HPV-negative Squamous Cell Carcinomas of the Cervix With Special Focus on Intraepithelial Precursor Lesions.
Recently, the World Health Organization (WHO) recognized human papilloma virus (HPV)-independent invasive cervical squamous cell carcinoma (SCC) without recognizing the existence of precursor lesions. This is a detailed characterization of 3 preinvasive lesions and 6 invasive SCC negative for HPV-DNA (32 genotypes), HPV-mRNA (14 genotypes) and genomic HPV sequencing. We evaluated histologic features, expression of p16ink4a, p53, CK7, and CK17, aberrations in 50 cancer genes and chromosomal copy number variations. HPV-negative preinvasive lesions were extensive basaloid or highly differentiated keratinizing intraepithelial proliferations of 3 to 20 cell layers thickness, partly with prominent cervical gland involvement. Overall, 2/3 intraepithelial lesions and the in situ component of 1/6 SCC showed p16ink4a block staining, while 1/6 in situ component revealed heterogenous p16ink4a staining. All invasive components of keratinizing SCC were p16ink4a-negative. Preinvasive and invasive SCC showed inconsistent CK7 and CK17 staining. Nuclear p53 overexpression was restricted to the TP53 gene mutated SCC. The highly vascularized peritumoral stroma showed a dense inflammatory infiltrate including plasma cells and intratumoral and peritumoral eosinophilic granulocytes. Inconsistent somatic gene mutations (PIK3CA, STK11, TP53, SMARC2B, and GNAS) occurred predominantly in nonhotspot locations at low mutational frequency in 3/6 SCC. Consistent aberrations included the pathogenic (angiogenic) germline polymorphism Q472H in the KDR gene (7/9 patients), and chromosome 3q gains (4/9 patients). In conclusion, HPV-negative intraepithelial cervical precancerous lesions exist, either as highly differentiated keratinized intraepithelial proliferations reminiscent of differentiated vulvar intraepithelial neoplasia, or undifferentiated basaloid intraepithelial lesions with occasional p16ink4a block staining resembling high-grade squamous intraepithelial lesion. Gains of chromosome 3q, angiogenic germline variants the inflammatory infiltrate may contribute to progression of HPV-negative cervical carcinogenesis.
Publication Date: 2021-08-14
Journal: The American journal of surgical pathology
High Prevalence of DICER1 Mutations and Low Frequency of Gene Fusions in Pediatric Follicular-Patterned Tumors of the Thyroid.
Follicular-patterned tumors of the thyroid in the adult population frequently harbor RAS mutations or PAX8-PPARG rearrangement, but little is known about molecular profiles in the pediatric patients with thyroid tumors, which is rare. To identify the molecular profile of pediatric follicular-patterned tumors, we enrolled 41 pediatric patients with follicular-patterned tumors from two institutions. We did next-generation sequencing using a mutation panel targeting 49 thyroid-tumor-related genes and a fusion panel targeting 88 types of thyroid-related gene fusions. We identified nonsynonymous mutations in at least one target gene in most of the tumors (28/41, 68%). Somatic DICER1 mutations (22%, n = 9) were the most common genetic alteration, followed by mutations of NRAS (15%), FGFR3 (15%), PTEN (12%), and STK11 (10%). Infrequent genetic alterations (≤ 5% of all cases) included mutations of HRAS, APC, TSHR, CTNNB1, TP53, EIF1AX, FGFR4, GNAS, RET, and SOS1, and gene fusion of THADA-IGF2BP3. DICER1 and RAS mutations were mutually exclusive. No patients had tumors related to the DICER1 syndrome or the Cowden syndrome. There was no significant difference in total mutation burden or distribution between follicular adenoma and follicular carcinoma. In the literature, the DICER1 mutation has been reported in 20 to 53% of pediatric patients with follicular-patterned tumors. In conclusion, our study reinforces the role of the DICER1 mutation in the development of pediatric thyroid tumors. Gene fusions rarely occur in pediatric follicular-patterned tumors. Mutation or gene fusion alone could not distinguish benign from malignant follicular-patterned tumors in pediatric patients.
Publication Date: 2021-07-28
Journal: Endocrine pathology
Early detection of pancreatic cancer using DNA-based molecular approaches.
