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Background and Aims: In patients who have undergone surgery for colorectal cancer (CRC), 3% have recurrence of (metachronous) CRC. We investigated whether tumor seeding during colonoscopy (iatrogenic implantation of tumor cells in damaged mucosa) increases risk for metachronous CRC., Methods: In a proof of principle study, we collected data from the Dutch National Pathology Registry for patients with a diagnosis of CRC from 2013 through 2015, with a second diagnosis of CRC within 6 months to 3.5 years after surgery. We reviewed pathology reports to identify likely metachronous CRC (histologically proven adenocarcinoma located elsewhere in the colon or rectum from the surgical anastomosis). For 22 patients fulfilling the inclusion criteria, we ascribed the most likely etiology to tumor seeding when endoscopic manipulations, such as biopsies or polypectomy, occurred at the location where the metachronous tumor was subsequently detected, after endoscopic manipulation of the primary tumor. We collected clinical data from patients and compared molecular profiles of the primary and metachronous colorectal tumors using next-generation sequencing. We then examined the source of seeded tumor. We tested whether tumor cells stay behind in the working channel of the endoscope after biopsies of colorectal tumors, and whether these cells maintain viability in organoid cultures., Results: In total, tumor seeding was suspected as the most likely etiology of metachronous CRC in 5 patients. Tumor tissues were available from 3 patients. An identical molecular signature was observed in the primary and metachronous colorectal tumors from all 3 patients. In 5 control cases with a different etiology of metachronous CRC, the molecular signature of the primary and metachronous tumor were completely different. Based on review of 2147 patient records, we estimated the risk of tumor seeding during colonoscopy to be 0.3%-0.6%. We demonstrated that the working channel of the colonoscope becomes contaminated with viable tumor cells during biopsy collection. Subsequent instruments introduced through this working channel also became contaminated. These cells were shown to maintain their proliferative potential., Conclusions: In an analysis of primary and secondary tumors from patients with metachronous CRC, we found that primary tumor cells might be seeded in a new location after biopsy of the primary tumor. Although our study does not eliminate other possibilities of transmission, our findings and experiments support the hypothesis that tumor seeding can occur during colonoscopy via the working channel of the endoscope. The possibility of iatrogenic seeding seems low. However, our findings compel awareness on this potentially preventable cause of metachronous CRC., (Copyright © 2019 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Germline mutations in CDKN2A result in Familial Atypical Multiple Mole Melanoma Syndrome (FAMMM), which is associated with an increased risk for pancreatic ductal adenocarcinoma and melanoma. CDKN2A is somatically inactivated in multiple neoplasms, raising the possibility that, although the data are not conclusive, germline CDKN2A mutation may also impose an increased risk for other neoplasms. We present a patient with a CDKN2A germline mutation (p16-Leiden mutation) and mosaicism for neurofibromatosis type 2, who presented with a small asymptomatic pancreatic lesion, detected during endoscopic ultrasound screening of the pancreas. After resection, the lesion was found to be a well-differentiated pancreatic neuroendocrine tumor (PanNET). Molecular analysis of the tumor showed somatic loss of the second allele, supporting a causal relation of the PanNET to the underlying FAMMM syndrome. Recent data, showing the association between certain single-nucleotide polymorphisms in the CDKN2A gene and an increased incidence for PanNET, further support a role for germline CDKN2A alterations in PanNET risk. We conclude that PanNETs can be a phenotypic expression of FAMMM syndrome. This can have implications for screening and for the diagnosis of pancreatic neoplasms in carriers of germline CDKN2A mutations.

Based on histological findings, calcifying fibrous tumor (CFT) may be a late (burned out) stage of inflammatory myofibroblastic tumor (IMT). This concept, however, has not been proven by molecular means. Five CFTs were analyzed for IMT-related rearrangements in ALK, ROS1 and RET using fluorescence in situ hybridization (FISH). Additionally, genome-wide methylation patterns were investigated and compared with IMT (n = 7), leiomyoma (n = 7), angioleiomyoma (n = 9), myopericytoma (n = 7) and reactive soft tissue lesions (n = 10) using unsupervised hierarchical cluster analysis and t distributed stochastic neighbor embedding. CFT patients, 4 females and 1 male, had a median age of 20 years ranging from 7 to 43 years. Two patients were younger than 18 years old. The tumors originated in the abdomen (n = 4) and axilla (n = 1). Histologically, all lesions were (multi) nodular and hypocellular consisting of bland looking (myo)fibroblasts embedded in a collagenous matrix with calcifications. FISH analysis brought up negative results for ALK, RET and ROS1 rearrangements. However, genome-wide methylation analysis revealed overlapping methylation patterns of CFT and IMT forming a distinct homogeneous methylation cluster with exception of one case clustering with myopericytoma/angioleiomyoma. In conclusion, DNA methylation profiling supports the concept that CFT and IMT represent both ends of a spectrum of one entity with CFT being the burn out stage of IMT., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Next-generation sequencing (NGS) is rapidly becoming the method of choice for mutation analysis in both research and diagnostics. The benefit of targeted NGS compared to whole-genome and whole-exome sequencing is that smaller amounts of input material can be used as well as qualitatively suboptimal tissue samples, like formalin-fixed, paraffin-embedded archival tissue.Here, we describe the protocol for targeted next-generation sequencing using the Ion Torrent PGM platform in combination with Ion Ampliseq NGS gene panels for formalin-fixed, paraffin-embedded tissues. Both the manual and the automated workflow are described as well as the bioinformatics for data analysis.

