Characterization of in vitro 3D cultures.
Over the past decade, 3D culture models of human and animal cells have found their way into tissue differentiation, drug development, personalized medicine and tumour behaviour studies. Embryoid bodies (EBs) are in vitro 3D cultures established from murine pluripotential stem cells, whereas tumoroids are patient-derived in vitro 3D cultures. This thesis aims to describe a new implication of an embryoid body model and to characterize the patient-specific microenvironment of the parental tumour in relation to tumoroid growth rate. In this thesis, we described a high-throughput monitoring method, where EBs are used as a dynamic angiogenesis model. In this model, digital image analysis (DIA) is implemented on immunohistochemistry (IHC) stained sections of the cultures over time. Furthermore, we have investigated the correlation between the genetic profile and inflammatory microenvironment of parental tumours on the in vitro growth rate of tumoroids. The EBs were cultured in spinner flasks. The samples were collected at days 4, 6, 9, 14, 18 and 21, dehydrated and embedded in paraffin. The histological sections were IHC stained for the endothelial marker CD31 and digitally scanned. The virtual whole-image slides were digitally analysed by Visiopharm® software. Histological evaluation showed vascular-like structures over time. The quantitative DIA was plausible to monitor significant increase in the total area of the EBs and an increase in endothelial differentiation. The tumoroids were established from 32 colorectal adenocarcinomas. The in vitro growth rate of the tumoroids was followed by automated microscopy over an 11-day period. The parental tumours were analysed by next-generation sequencing for KRAS, TP53, PIK3CA, SMAD4, MAP2K1, BRAF, FGFR3 and FBXW7 status. The tumoroids established from KRAS-mutated parental tumours showed a significantly higher growth rate compared to their wild-type counterparts. The density of CD3+ T lymphocytes and CD68+ macrophages was calculated in the centre of the tumours and at the invasive margin of the tumours. The high density of CD3+ cells and the low density of CD68+ cells showed a significant correlation with a higher growth rate of the tumoroids. In conclusion, a novel approach for histological monitoring of endothelial differentiation is presented in the stem cell-derived EBs. Furthermore, the KRAS status and density of CD3+ T cells and macrophages in the parental tumour influence the growth rate of the tumoroids. Our results indicate that these parameters should be included when tumoroids are to be implemented in personalized medicine.
I løbet af det seneste årti er man begyndt at bruge 3D cellekulturer med humane og dyreceller til vaevsdifferentieringsstudier, til medicinudvikling, til personaliseret medicin og til cancer mikromiljøstudier. Embryoid bodies (EBer) er in vitro 3D cellekulturer etableret fra muse stamceller, hvorimod tumoroider er 3D kulturer etableret fra patienters tumorceller. Formålet med denne PhD var at kortlaegge en ny anvendelse af ”embryoid body” modellen samt at karakterisere sammenhaengen mellem det patient-specifikke tumormiljø og tumoroidernes efterfølgende vaekst in vitro. I denne PhD har vi kortlagt en high throughput monitoreringsmetode, hvor EBerne er anvendt som en dynamisk angiogenesemodel, og hvor immunfarvede snit af EBerne er analyseret digitalt. Herudover har vi undersøgt sammenhaengen mellem den genetiske profil og det inflammatoriske mikromiljø i primaertumorerne med vaekstraten af de fremdyrkede tumoroider. EBerne blev dyrket i spinnerflasker og høstet på dag 4, 6, 9, 14, 18, og 21, dehydreret og indstøbt i paraffin. De histologiske snit blev farvet for en endothel markør (CD31) og skannet digitalt. Disse hel-slide billeder blev analyseret ved hjaelp af Visiopharm® software. Den histologiske evaluering viste, at der blev dannet vaskulaer-lignende strukturer med tiden. Den kvantitative digitale billedanalyse fandt vi velegnet til at monitorere et vaekstareal samt endotheldifferentieringen i EBerne. Tumoroider blev etableret fra 32 colorektal adenokarcinomer. In vitro vaeksten af tumoroiderne blev fulgt i 11 dage ved hjaelp af et automatiseret mikroskop. De oprindelige tumorer blev analyseret ved hjaelp af next generation sekventering for KRAS, TP53, PIK3CA, SMAD4, MAP2K1, BRAF, FGFR3 og FBXW7 mutationer. Tumoroider, som blev etableret fra primaertumorer indeholdende en KRAS mutation, viste en signifikant højere vaekstrate sammenlignet med wild-type tumorer. CD3+ T celler og CD68+ makrofagers densitet blev beregnet i tumormidten samt i den invasive margin af tumorerne. Vi fandt en statistisk signifikant sammenhaeng mellem vaeksten af tumoroiderne in vitro og taetheden af CD3+ immunceller og lav taethed af CD68+ immunceller i primaertumoren. Vi konkluder, at en kombination af immunfarvning, digital slideskanning og digital billedeanalyse er en ny tilgang til at følge endothel uddifferentiering i en EB model. Derudover påvirker KRAS mutationsstatus samt antallet af CD3+ T celler og CD68+ makrofager i primaervaevet tumoroidernes efterfølgende vaekst. Vores resultater indikerer, at disse parametre bør analyseres, når tumoroider ønskes anvendt til personaliseret medicin.