Due to its poor prognosis and the late stage at which it is typically diagnosed, early detection of pancreatic cancer is a pressing clinical problem. Advances in genomic analysis of human pancreatic tissue and other biospecimens such as pancreatic cyst fluid, pancreatic juice and blood have opened the possibility of DNA-based molecular approaches for early detection of pancreatic cancer. In this Review, we discuss and focus on the pathological and molecular features of precancerous lesions of the pancreas, including pancreatic intraepithelial neoplasia, intraductal papillary mucinous neoplasm and mucinous cystic neoplasm, which are target lesions of early detection approaches. We also discuss the most prevalent genetic alterations in these precancerous lesions, including somatic mutations in the oncogenes KRAS and GNAS as well as tumour suppressor genes CDKN2A, TP53 and SMAD4. We highlight the latest discoveries related to genetic heterogeneity and multifocal neoplasia in precancerous lesions. In addition, we review specific approaches, challenges and clinically available assays for early detection of pancreatic cancer using DNA-based molecular techniques. Although detection and risk stratification of precancerous pancreatic neoplasms are difficult problems, progress in this field highlights the promise of molecular approaches for improving survival of patients with this disease.
Publication Date: 2021-06-09
Journal: Nature reviews. Gastroenterology & hepatology
Liquid biopsy-based analysis by ddPCR and CAPP-Seq in melanoma patients.
The development of BRAF/MEK inhibitors in patients with metastatic melanoma harboring BRAF mutations has garnered attention for liquid biopsy to detect BRAF mutations in cell-free DNA (cfDNA) using droplet digital PCR (ddPCR) or next-generation sequencing methods.
To investigate gene mutations in tumor DNA and cfDNA collected from 43 melanoma patients and evaluate their potential as biomarkers.
ddPCR and CAncer Personalized Profiling by deep Sequencing (CAPP-Seq) techniques were performed to detect gene mutations in plasma cfDNA obtained from patients with metastatic melanoma.
Gene variants, including BRAF, NRAS, TP53, GNAS, and MET, were detectable in the plasma cfDNA, and the results were partially consistent with the results of those identified in the tissues. Among the variants examined, copy numbers of MET mutations were consistent with the disease status in two melanoma patients.
Liquid biopsy using CAPP-Seq and ddPCR has the potential to detect tumor presence and mutations, especially when tissue biopsies are unavailable. MET mutations in cfDNA may be a potential biomarker in patients with metastatic melanoma.
Publication Date: 2021-05-30
Journal: Journal of dermatological science
Serine/Threonine Kinase 11 Plays a Canonical Role in Malignant Progression of KRAS-mutant and GNAS-wild-type Intraductal Papillary Mucinous Neoplasms of the Pancreas.
We aimed to elucidate the clinicopathobiological significance of Serine/Threonine Kinase 11 (STK11) in pancreatic intraductal papillary mucinous neoplasms (IPMNs).
STK11 is a tumor suppressor involved in certain IPMNs, however, its significance is not well known.
In 184 IPMNs without Peutz-Jeghers syndrome, we analyzed expression of STK11 and phosphorylated-AMPKα in all cases, and p16, p53, SMAD4, and β-catenin in 140 cases by immunohistochemistry; and we analyzed mutations in 37 genes, including whole coding exons of STK11, CDKN2A, TP53, and SMAD4, and hotspots of KRAS, BRAF, and GNAS in 64 cases by targeted sequencing. KRAS and GNAS were additionally analyzed in 86 STK11-normal IPMNs using digital-PCR.
Consistent loss or reduction of STK11 expression was observed in 26/184 (14%) IPMNs. These STK11-aberrant IPMNs were 17/45 (38%) pancreatobiliary, 8/27 (30%) oncocytic, 1/54 (2%) gastric, and 0/58 (0%) intestinal subtypes (P = 8.5E-11), and 20/66 (30%) invasive, 6/74 (8%) high-grade, and 0/44 (0%) low-grade (P = 3.9E-06). Sixteen somatic STK11 mutations (5 frameshift, 6 nonsense, 1 splicing, and 4 missense) were detected in 15/26 STK11-aberrant IPMNs (P = 4.1E-06). All STK11-aberrant IPMNs were GNAS-wild-type and 96% of them were KRAS or BRAF-mutant. Morphologically, STK11-aberrant IPMNs presented "fern-like" arborizing papillae with thin fibrovascular core. Phosphorylated-AMPKα was downregulated in STK11-aberrant IPMNs (92%, P = 6.8E-11). Patients with STK11-aberrant IPMNs showed poorer survival than patients with STK11-normal IPMNs (P = 3.6E-04 overall; P = 6.1E-04 disease-free).
