop less frequently and in older patients than CRCs with variants in other mismatch repair genes. Methods We analyzed 20 CRCs associated with germline variants in PMS2 , 22 sporadic CRCs, 18 CRCs with germline variants in MSH2 , and 24 CRCs from patients with germline variants in MLH1. Tumor tissue blocks were collected from Dutch pathology departments in 2017. After extraction of tumor DNA, we used a platform designed to detect approximately 3,000 somatic hotspot variants in 55 genes (including KRAS , APC , CTNNB1 , and TP53). Somatic variant frequencies were compared using the Fisher exact test. Results None of the PMS2 -associated CRCs contained any somatic variants in the catenin-β 1 gene (CTNNB1), which encodes β-catenin, whereas 14 of 24 MLH1 -associated CRCs (58%) contained variants in CTNNB1. Half the PMS2 -associated CRCs contained KRAS variants, but only 20% of these were in hotspots that encoded G12D or G13D. These hotspot variants occurred more frequently in CRCs associated with variants in MLH1 (37.5%; P =.44) and MSH2 (71.4%; P =.035) than in those associated with variants in PMS2. Conclusions In a genetic analysis of 84 colorectal tumors, we found tumors from patients with PMS2 -associated Lynch syndrome to be distinct from colorectal tumors associated with defects in other mismatch repair genes. This might account for differences in development and less frequent occurrence. [ABSTRACT FROM AUTHOR]

Background & Aims Statin use has been associated with a reduced incidence of colorectal cancer and might also affect survival of patients diagnosed with colon cancer. Statins are believed to inhibit Ras signaling and may also activate the bone morphogenetic protein (BMP) signaling pathway in colorectal cancer cells. We investigated the effects of statins on overall survival of patients with a diagnosis of colon cancer, and whether their effects were associated with changes in KRAS or the BMP signaling pathways. Methods Data were derived from the PHARMO database network (Netherlands) and linked to patients diagnosed with colon cancer from 2002 through 2007, listed in the Eindhoven Cancer Registry. We obtained information on causes of death from statistics Netherlands. We constructed a tissue microarray of 999 colon cancer specimens from patients who underwent surgical resection from 2002 through 2008. Survival was analyzed with statin user status after diagnosis as a time-dependent covariate. Multivariable Poisson regression survival models and Cox analyses were used to study the effect of statins on survival. Tumor tissues were analyzed by immunohistochemistry for levels of SMAD4, BMPR1A, BMPR1B, and BMPR2 proteins. Tumor tissues were considered to have intact BMP signaling if they contained SMAD4 plus BMPR1A, BMPR1B, or BMPR2. DNA was isolated from tumor tissues and analyzed by quantitative polymerase chain reaction to detect mutations in KRAS. The primary outcome measures were overall mortality and cancer-specific mortality. Results In this cohort, 21.0% of the patients (210/999) were defined as statin users after diagnosis of colon cancer. Statin use after diagnosis was significantly associated with reduced risk of death from any cause (adjusted relative risk [RR], 0.67; 95% confidence interval [CI], 0.51–0.87; P = .003) and death from cancer (adjusted RR, 0.66; 95% CI, 0.49–0.89; P = .007). Statin use after diagnosis was associated with reduced risk of death from any cause or from cancer for patients whose tumors had intact BMP signaling (adjusted RR, 0.39; 95% CI, 0.22–0.68; P = .001), but not for patients whose tumors did not have BMP signaling (adjusted RR, 0.81; 95% CI, 0.55–1.21; P = .106; P < .0001 for the interaction). Statin use after diagnosis was not associated with reduced risk of death from any cause or from cancer for patients whose tumors did not contain KRAS mutations (adjusted RR, 0.81; 95% CI, 0.56–1.18; P = .273) or whose tumors did have KRAS mutations (adjusted RR, 0.59; 95% CI 0.35–1.03; P = .062; P = .90 for the interaction). Conclusions In an analysis of 999 patients with a diagnosis of colon cancer, we associated statin with reduced risk of death from any cause or from cancer. The benefit of statin use is greater for patients whose tumors have intact BMP signaling, independent of KRAS mutation status. Randomized controlled trials are required to confirm these results. [ABSTRACT FROM AUTHOR]