Publication Date: 2021-08-17
Journal: APMIS : acta pathologica, microbiologica, et immunologica Scandinavica
Emerging roles of miRNAs in the development of pancreatic cancer.
Pancreatic cancer is a fatal cancer which is expected to exceed breast cancer as the third foremost source of cancer mortality by 2025. This cancer has been associated with several somatic genetic aberrations including mutations in the KRAS, CDKN2A/p16, TP53, and SMAD4. In addition, epigenetic alterations have been shown to affect development of this cancer. miRNAs are among the mostly appreciated epigenetic factors in this regard. Several oncomiRs such as miR-212, miR 506, miR-196b, miR-221-3p, miR-301a-3p, miR-23a and miR-29a have been found to promote proliferation of pancreatic cancer cells and block apoptotic pathways in these cells. On the other hand, miR-451a, miR-506, miR-142, miR-216b, miR-519d-3p, miR-1181, miR-340, miR-143-3p, miR-203a-3p, miR-455, miR-15a, miR-135a and miR-202 are among tumor suppressor miRNAs that modulate proliferation and cell cycle transition in these cells. In the current paper, we will discuss the role of oncomiRs and tumor suppressor miRNAs in the evolution of pancreatic cancer. Moreover, we will summarize the application of miRNAs as diagnostic and prognostic markers in pancreatic cancer. These studies have shown the ability of miRNAs to be served as non-invasive markers for pancreatic cancer.
Publication Date: 2021-07-31
Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie
Combined tumor epithelial and stromal histopathology with keratin 81 expression predicts prognosis for pancreatic ductal adenocarcinoma.
Development of a pragmatic pathologic classifier of pancreatic ductal adenocarcinoma (PDAC) that reflects biological behavior is needed.
The tumor epithelial and stromal features of PDAC and molecular subtype-related markers were evaluated in three independent cohorts.
In the non-neoadjuvant therapy cohort (n = 108), regarding tumor-epithelial feature, non-gland-forming type showed worse prognosis compared to gland-forming type (P < .001). For tumor-stromal feature, in gland-forming type, the prognosis was good in order of inactivated stroma-rich, stroma-poor, and activated stroma-rich (P = .027). Whereas, non-gland-forming type revealed no difference of prognosis according to tumor stroma. Of molecular subtype-related markers, keratin 81 expression was correlated with non-gland-forming type and poor prognosis (P = .005 and P = .021, respectively). Other markers (HNF1A, c-MET, and p53) showed no significant differences in prognosis. In the neoadjuvant therapy cohort (n = 68), non-gland-forming type was correlated with high residual tumor volume (≥20%) (P < .001) and gland-forming/stroma-poor type was not present. In the next-generation sequencing cohort (n = 55), non-gland-forming type was correlated with a higher number of the KRAS, TP53, CDKN2A, and SMAD4 mutations (P = .038).
Combined tumor epithelial and stromal histopathology with keratin 81 expression is suggested to be useful for predicting prognosis of PDAC.
Publication Date: 2021-07-29
Journal: Journal of hepato-biliary-pancreatic sciences
Detection of circulating tumor DNA without a tumor-informed search using next-generation sequencing is a prognostic biomarker in pancreatic ductal adenocarcinoma.