STK11 may play a canonical role in malignant progression and poor survival of patients with IPMNs. Aberrant STK11-driven phosphorylated AMPK downregulation may provide therapeutic opportunities with mTOR inhibitors/AMPK activators.
Publication Date: 2021-04-30
Journal: Annals of surgery
KRAS/GNAS-testing by highly sensitive deep targeted next generation sequencing improves the endoscopic ultrasound-guided workup of suspected mucinous neoplasms of the pancreas.
Pancreatic cysts or dilated pancreatic ducts are often found by cross-sectional imaging, but only mucinous lesions can become malignant. Therefore, distinction between mucinous and non-mucinous lesions is crucial for adequate patient management. We performed a prospective study including targeted next generation sequencing (NGS) of cell-free DNA in the diagnostic endoscopic ultrasound (EUS)-guided workup. Pancreatic cyst(s) or main duct fluid obtained by EUS-guided FNA was analysed by carcinoembryonic antigen (CEA), cytology and deep targeted NGS of 14 known gastrointestinal cancer genes (AKT1, BRAF, CTNNB1, EGFR, ERBB2, FBXW7, GNAS, KRAS, MAP2K1, NRAS, PIK3CA, SMAD4, TP53, APC) with a limit of detection down to variant allele frequency of 0.01%. Results were correlated to histopathology and clinical follow-up. One hundred and thirteen patients with pancreatic cyst(s) and/or a dilated pancreatic main duct (≥5 mm) were screened. Sixty-six patients had to be excluded, mainly due to inoperability or small cyst size (≤10 mm). Forty-seven patients were enrolled for further analysis. A final diagnosis was available in 27 cases including 8 negative controls. In 43/47 (91.5%) of patients a KRAS- and/or GNAS-mutation was diagnosed by NGS. 27.0% of the KRAS-mutated and 10.0% of the GNAS-mutated lesions harbored multiple mutations. KRAS/GNAS-testing by NGS, cytology, and CEA had a sensitivity and specificity of 94.7/100%, 38.1/100%, and 42.1/75.0%, respectively. KRAS/GNAS-testing was significantly superior to CEA (P = .0209) and cytology (P = .0016). In conclusion, KRAS/GNAS-testing by deep targeted NGS is a suitable method to distinguish mucinous from non-mucinous pancreatic lesions, suggesting its usage as a single diagnostic test. Results must be confirmed in a larger cohort.
Publication Date: 2021-03-10
Journal: Genes, chromosomes & cancer
High prevalence of clonal hematopoiesis-type genomic abnormalities in cell-free DNA in invasive gliomas after treatment.
Plasma cell-free DNA (cfDNA) is emerging as an important diagnostic tool in cancer. However, cfDNA alterations may differ from those in tissue and sometimes may reflect processes unrelated to the cancer, including clonal hematopoiesis (CH). We examined plasma cfDNA, tested by next-generation sequencing (NGS), for characterized alterations (excluding variants of unknown significance) in 135 patients with invasive glioma. Overall, 21% (28/135) had ≥1 alteration; 17% (23/135) had CH-type cfDNA mutations. Temozolomide (a mutagenic alkylating agent) with concurrent radiation therapy prior to blood draw was significantly associated with an increase in CH-type mutations, even after age, race/ethnicity, and WHO-grade were considered as confounders (odds ratio [95% confidence interval, CI] 8.98 [1.13-71.46]; P = .04; multivariable analysis). Further, of 18 patients with invasive glioma who had both cfDNA and tissue DNA NGS and had ≥1 cfDNA alteration, 16 (89%) had ≥1 cfDNA alteration not found in their tissue DNA, including CH-type alterations in genes such as TP53 (most common), ATM, GNAS, and JAK2. Altogether, 87% of cfDNA alterations (20/23) observed in the 18 patients were implicated in CH. Finally, examining all 135 patients, CH-type cfDNA mutations were an independent prognostic factor for shorter survival (hazard ratio [95% CI] 3.28 [1.28-8.40]; P = .01). These findings emphasize that not all characterized cfDNA alterations detected in patients with solid tumors are cancer-related. Importantly, in patients with invasive gliomas who have had prior temozolomide and radiation, CH-related alterations in cfDNA are frequent and correlate with poor outcomes.