We investigated the presence and patterns of mosaicism in the APC gene in patients with colon neoplasms not associated with any other genetic variants; we performed deep sequence analysis of APC in at least 2 adenomas or carcinomas per patient. We identified mosaic variants in APC in adenomas from 9 of the 18 patients with 21 to approximately 100 adenomas. Mosaic variants of APC were variably detected in leukocyte DNA and/or non-neoplastic intestinal mucosa of these patients. In a comprehensive sequence analysis of 1 patient, we found no evidence for mosaicism in APC in non-neoplastic intestinal mucosa. One patient was found to carry a mosaic c.4666dupA APC variant in only 10 of 16 adenomas, indicating the importance of screening 2 or more adenomas for genetic variants. [ABSTRACT FROM AUTHOR]

Background & Aims Most colorectal cancer (CRC) cells with high levels of microsatellite instability (MSI-H) accumulate mutations at a microsatellite sequence in the gene encoding transforming growth factor β receptor II ( TGFBR2 ). TGFβ signaling therefore is believed to be defective in these tumors, although CRC cells with TGFBR2 mutations have been reported to remain sensitive to TGFβ. We investigated how TGFβ signaling might continue in MSI-H CRC cells. Methods We sequenced the 10-adenines microsatellite sequence in the TGFBR2 gene of 32 MSI-H colon cancer tissues and 6 cell lines (HCT116, LS180, LS411N, RKO, SW48, and SW837). Activation of TGFβ signaling was detected by SMAD2 phosphorylation and through use of a TGFβ–responsive reporter construct in all CRC cell lines. Transcripts of TGFBR2 were knocked-down in CRC cells using short hairpin RNA. Full-length and mutant forms of TGFBR2 were expressed in LS411N cells, which do not respond to TGFβ, and their activities were measured. Results SMAD2 was phosphorylated in most MSI-H CRC tissues (strong detection in 44% and weak detection in 34% of MSI-H tumors). Phosphorylation of SMAD2 in MSI-H cells required TGFBR2 —even the form encoding a frameshift mutation. Transcription and translation of TGFBR2 with a 1-nucleotide deletion at its microsatellite sequence still produced a full-length TGFBR2 protein. However, protein expression required preservation of the TGFBR2 microsatellite sequence; cells in which this sequence was replaced with a synonymous nonmicrosatellite sequence did not produce functional TGFBR2 protein. Conclusion TGFβ signaling remains active in some MSI-H CRC cells despite the presence of frameshift mutations in the TGFBR2 gene because the mutated gene still expresses a functional protein. Strategies to reactivate TGFβ signaling in colorectal tumors might not be warranted, and the functional effects of mutations at other regions of microsatellite instability should be evaluated. [ABSTRACT FROM AUTHOR]