The confounding effects of next-generation sequencing (NGS) noise on detection of low frequency circulating tumor DNA (ctDNA) without a priori knowledge of solid tumor mutations has limited the applications of circulating cell-free DNA (ccfDNA) in clinical oncology. Here, we use a 118 gene panel and leverage ccfDNA technical replicates to eliminate NGS-associated errors while also enhancing detection of ctDNA from pancreatic ductal adenocarcinomas (PDACs). Pre-operative ccfDNA and tumor DNA were acquired from 14 patients with PDAC (78.6% stage II-III). Post-operative ccfDNA was also collected from 11 of the patients within 100 days of surgery. ctDNA detection was restricted to variants corresponding to pathogenic mutations in PDAC present in both replicates. PDAC-associated pathogenic mutations were detected in pre-operative ccfDNA in four genes (KRAS, TP53, SMAD4, ALK) from five patients. Of the nine ctDNA variants detected (variant allele frequency: 0.08%-1.59%), five had a corresponding mutation in tumor DNA. Pre-operative detection of ctDNA was associated with shorter survival (312 vs. 826 days; χ
Publication Date: 2021-07-24
Journal: Neoplasia (New York, N.Y.)
Characterization of genomic alterations in Chinese colorectal cancer patients with liver metastases.
The exploration of genomic alterations in Chinese colorectal liver metastasis (CRLM) is limited, and corresponding genetic biomarkers for patient's perioperative management are still lacking. This study aims to understand genome diversification and complexity that developed in CRLM.
A custom-designed IDT capture panel including 620 genes was performed in the Chinese CRLM cohort, which included 396 tumor samples from metastatic liver lesions together with 133 available paired primary tumors.
In this Chinese CRLM cohort, the top-ranked recurrent mutated genes were TP53 (324/396, 82%), APC (302/396, 76%), KRAS (166/396, 42%), SMAD4 (54/396, 14%), FLG (52/396, 13%) and FBXW7 (43/396, 11%). A comparison of CRLM samples derived from left- and right-sided primary lesions confirmed that the difference in survival for patients with different primary tumor sites could be driven by variations in the transforming growth factor β (TGF-β), phosphatidylinositol 3-kinase (PI3K) and RAS signaling pathways. Certain genes had a higher variant rate in samples with metachronous CRLM than in samples with simultaneous metastasis. Overall, the metastasis and primary tumor samples displayed highly consistent genomic alterations, but there were some differences between individually paired metastases and primary tumors, which were mainly caused by copy number variations.
We provide a comprehensive depiction of the genomic alterations in Chinese patients with CRLM, providing a fundamental basis for further personalized therapy applications.
Publication Date: 2021-07-21
Journal: Journal of translational medicine
Identification of LIPH as an unfavorable biomarkers correlated with immune suppression or evasion in pancreatic cancer based on RNA-seq.
It is widely considered that pancreatic cancer (PC) is an immunosuppressive cancer. Immune-based therapies remain promising therapeutic strategies for PC. Overexpression of lipase H (LIPH) was reported to be related to immunity in cattle and has also been demonstrated to promote tumor progression in several tumors, but its role in pancreatic carcinogenesis remains unclear. Study on LIPH in PC might provide a new insight into the immunosuppression in PC.
The potential biological and clinical significance of LIPH was evaluated by bioinformatics analysis. We further investigated potential associations between the expression of LIPH and tumor immune infiltration using the CIBERSORT algorithm, the ESTIMAT algorithm, and single sample gene set enrichment analysis (ssGSEA).
LIPH was significantly overexpressed in tumor tissues compared with normal tissues. LIPH overexpression correlated with tumor recurrence, advanced histologic grade, and poorer overall survival (OS). Four of the most common somatic mutation, including KRAS, TP53, CDKN2A, and SMAD4, in PC were all correlated with high LIPH expression. And high LIPH expression was significantly correlated with KRAS activation and SMAD4 inactivation. Besides, LIPH expression was involved in various biological pathways such as negative regulation of cell-cell adhesion, actin cytoskeleton, EMT, angiogenesis, and signaling by MST1. And LIPH overexpression caused high infiltration of TAMs, Treg cells, and Th2/Th1, but reduced the infiltration of CD8
Our findings demonstrated that LIPH correlated with immune suppression or evasion and may function as a novel unfavorable prognostic biomarker in PC.
Publication Date: 2021-07-20
Journal: Cancer immunology, immunotherapy : CII
The germline/somatic DNA damage repair gene mutations modulate the therapeutic response in Chinese patients with advanced pancreatic ductal adenocarcinoma.