Publication Date: 2021-01-27
Journal: International journal of cancer
Mutation profile of non-small cell lung cancer revealed by next generation sequencing.
Precision therapy for lung cancer requires comprehensive genomic analyses. Specific effects of targeted therapies have been reported in Asia populations, including Taiwanese, but genomic studies have rarely been performed in these populations.
We enrolled 72 patients with non-small cell lung cancer, of whom 61 had adenocarcinoma, 10 had squamous cell carcinoma, and 1 had combined adenocarcinoma and squamous cell carcinoma. Whole-exome or targeted gene sequencing was performed. To identify trunk mutations, we performed whole-exome sequencing in two tumor regions in four patients.
Nineteen known driver mutations in EGFR, PIK3CA, KRAS, CTNNB1, and MET were identified in 34 of the 72 tumors evaluated (47.22%). A comparison with the Cancer Genome Atlas dataset showed that EGFR was mutated at a much higher frequency in our cohort than in Caucasians, whereas KRAS and TP53 mutations were found in only 5.56% and 25% of our Taiwanese patients, respectively. We also identified new mutations in ARID1A, ARID2, CDK12, CHEK2, GNAS, H3F3A, KDM6A, KMT2C, NOTCH1, RB1, RBM10, RUNX1, SETD2, SF3B1, SMARCA4, THRAP3, TP53, and ZMYM2. Moreover, all ClinVar pathogenic variants were trunk mutations present in two regions of a tumor. RNA sequencing revealed that the trunk or branch genes were expressed at similar levels among different tumor regions.
We identified novel variants potentially associated with lung cancer tumorigenesis. The specific mutation pattern in Taiwanese patients with non-small cell lung cancer may influence targeted therapies.
Publication Date: 2021-01-08
Journal: Respiratory research
Spectrum of Somatic Cancer Gene Variations Among Adults With Appendiceal Cancer by Age at Disease Onset.
The incidence of appendiceal cancer (AC) is rising, particularly among individuals younger than 50 years (early-onset AC), with unexplained etiologies. The unique spectrum of somatic cancer gene variations among patients with early-onset AC is largely undetermined.
To characterize the frequency of somatic variations and genomic patterns among patients with early-onset (age <50 years) vs late-onset (age ≥50 years) AC.
This cohort study included individuals aged 18 years and older diagnosed with pathologically verified AC. Cases with clinical-grade targeted sequencing data from January 1, 2011, to December 31, 2019, were identified from the international clinicogenomic data-sharing consortium American Association for Cancer Research Project Genomics Evidence Neoplasia Information Exchange (GENIE). Data analysis was conducted from May to September 2020.
Age at disease onset.
Somatic variation prevalence and spectrum in AC patients was determined. Variation comparisons between early-onset and late-onset AC were evaluated using multivariable logistic regression with adjustment for sex, race/ethnicity, histological subtype, sequencing center, and sample type.
In total 385 individuals (mean [SD] age at diagnosis, 56.0 [12.4] years; 187 [48.6%] men; 306 [79.5%] non-Hispanic White individuals) with AC were included in this study, and 109 patients (28.3%) were diagnosed with early-onset AC. Race/ethnicity differed by age at disease onset; non-Hispanic Black individuals accounted for a larger proportion of early-onset vs late-onset cases (9 of 109 [8.3%] vs 11 of 276 [4.0%]; P = 0.04). Compared with patients aged 50 years or older at diagnosis, patients with early-onset AC had significantly higher odds of presenting with nonsilent variations in PIK3CA, SMAD3, and TSC2 (PIK3CA: odds ratio [OR], 4.58; 95% CI, 1.72-12.21; P = .002; SMAD3: OR, 7.37; 95% CI, 1.24-43.87; P = .03; TSC2: OR, 12.43; 95% CI, 1.03-149.59; P = .047). In contrast, patients with early-onset AC had a 60% decreased odds of presenting with GNAS nonsilent variations compared with patients with late-onset AC (OR, 0.40; 95% CI, 0.21-0.76, P = .006). By histological subtype, young patients with mucinous adenocarcinomas of the appendix had 65% decreased odds of variations in GNAS compared with late-onset cases in adjusted models (OR, 0.35; 95% CI, 0.15-0.79; P = .01). Similarly, patients with early-onset nonmucinous appendiceal adenocarcinomas had 72% decreased odds of presenting with GNAS variations vs late-onset cases, although these findings did not reach significance (OR, 0.28; 95% CI, 0.07-1.14; P = .08). GNAS and TP53 variations were mutually exclusive in ACs among early-onset and late-onset cases (P < .05).