Acinar cell carcinoma (ACC) is a rare pancreatic neoplasm with dismal prognosis. Insights into the molecular basis of ACC can pave the way for the application of more effective, personalized therapies and detection of patients with hereditary predisposition. Molecular analysis revealed a germline BRCA2 (and CHEK2 ) mutation in a patient with a rare pancreatic ACC with extensive intraductal growth. Somatic loss of the wild-type BRCA2 allele in the tumor indicated the causal relationship of ACC with the germline defect. A thorough literature review identified another nine ACCs associated with germline BRCA2 mutation and two ACCs associated with germline BRCA1 mutation, resulting in a prevalence of BRCA1/2 germline mutations in almost 7% of ACCs. Moreover, somatic BRCA1/2 alterations are reported in 16% of sporadic ACCs. Overall, about one fifth (22%) of all pancreatic ACCs exhibit BRCA1/2 deficiency. This study underscores the important role of BRCA1/2 mutations in pancreatic ACC. All ACC patients should undergo genetic testing for BRCA1/2 mutations to identify carriers of pathogenic variants. This will allow to select patients that can benefit from targeted therapies directed against BRCA1/2 -deficient tumors and is also crucial as a referral to genetic screening for the relatives of affected individuals carrying germline BRCA1/2 alterations. Abbreviations: ACC: acinar cell carcinoma; HBOC: Hereditary Breast and Ovarian Cancer; LOH: loss of heterozygosity; PARP: poly (ADP-ribose) polymerase; PDAC: pancreatic ductal adenocarcinoma; PP: pancreatic panniculitis; SD: standard deviation; WES: whole-exome sequencing.

Desmoid-type fibromatosis, also called desmoid tumor, is a locally aggressive myofibroblastic neoplasm that usually arises in deep soft tissue with significant potential for local recurrence. It displays an unpredictable clinical course. β-Catenin, the genetic key player of desmoid tumors shows nuclear accumulation due to mutations that prevent its degradation leading to activation of Wnt signaling and myofibroblastic cell proliferation. The corresponding hot spot mutations are located in exon 3 of the CTNNB1 gene or alternatively, in the APC tumor suppressor gene, most often as a germline mutation. Multifocal desmoid tumors are very rare and clinical characteristics are poorly understood. Here we present six sporadic and one familial case of multifocal desmoid tumors. Four female and three male patients, aged between 7 and 30 years (mean 18.4 years) were identified in a cohort of 1392 cases. Tumors were located in (distal) extremities, thorax, breast, abdominal wall, shoulder, and neck. Four cases showed a CTNNB1 mutation and one an APC germline mutation. In two sporadic cases no CTNNB1 mutation was identified. Four patients showed (multiple) recurrences and one patient was lost to follow-up. In conclusion, multifocal desmoid tumors are a very rare disease and may occur in sporadic cases that are characterized by recurrent CTNNB1 mutations. However, the underlying pathogenesis of multifocal desmoid tumors remains poorly understood with often aggressive clinical behavior and challenging therapeutical management., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Colorectal cancer (CRC) accounts for over 8% of all deaths annually worldwide. Between 2 and 5% of all CRCs occur due to inherited syndromes, including Lynch syndrome, familial adenomatous polyposis, MUTYH-associated polyposis, Peutz-Jeghers syndrome, juvenile polyposis and Cowden/PTEN hamartoma syndrome. In addition, serrated polyposis is a clinically defined condition characterised by multiple colorectal serrated polyps and an increased risk of CRC but the genetics are not known. In most hereditary CRC syndromes, polyps undergo carcinogenesis, but the exact route to carcinoma seems to differ between the conditions. Discovery of the key germline mutations in these syndromes has been instrumental to our understanding of the underlying molecular mechanisms of colorectal carcinogenesis. This review summarises the genetic and pathological alterations in hereditary CRC syndromes., (Copyright © 2017 Royal College of Pathologists of Australasia. Published by Elsevier B.V. All rights reserved.)