Pancreatic ductal adenocarcinoma (PDAC) is a fatal disease with molecular heterogeneity, inducing differences in biological behavior, and therapeutic strategy. NGS profiles of pathogenic alterations in the Chinese PDAC population are limited. We conducted a retrospective study to investigate the predictive role of DNA damage repair (DDR) mutations in precision medicine.
The NGS profiles were performed on resected tissues from 195 Chinese PDAC patients. Baseline clinical or genetic characteristics and survival status were collected. The Kaplan-Meier survival analyses were performed by the R version 3.6.1.
The main driver genes were KRAS, TP53, CDKN2A, and SMAD4. Advanced patients with KRAS mutation showed a worse OS than KRAS wild-type (p = 0.048). DDR pathogenic deficiency was identified in 30 (15.38%) of overall patients, mainly involving BRCA2 (n = 9, 4.62%), ATM (n = 8, 4.10%) and RAD50 genes (n = 3, 1.54%). No significance of OS between patients with or without DDR mutations (p = 0.88). But DDR mutation was an independent prognostic factor for survival analysis of advanced PDAC patients (p = 0.032). For DDR mutant patients, treatment with platinum-based chemotherapy (p = 0.0096) or olaparib (p = 0.018) respectively improved the overall survival. No statistical difference between tumor mutation burden (TMB) and DDR mutations was identified. Treatment of PD-1 blockades did not bring significantly improved OS to DDR-mutated patients than the naive DDR group (p = 0.14).
In this retrospective study, we showed the role of germline and somatic DDR mutation in predicting the efficacy of olaparib and platinum-based chemotherapy in Chinese patients. However, the value of DDR mutation in the prediction of hypermutation status and the sensitivity to the PD-1 blockade needed further investigation.
Publication Date: 2021-07-13
Journal: Journal of translational medicine
Genomic Sequencing and Insight into Clinical Heterogeneity and Prognostic Pathway Genes in Patients with Metastatic Colorectal Cancer.
An understanding of signaling pathways has not been fully incorporated into prognostication and therapeutic options. We evaluated the hypothesis that information about cancer-related signaling pathways can improve prognostic stratification and explain some of the clinical heterogeneity in patients with metastatic colorectal cancer.
We analyzed prognostic relevance of signaling pathways in patients undergoing resection of colorectal liver metastases (CLM) from 2004-2017, and clinical actionability of gene alterations in 7 signaling pathways: p53, Wnt, RTK-RAS, PI3K, TGFβ, Notch, and cell cycle. To assess the wide applicability, the results were validated in an external retrospective cohort including patients with unresectable metastatic colorectal cancer.
Of 579 patients, the numbers of patients with pathway alterations were as follows: p53, n = 420 (72.5%); Wnt, 340 (58.7%); RTK-RAS, 333 (57.5%); PI3K, 110 (19.0%); TGFβ, 65 (11.2%); Notch, 41 (7.1%); and cell cycle, 15 (2.6%). More than 80% of alterations in each pathway occurred in a single predominant gene TP53, APC, KRAS, PIK3CA, FBXW7, and RB1 in p53, Wnt, RTK-RAS, PI3K, Notch, and cell cycle pathways, respectively. Alterations of 4 pathways (p53, RTK-RAS, TGFβ, and Notch) and corresponding predominant genes (TP53, RAS/BRAF, SMAD4, and FBXW7) were significantly associated with worse overall survival (OS), and alterations of Wnt pathway (APC) were associated with better OS in the median follow-up duration of 3.8 years. Similarly, in the external cohort, alterations of p53 (TP53) and RTK-RAS (RAS/BRAF) were significantly associated with worse OS, whereas alteration of Wnt (APC) was associated with better OS in the median follow-up duration of 2.6 years.
Genomic sequencing provides insights into clinical heterogeneity and permits finer prognostic stratification in patients with metastatic colorectal cancer.
Publication Date: 2021-06-11
Journal: Journal of the American College of Surgeons
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
The pancreatic cancer genome revisited.