In the study, AC diagnosed among younger individuals harbored a distinct genomic landscape compared with AC diagnosed among older individuals. Development of therapeutic modalities that target these unique molecular features may yield clinical implications specifically for younger patients.
Publication Date: 2020-12-10
Journal: JAMA network open
Primary Cutaneous Desmoplastic Melanoma With Collagen Rosettes and Pseudoglandular Features.
Primary cutaneous desmoplastic melanoma (DM) is a group of rare melanocytic tumors arising on severely sun-damaged skin, histologically characterized by the proliferation of spindled melanocytes in a prominent desmoplastic stroma, with a range of morphological presentations. In this article, we report a unique case of primary cutaneous DM composed of a nodular proliferation of highly pleomorphic spindled and epithelioid cells, pseudoglandular structures, clear cell change, and unusual collagen rosettes. Immunohistochemical analysis showed a strong and diffuse positivity for S-100 protein, SOX-10, nestin, p75 (nerve growth factor receptor), WT1, and p53. Molecular analysis detected a mutation in the NF1 gene [c.4084C > T, p.(Arg1362Ter)], 2 different pathogenic mutations in TP53 [c.742C > T, p.(Arg248Trp), AF:12%, COSM1640831 and c.528C > G, p.(Cys176Trp), AF:12%, COSM11114], and a mutation in GNAS [c.601C > T, p.(Arg201Cys), AF: 9%, COSM123397]. To the best of our knowledge, this is the first case reporting collagen rosettes and pseudoglandular features in primary cutaneous DM.
Publication Date: 2020-11-18
Journal: The American Journal of dermatopathology
Diffuse expression of MUC6 defines a distinct clinicopathological subset of pulmonary invasive mucinous adenocarcinoma.
Invasive mucinous adenocarcinoma (IMA) of the lung is a unique variant of lung adenocarcinoma. Aberrant mucin expression is associated with cancer development and metastasis. However, the clinicopathological significance of mucin expression in IMA is not fully understood. Herein, we evaluated the clinicopathological, immunohistochemical, and molecular characteristics of 70 IMA tumors. EGFR, KRAS, GNAS, and TP53 mutations were assessed by PCR-based sequencing. Next-generation sequencing was used to assess cases without EGFR/KRAS mutations. A NanoString-based screening for fusions was performed in all IMAs without mitogenic driver mutations. Expression of mucins (MUC1, MUC2, MUC4, MUC5AC, and MUC6) was evaluated by immunohistochemistry and categorized as follows: negative (<10% of tumor cells), patchy expression (<90% of tumor cells), or diffuse expression (≥90% of tumor cells). Immunohistochemical testing for transcription factors (TTF-1, CDX2, HNF1β, HNF3α, HNF3β, and HNF4α) was also performed. As expected, KRAS mutations were the most common (in 67% of cases), followed by small numbers of other alterations. Patchy or diffuse expression of MUC1, MUC2, MUC4, MUC5AC, and MUC6 was observed in 52% or 6%, 3% or 0%, 30% or 3%, 26% or 73%, and 59% or 27% of cases, respectively. Furthermore, all IMAs were generally positive for HNF1β (100%), HNF3α (100%), HNF3β (100%), and HNF4α (99%) but were positive less often for TTF-1 (6%) and CDX2 (9%). Overall, there was no significant correlation between mucin expression and transcription factor expression. Unexpectedly, diffuse expression of MUC6 was significantly associated with KRAS-wild-type tumors (p = 0.0008), smaller tumor size (p = 0.0073), and tumors in female patients (p = 0.0359) in multivariate analyses. Furthermore, patients with tumors exhibiting diffuse MUC6 expression had significantly favorable outcomes. Notably, none of these patients died of the disease. Our data suggested that diffuse expression of MUC6 defines a distinct clinicopathological subset of IMA characterized by wild-type KRAS and possibly less aggressive clinical course.
Publication Date: 2020-10-08
Journal: Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc
Molecular and clinicopathological features of appendiceal mucinous neoplasms.