Head and neck squamous cell carcinomas (HNSCC) form a large heterogeneous group of tumors and have a relatively poor outcome in advanced cases. Revealing the underlying genetic mutations in HNSCC facilitates the development of diagnostic biomarkers, which might lead to improved diagnosis and post treatment surveillance. We retrospectively analyzed mutational hotspots using targeted next-generation sequencing (NGS) of 239 HNSCC tumor samples in order to examine the mutational profile of HNSCC. Furthermore, we assessed prevalence, co-occurrence, and synonymy of gene mutations in (matched) tumor samples. TP53 was found mutated the most frequent with mutation rates of up to 83% in all tumors, compared to mutation rates of between 0 and 21% of CDKN2A, PIK3CA, HRAS, CDK4, FBXW7 and RB1. Mutational co-occurrence predominantly existed between TP53 and PIK3CA, TP53 and CDKN2A, and HRAS and PIK3CA. Mutational synonymy between primary tumor and associated metastasis and recurrence was present in respectively 88% and 89%. TP53 mutations were concordantly mutated in 95% of metastases and in 91% of recurrences. This indicates TP53 mutations to be highly prevalent and concordant in primary tumors and associated locoregional metastases and recurrences. In turn, this provides ground for further investigating the use of TP53 mutations as diagnostic biomarkers in HNSCC patients., Competing Interests: The authors declare no conflicts of interest.

Hereditary diffuse gastric cancer is an autosomal dominant cancer syndrome characterized by highly penetrant diffuse gastric cancer. It is caused by germ line mutations in CDH1, encoding the cell-cell adhesion protein E-cadherin. Pancreatic ductal adenocarcinoma is one of the most dismal malignancies in humans. Although absent E-cadherin expression in pancreatic ductal adenocarcinoma is related to a higher tumor grade and a worse prognosis, there have been no reports of pancreatic ductal adenocarcinoma associated with hereditary diffuse gastric cancer. Here, we describe a patient with hereditary diffuse gastric cancer who was subsequently diagnosed with pancreatic ductal adenocarcinoma. To investigate if the previously identified CDH1 germ line mutation initiated pancreatic ductal adenocarcinoma development, we performed mutational and proteomic analyses. We conclude that the pancreatic ductal adenocarcinoma did not occur in the context of the germ line CDH1 mutation but rather appeared as a sporadic event. Immunohistochemistry ultimately proved to be the most valuable tool of investigation as persistent CDH1 staining in the pancreatic ductal adenocarcinoma unequivocally revealed E-cadherin expression., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: Juvenile polyps are distinct hamartomatous malformations of the gastrointestinal tract that may occur in the heritable juvenile polyposis syndrome (JPS) or sporadically. Histologically, juvenile polyps are characterized by a marked increase of the stromal cell compartment, but an epithelial phenotype has also been reported. JPS has an increased risk of colorectal cancer but sporadic juvenile polyps do not. In 50% to 60% of patients with JPS, a germline mutation of the transforming growth factor-β/bone morphogenetic protein (BMP) pathway genes SMAD4 or BMPR1A is found. This study compares the histologic phenotype of juvenile polyps with a SMAD4 or BMPR1A germline mutation and sporadic juvenile polyps., Methods: Hematoxylin and Eosin-stained slides of 65 JPS polyps and 25 sporadic juvenile polyps were reviewed for histologic features and dysplasia. Systematic random crypt and stroma counts were obtained by count stereology, and a crypt-stroma ratio was determined. All polyps were subsequently categorized as type A (crypt-stroma ratio <1.00) or type B (crypt-stroma ratio ≥1.00), the latter referring to the epithelial phenotype. Cell cycle activity was assessed using immunohistochemistry ofthe proliferation marker Ki67, and mutation analysis was carried out for KRAS and APC to determine the involvement of the adenoma-carcinoma sequence., Results: Juvenile polyps with a SMAD4 germline mutation were predominantly type B, whereas type A was more common among juvenile polyps with a BMPR1A germline mutation. However, this distinction could not be ascribed to differences in cell cycle activity. Dysplasia was equally common in JPS polyps with either a SMAD4 or BMPR1A germline mutation, in which the involvement of the adenoma-carcinoma sequence does not seem to play a distinct role., Conclusion: Juvenile polyps in the setting of JPS exhibit distinct phenotypes correlating with the underlying genetic defect.