Background & Aims: SMAD4 frequently is lost from colorectal cancers (CRCs), which is associated with the development of metastases and a poor prognosis. SMAD4 loss is believed to alter transforming growth factor β signaling to promote tumor progression. However, SMAD4 is also a central component of the bone morphogenetic protein (BMP) signaling pathway, implicated in CRC pathogenesis by human genetic studies. We investigated the effects of alterations in BMP signaling on the invasive and metastatic abilities of CRC cells and changes in members in this pathway in human tumor samples. Methods: We activated BMP signaling in SMAD4-positive and SMAD4-negative CRC cells (HCT116, HT-29, SW480, and LS174T); SMAD4 was stably expressed or knocked down using lentiviral vectors. We investigated the effects on markers of epithelial–mesenchymal transition and on cell migration, invasion, and formation of invadopodia. We performed kinase activity assays to characterize SMAD4-independent BMP signaling and used an inhibitor screen to identify pathways that regulate CRC cell migration. We investigated the effects of the ROCK inhibitor Y-27632 in immunocompromised (CD-1 Nu) mice with orthotopic metastatic tumors. Immunohistochemistry was used to detect BMPR1a, BMPR1b, BMPR2, and SMAD4 in human colorectal tumors; these were related to patient survival times. Results: Activation of BMP signaling in SMAD4-negative cells altered protein and messenger RNA levels of markers of epithelial–mesenchymal transition and increased cell migration, invasion, and formation of invadopodia. Knockdown of the BMP receptor in SMAD4-negative cells reduced their invasive activity in vitro. SMAD4-independent BMP signaling activated Rho signaling via ROCK and LIM domain kinase (LIMK). Pharmacologic inhibition of ROCK reduced metastasis of colorectal xenograft tumors in mice. Loss of SMAD4 from colorectal tumors has been associated with reduced survival time; we found that this association is dependent on the expression of BMP receptors but not transforming growth factor β receptors. Conclusions: Loss of SMAD4 from colorectal cancer cells causes BMP signaling to switch from tumor suppressive to metastasis promoting. Concurrent loss of SMAD4 and normal expression of BMP receptors in colorectal tumors was associated with reduced survival times of patients. Reagents that interfere with SMAD4-independent BMP signaling, such as ROCK inhibitors, might be developed as therapeutics for CRC. [Copyright &y& Elsevier]

Since the approval of neurotrophic tropomyosin receptor kinase (NTRK) tyrosine kinase inhibitors for fist-line advanced stage pan-cancer therapy, pathologists and molecular biologists have been facing a complex question: how should the large volume of specimens be screened for NTRK fusions? Immunohistochemistry is fast and cheap, but the sensitivity compared to RNA NGS is unclear. We performed RNA-based next-generation sequencing on 1,329 cases and stained 24 NTRK -rearranged cases immunohistochemically with pan-TRK (ERP17341). Additionally, we performed a meta-analysis of the literature. After screening 580 studies, 200 additional NTRK -rearranged cases from 13 studies, analysed with sensitive molecular diagnostics as well as pan-TRK IHC, were included. In the included 224 NTRK-rearranged solid tumours, the sensitivity for pan-TRK IHC was 82% and the false-negative rate was 18%. NTRK3 fusions had more false negatives (27%) compared to NTRK1 (6%) and NTRK2 (14%) (p = 0.0006). Membranous, nuclear and peri-nuclear staining patterns strongly correlated with different fusion products, with membranous staining being more prevalent in NTRK1 and NTRK2 , nuclear in NTRK3 , and perinuclear in NTRK1. Despite a reduction in the number of molecular analysis, using pan-TRK immunohistochemistry as a prescreening method to detect NTRK fusions in solid tumours will miss 18% of all NTRK -fused cases (especially involving NTRK3). Therefore, the most comprehensive and optimal option to detect NTRK fusions is to perform molecular testing on all eligible cases. However, in case of financial or logistical limitations, an immunohistochemistry-first approach is defensible in tumours with a low prevalence of NTRK fusions. • Neurotrophic tropomyosin receptor kinase (NTRK) inhibitors were recently accepted as first-line pan-tumour treatment. • Laboratories worldwide are faced with the setup of an NTRK screening strategy. • This meta-analysis includes 7000 cases of which 224 harbour NTRK fusions. • Sensitivity of pan-TRK immunohistochemistry is 82%, missing 18% of NTRK fusions. • NTRK screening should be performed with molecular methods, if possible. [ABSTRACT FROM AUTHOR]