Appendiceal mucinous tumors (AMTs) include low-grade mucinous appendiceal neoplasms (LAMNs), high-grade mucinous appendiceal neoplasms (HAMNs), and mucinous adenocarcinomas (MACs). We collected 51 AMT samples (LAMN: 34, HAMN: 8, MAC: 9). Three of the eight HAMN cases contained LAMN components, and four out of nine MAC cases contained LAMN and/or HAMN components within the tumor. A next-generation sequencing (NGS) cancer hotspot panel was used to analyze 11 pure LAMN, 4 HAMN, and 3 MAC cases. The results revealed KRAS and GNAS as the most frequently mutated genes. Sanger sequencing was then performed to detect KRAS, GNAS, and TP53 mutations in the remaining 31 cases and RNF43 mutations in all cases. KRAS/GNAS and TP53 mutations occurred exclusively in pure LAMNs; however, five LAMN cases had mutations in both KRAS and GNAS. RNF43 mutations almost exclusively occurred with KRAS/GNAS mutations in pure LAMNs. In MAC and HAMN, KRAS/GNAS mutation status was nearly preserved between lower-grade areas. Most of the detected RNF43 mutations was missense type. RNF43 mutations were detected in both components of MAC with lower-grade area; however, RNF43 mutations detected in these two lesions were entirely different. RNF43 mutations were detected in only one of the eight HAMN patients, which was the sole case without pseudomyxoma peritonei (PMP). None of the four MAC patients with RNF43 mutation showed PMP. These findings suggest that RNF43 mutations occur at a later stage of MAC development and do not associate with PMP. Furthermore, a gradual transition from LAMN to MAC via HAMN could be considered.
Publication Date: 2020-08-22
Journal: Virchows Archiv : an international journal of pathology
Activation of the RAS pathway through uncommon BRAF mutations in mucinous pancreatic cysts without KRAS mutation.
Diagnostic testing of pancreatic cyst fluid obtained by endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) has traditionally utilized elevated carcinoembryonic antigen (CEA) (≥192 ng/ml) and cytomorphologic examination to differentiate premalignant mucinous from benign pancreatic cystic lesions (PCLs). Molecular testing for KRAS/GNAS mutations has been shown to improve accuracy of detecting mucinous PCLs. Using a targeted next-generation sequencing (NGS) panel, we assess the status of PCL-associated mutations to improve understanding of the key diagnostic variables. Molecular analysis of cyst fluid was performed on 108 PCLs that had concurrent CEA and/or cytological analysis. A 48-gene NGS assay was utilized, which included genes commonly mutated in mucinous PCLs such as GNAS, KRAS, CDKN2A, and TP53. KRAS and/or GNAS mutations were seen in 59 of 68 (86.8%) cases with multimodality diagnosis of a mucinous PCL. Among 31 patients where surgical histopathology was available, the sensitivity, specificity, and diagnostic accuracy of NGS for the diagnosis of mucinous PCL was 88.5%, 100%, and 90.3%, respectively. Cytology with mucinous/atypical findings were found in only 29 of 62 cases (46.8%), with fluid CEA elevated in 33 of 58 cases (56.9%). Multiple KRAS mutations at different variant allele frequencies were seen in seven cases favoring multiclonal patterns, with six of them showing at least two separate PCLs by imaging. Among the 6 of 10 cases with GNAS + /KRAS- results, uncommon, non-V600E exon 11/15 hotspot BRAF mutations were identified. The expected high degree of accuracy of NGS detection of KRAS and/or GNAS mutations for mucinous-PCLs, as compared with CEA and cytological examination, was demonstrated. Multiple KRAS mutations correlated with multifocal cysts demonstrated by radiology. In IPMNs that lacked KRAS mutations, the concurring BRAF mutations with GNAS mutations supports an alternate mechanism of activation in the Ras pathway.
Publication Date: 2020-08-15
Journal: Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc
Integrated clinico-molecular profiling of appendiceal adenocarcinoma reveals a unique grade-driven entity distinct from colorectal cancer.
Appendiceal adenocarcinoma (AA) is an orphan disease with unique clinical attributes but often treated as colorectal cancer (CRC). Understanding key molecular differences between AA and CRC is critical.
We performed retrospective analyses of AA patients (N = 266) with tumour and/or blood next-generation sequencing (NGS) (2013-2018) with in-depth clinicopathological annotation. Overall survival (OS) was examined. For comparison, CRC cohorts annotated for sidedness, consensus molecular subtypes (CMS) and mutations (N = 3283) were used.