Purpose: Juvenile polyposis syndrome (JPS) can be caused by a germline defect of the SMAD4 gene. Somatic inactivation of SMAD4 occurs in pancreatic and colorectal cancers and is reflected by loss of SMAD4 immunohistochemistry. Here, SMAD4 immunohistochemistry as a marker of SMAD4 gene status and the role of SMAD4 in the adenoma-carcinoma sequence in neoplastic progression in JPS are studied., Experimental Design: Twenty polyps with a SMAD4 germline defect and 38 control polyps were studied by SMAD4 immunohistochemistry. Inactivation of the SMAD4 wild-type allele was studied in dysplastic epithelium and in areas with aberrant SMAD4 expression. APC, beta-catenin, p53, and K-ras were studied to evaluate the adenoma-carcinoma sequence., Results: Nine of 20 polyps with a SMAD4 germline defect showed loss of epithelial SMAD4 expression. Loss of heterozygosity of SMAD4 was found in five polyps and a somatic stop codon mutation was found in two polyps without loss of heterozygosity. Remarkably, somatic inactivation of epithelial SMAD4 did not always coincide with dysplasia and aberrant p53 staining was found in four of six dysplastic polyps with normal SMAD4 staining. One K-ras mutation was found in nine juvenile polyps with dysplasia. No evidence for Wnt activation was found., Conclusions: SMAD4 immunohistochemistry mirrors genetic status and provides a specific adjunct in the molecular diagnosis of JPS. However, epithelial SMAD4 inactivation is not required for polyp formation and is not obligatory for neoplastic progression in JPS. Instead, different routes to neoplasia in JPS caused by germline SMAD4 mutation seem to be operative, including somatic loss of SMAD4 and p53 inactivation without somatic loss of SMAD4.

Colorectal cancer is one of the leading causes of cancer-related death in the Western society, and the incidence is rising. Rare hereditary gastrointestinal polyposis syndromes that predispose to colorectal cancer have provided a model for the investigation of cancer initiation and progression in the general population. Many insights in the molecular genetic basis of cancer have emerged from the study of these syndromes. This review discusses the genetics and clinical manifestations of the three most common syndromes with gastrointestinal polyposis and an increased risk of colorectal cancer: familial adenomatous polyposis (FAP), juvenile polyposis (JP) and Peutz-Jeghers syndrome (PJS).

Purpose: Peutz-Jeghers syndrome (PJS) is a hamartomatous polyposis disorder with a high cancer risk. Debate exists about the premalignant potential of hamartomas. Also, treatment options other than surveillance are not available. Therefore, molecular alterations in hamartomas and PJS carcinomas were studied. The objective was (a) to evaluate expression of cyclooxygenase (COX)-2 as target for chemopreventive treatment and (b) to define the neoplastic potential of hamartomas at the molecular level., Experimental Design: Paraffin-embedded samples of 24 PJS hamartomas, including 2 hamartomas with dysplastic changes, and 11 PJS carcinomas were available. Slides were stained with antibodies against COX-2, beta-catenin, cyclin D1, p21(waf1/cip1), Ki-67, and p53. DNA was studied for loss of heterozygosity (LOH) at 19p (STK11), 5q (APC), and 17p (TP53); mutations in beta-catenin, APC, and K-RAS; and microsatellite instability., Results: Moderate or strong epithelial COX-2 was present in 25% of hamartomas, including two hamartomas with dysplastic changes, and 64% of carcinomas. Several hamartomas showed focal nuclear beta-catenin (18%) and cyclin D1 overexpression (29%), both unrelated to dysplasia at histological examination. Disturbed topographical expression of Ki-67 in relation to p21(waf1/cip1) was focally present in 27% of hamartomas, including those with dysplastic changes. Most carcinomas showed nuclear beta-catenin (71%), cyclin D1 overexpression (71%), and aberrant Ki-67 staining (100%). There was LOH at 19p in 32% of hamartomas and 82% of carcinomas. p53 staining was present in four (36%) carcinomas, one of which showed LOH at 17p. No beta-catenin mutations were found. APC mutations were present in two carcinomas, but LOH at 5q was not found. Two carcinomas had K-RAS mutations, and one carcinoma had microsatellite instability., Conclusions: The presence of COX-2 expression in PJS carcinomas and dysplastic hamartomas provides a rationale for chemoprevention with nonsteroidal anti-inflammatory drugs or COX-2 inhibitors. Focal immunohistochemical changes, which may indicate a premalignant potential, were present in some nondysplastic PJS hamartomas. Molecular changes in carcinomas and dysplastic hamartomas in PJS are distinct from the usual adenoma-carcinoma sequence.