Expression of human leukocyte antigen-G (HLA-G) is a suggested mechanism used by tumor cells to escape from host immune recognition and destruction. Advances in the field have made it evident that HLA-G is expressed in different types of malignancies including colorectal cancer (CRC). We analyzed HLA-G expression in 21 low passage CRC cell lines. The level of DNA methylation of the HLA-G gene and the presence of mRNA encoding HLA-G was measured. Moreover, HLA-G protein expression was determined by flow cytometry and immunohistochemistry (IHC). IHC was performed with three different monoclonal antibodies (mAbs) (4H84, MEM-G/1 and MEM-G/2). In addition, HLA-G protein expression was measured in matching primary tumor tissues. RNA analysis using RT-PCR followed by sequencing in 6 samples indicated strong homology of the PCR product with HLA-G3 in 5 samples. In accordance, in none of the cell lines, HLA-G1 expression was detected by flow-cytometry. Furthermore, no association between HLA-G DNA methylation patterns and HLA-G mRNA expression was observed. In addition, different immunohistochemical staining profiles among various anti-HLA-G mAbs were observed. In conclusion, the results of this study show that the HLA-G3 isoform was expressed in some of the CRC cell lines irrespective of the level of DNA methylation of HLA-G . [ABSTRACT FROM AUTHOR]

Primary cutaneous follicle center lymphoma (PCFCL) is characterized by lesions on the trunk and head-and-neck region consisting of centrocytes and admixed centroblasts. In general, PCFCL has an indolent behaviour with a 5-year disease-specific survival of 95%. Local radiotherapy results in a complete remission in 99% of the patients, however, cutaneous relapses are frequent (30%) and 10% of the patients develop progressive disease with extracutaneous dissemination or large-cell transformation. As it is difficult to identify these patients, the aim of this study was to identify 'clues' in clinical presentation, histopathology, immune phenotype, and genetic profile of PCFCL patients at time of diagnosis that may help to identify patients at risk for disease progression. In total, 15 patients with progressive PCFCL and 15 patients with indolent PCFCL (without disease progression and a follow-up duration of at least 5 years) were selected from the Dutch Registry of Cutaneous Lymphoma. Clinical presentation and histopathology were reviewed and immune phenotyping and molecular analysis, including targeted next-generation sequencing (NGS) with a 200 B-cell lymphoma-related gene panel, were performed. At diagnosis, all patients presented with classic PCFCL lesions on the trunk or head-and-neck region. Histology showed a characteristic predominance of centrocytes with admixed centroblasts. The median follow-up duration was 65 (range, 30 to 193) months with a progression-free survival of 26 (range, 4 to 147) months, during which five patients died of progressive PCFCL and one patient died of cardiac disease. In patients with progressive disease, IgM was significantly more often expressed compared with patients with indolent disease (53% vs. 7%; p=0.005). Targeted NGS demonstrated one or multiple PCFCL-like mutations in TNFRSF14, IRF8, CREBBP, EP300, EZH2, SETD2, SOCS1, STAT6 in 6 of 11 (55%) patients with indolent disease and in 9 of 12 (75%) patients with progressive disease. In 7 of 9 of these patients with progressive disease, however, additional non-PCFCL-like alterations were detected that were absent in patients with indolent disease, including MYC rearrangements in two patients and ERBB4, ZEB2, ARID1A, and ITBKB mutations in four patients as well as MYD88, CD79B, PIM1, and TBL1XR1 mutations in one of these patients. In three patients with progressive disease but without a PCFCL-like profile, we detected mutations in MYD88, PIM1, and TBL1XR1 in one patient, a MYD88 non-L265P mutation amongst others in one patient, and mutations in BRAF and CIITA in one patient, of which CIITA was also detected in one of the indolent patients. In conclusion, expression of IgM at diagnosis might represent a clue for a progressive disease course in PCFCL and may warrant additional genetic analysis and closer clinical follow-up of these patients. As this observation was made on a limited number of patients, these results need validation in an independent cohort. [ABSTRACT FROM AUTHOR]