Blood-NGS identified less RAS/GNAS mutations compared to tissue-NGS (4.2% vs. 60.9%, P < 0.0001) and showed poor concordance with tissue for well-/moderately differentiated tumours. RAS (56.2%), GNAS (28.1%) and TP53 (26.9%) were most frequent mutations. Well/moderately differentiated tumours harboured more RAS (69.2%/64.0% vs. 40.5%) and GNAS (48.7%/32.0% vs. 10.1%) while moderate/poorly differentiated tumours had more TP53 (26.0%/27.8% vs. 7.7%) mutations. Appendiceal adenocarcinoma (compared to CRC) harboured significantly fewer APC (9.1% vs. 55.4%) and TP53 (26.9% vs. 67.5%) and more GNAS mutations (28.1% vs. 2.0%) (P < 0.0001). Appendiceal adenocarcinoma mutation profile did not resemble either right-sided CRC or any of the four CMS in CRC. High grade, but no mutation, was independently predictive of survival.
Integrated clinico-molecular profiling of AA identified key molecular drivers distinct from CRC. Appendiceal adenocarcinoma has a predominantly grade-driven biology that trumps mutations.
Publication Date: 2020-08-01
Journal: British journal of cancer
Genetic characteristics of gastric-type mucinous carcinoma of the uterine cervix.
Gastric-type mucinous carcinoma (GAS) is a recently established variant of endocervical mucinous adenocarcinoma that is characterized as being unrelated to HPV and having aggressive behavior and chemoresistance. GAS has a distinct morphology resembling nonneoplastic gastric glands or pancreaticobiliary adenocarcinoma, and their possible genetic similarity has been posed. In this study, next-generation sequencing was performed in 21 GAS cases using a customized panel including 94 cancer-associated genes. A total of 54 nonsynonymous somatic mutations were detected with an average mutation rate of 2.6 per lesion (range: 0-9). The most frequently mutated gene was TP53 (11/21, 52.4%), followed by STK11, HLA-B, PTPRS (4/21, 19.0%), FGFR4 (3/21, 14.3%), GNAS, BRCA2, ELF3, ERBB3, KMT2D, SLX4 (2/21, 9.5%), CDH1, EPCAM, KRAS, MLH1, RNF43, SNAI1, TWIST1, ZEB1, ZEB2, and so on (1/21, 4.8%). The mutated genes were mostly involved in signal transduction, DNA damage repair, and epithelial-mesenchymal transition (EMT). Correlation of TP53 mutation and p53 protein expression demonstrated that 31.3% with abnormal p53 expression harbored wild-type TP53. Compared to genetic features of gastric and pancreaticobiliary adenocarcinoma, TP53 mutations were frequent in both GAS and gastrointestinal adenocarcinoma. While KMT2D, ERBB3, and RNF43 mutations were shared between GAS and gastric adenocarcinoma, highly mutated genes in pancreatic ductal adenocarcinoma such as KRAS, SMAD4, and CDKN2A were rarely mutated in GAS. Of frequently mutated genes in cholangiocarcinoma, BAP1 and HLA-B were identified in GAS. Frequent EMT-related gene mutations suggested a possible role of EMT-related pathways in tumor dissemination and chemoresistance of GAS. In addition, GAS shared some genetic features with gastrointestinal adenocarcinoma. These findings provide a clue in understanding the biological basis of GAS.
Publication Date: 2020-07-10
Journal: Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc
Next-generation sequencing of residual cytologic fixative preserved DNA from pancreatic lesions: A pilot study.
Endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) is a sensitive and specific tool in the risk stratification of pancreatic lesions, including cysts. The sensitivity and specificity of EUS-FNA has been shown to improve when cytology is combined with next-generation sequencing (NGS). Ideally, fresh cyst fluid is used for NGS. In this pilot study, we explore the possibility of sequencing DNA derived from residual alcohol-fixed pancreatic aspirates.
Residual cytologic fixatives (n = 42) from 39 patients who underwent EUS-FNA for pancreatic lesions were collected along with demographics, imaging, and laboratory studies. Samples were designated as nonneoplastic/nonmucinous benign (NB), mucinous cyst (MC), pancreatic ductal adenocarcinoma (PDAC), or well-differentiated neuroendocrine tumor (NET) on the basis of cytopathologic evaluation and sequenced on the Oncomine platform (ThermoFisher Scientific, Waltham, Massachusetts).
Ten of 14 (71.4%) MCs exhibited clinically significant variants, including KRAS, GNAS, and TP53. Ten of 15 (66.7%) PDACs had KRAS alterations, and 9 of 15 (60%) showed variants in TP53. No variants were detected in any NETs. Only 1 of 9 (11.1%) NB aspirates showed variants in KRAS and MAP2K. Sequencing of formalin-fixed, paraffin-embedded tissue revealed variants identical to those detected in fixative-derived DNA in 4 of 5 cases (80%).
Residual DNA from alcohol-fixed aspirates are an underutilized source for NGS. Sequencing residual fixative-derived DNA has the potential to be integrated into the workup of pancreatic aspirates, possibly impacting management.
Publication Date: 2020-07-01
Journal: Cancer cytopathology
Transcriptomic silencing as a potential mechanism of treatment resistance.
Next-generation sequencing (NGS) has not revealed all the mechanisms underlying resistance to genomically matched drugs. Here, we performed in 1417 tumors whole-exome tumor (somatic)/normal (germline) NGS and whole-transcriptome sequencing, the latter focusing on a clinically oriented 50-gene panel in order to examine transcriptomic silencing of putative driver alterations. In this large-scale study, approximately 13% of the somatic single nucleotide variants (SNVs) were unexpectedly not expressed as RNA; 23% of patients had ≥1 nonexpressed SNV. SNV-bearing genes consistently transcribed were TP53, PIK3CA, and KRAS; those with lower transcription rates were ALK, CSF1R, ERBB4, FLT3, GNAS, HNF1A, KDR, PDGFRA, RET, and SMO. We also determined the frequency of tumor mutations being germline, rather than somatic, in these and an additional 462 tumors with tumor/normal exomes; 33.8% of germline SNVs within the gene panel were rare (not found after filtering through variant information domains) and at risk of being falsely reported as somatic. Both the frequency of silenced variant transcription and the risk of falsely identifying germline mutations as somatic/tumor related are important phenomena. Therefore, transcriptomics is a critical adjunct to genomics when interrogating patient tumors for actionable alterations, because, without expression of the target aberrations, there will likely be therapeutic resistance.
Publication Date: 2020-06-05
Journal: JCI insight
Intraductal papillary neoplasms of the bile duct consist of two distinct types specifically associated with clinicopathological features and molecular phenotypes.
Intraductal papillary neoplasm of the bile duct (IPNB) is a grossly visible papillary biliary neoplasm with morphological variations and occasional invasion. Recently a new classification of IPNB into type 1 and type 2 was proposed in which the type 1 IPNBs consist of fine papillary neoplastic glands and the type 2 IPNBs consist of complex branching glands, seldom with foci of solid-tubular components. However, clinicopathological and molecular characteristics of these types of IPNBs are yet to be identified. We aimed to uncover clinicopathological and molecular characteristics of the types of IPNBs. Thirty-six IPNBs were studied retrospectively. Clinicopathological features as well as molecular alterations of 31 genes were evaluated by means of targeted next-generation sequencing and immunohistochemical examination of expression of mucin and cancer-associated molecules. The 36 IPNBs were classified into 22 of type 1 and 14 of type 2. The type 1 IPNBs were associated with a non-invasive phenotype, intestinal and oncocytic subtypes, development in the intrahepatic bile duct, overt mucin production, and a relatively good prognosis. The type 2 IPNBs were associated with an invasive phenotype, the pancreatobiliary subtype, development within the extrahepatic bile duct, and worse prognosis compared with the type 1 IPNBs. In the molecular analysis, recurrent mutations were found in TP53 (34.3%), KRAS (31.4%), STK11 (25.7%), CTNNB1 (17.1%), APC (14.3%), SMAD4 (14.3%), GNAS (11.4%), PBRM1 (11.4%), ELF3 (8.6%), KMT2C (8.6%), NF1 (8.6%), PIK3CA (8.6%), ARID1A (5.7%), ARID2 (5.7%), BAP1 (5.7%), BRAF (5.7%), EPHA6 (5.7%), ERBB2 (5.7%), ERBB3 (5.7%), KMT2D (5.7%), and RNF43 (5.7%). Mutations in KRAS and GNAS were enriched in the type 1 IPNBs, whereas mutations in TP53, SMAD4, and KMT2C were enriched in the type 2 IPNBs. These results indicate that IPNBs consist of two distinct types of neoplasms specifically associated with clinicopathological features and molecular phenotypes. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Publication Date: 2020-02-27
Journal: The Journal of pathology