Pancreatic cancer is a genetic disease, and the recurrent genetic alterations characteristic of pancreatic cancer indicate the cellular processes that are targeted for malignant transformation. In addition to somatic alterations in the most common driver genes (KRAS, CDKN2A, TP53 and SMAD4), large-scale studies have revealed major roles for genetic alterations of the SWI/SNF and COMPASS complexes, copy number alterations in GATA6 and MYC that partially define phenotypes of pancreatic cancer, and the role(s) of polyploidy and chromothripsis as factors contributing to pancreatic cancer biology and progression. Germline variants that increase the risk of pancreatic cancer continue to be discovered along with a greater appreciation of the features of pancreatic cancers with mismatch repair deficiencies and homologous recombination deficiencies that confer sensitivity to therapeutic targeting. Wild-type KRAS pancreatic cancers, some of which are driven by alternative oncogenic events affecting NRG1 or NTRK1 - for which targeted therapies exist - further underscore that pancreatic cancer is formally entering the era of precision medicine. Given the vast developments within this field, here we review the wide-ranging and most current information related to pancreatic cancer genomics with the goal of integrating this information into a unifying description of the life history of pancreatic cancer.
Publication Date: 2021-06-06
Journal: Nature reviews. Gastroenterology & hepatology
NGS-based liquid biopsy profiling identifies mechanisms of resistance to ALK inhibitors: a step toward personalized NSCLC treatment.
Despite impressive and durable responses, nonsmall cell lung cancer (NSCLC) patients treated with anaplastic lymphoma kinase (ALK) inhibitors (ALK-Is) ultimately progress due to development of resistance. Here, we have evaluated the clinical utility of circulating tumor DNA (ctDNA) profiling by next-generation sequencing (NGS) upon disease progression. We collected 26 plasma and two cerebrospinal fluid samples from 24 advanced ALK-positive NSCLC patients at disease progression to an ALK-I. These samples were analyzed by NGS and digital PCR. A tool to retrieve variants at the ALK locus was developed (VALK tool). We identified at least one resistance mutation in the ALK locus in ten (38.5%) plasma samples; the G1269A and G1202R mutations were the most prevalent among patients progressing to first- and second-generation ALK-Is, respectively. Overall, 61 somatic mutations were detected in 14 genes: TP53, ALK, PIK3CA, SMAD4, MAP2K1 (MEK1), FGFR2, FGFR3, BRAF, EGFR, IDH2, MYC, MET, CCND3, and CCND1. Specifically, a deletion in exon 19 in EGFR, a non-V600 BRAF mutation (G466V), and the F129L mutation in MAP2K1 were identified in four patients who showed no objective survival benefit from ALK-Is. Potential ALK-I-resistance mutations were also found in PIK3CA and IDH2. Finally, a c-MYC gain, along with a loss of CCND1 and FGFR3, was detected in a patient progressing on a first-line treatment with crizotinib. We conclude that NGS analysis of liquid biopsies upon disease progression identified different putative ALK-I-resistance mutations in most cases and could be a valuable approach for therapy decision making.
Publication Date: 2021-06-01
Journal: Molecular oncology
Genetic regressive trajectories in colorectal cancer: A new hallmark of oligo-metastatic disease?
Colorectal cancer (CRC) originates as consequence of multiple genetic alterations. Some of the involved genes have been extensively studied (APC, TP53, KRAS, SMAD4, PIK3CA, MMR genes) in highly heterogeneous and poly-metastatic cohorts. However, about 10% of metastatic CRC patients presents with an indolent oligo-metastatic disease differently from other patients with poly-metastatic and aggressive clinical course. Which are the genetic dynamics underlying the differences between oligo- and poly-metastatic CRC? The understanding of the genetic trajectories (primary→metastatic) of CRC, in patients selected to represent homogenous clinical models, is crucial to make genotype/phenotype correlations and to identify the molecular events pushing the disease towards an increasing malignant phenotype. This information is crucial to plan innovative therapeutic strategies aimed to reverse or inhibit these phenomena. In the present study, we review the genetic evolution of CRC with the intent to give a developmental perspective on the border line between oligo- and poly-metastatic diseases.
Publication Date: 2021-05-26
Journal: Translational oncology
Genomic analysis for the prediction of prognosis in small-bowel cancer.
The current understanding of clinicopathological features and genomic variants of small-bowel cancer is limited, in part due to the rarity of the disease. However, understanding of these factors is necessary for the development of novel therapeutic agents for small-bowel cancer. Thus, we aimed to identify the clinicopathological features and genomic variants associated with its prognosis and recurrence. We retrospectively examined 24 consecutive patients with primary small-bowel cancer surgically treated between May 2005 and August 2018 and collected 29 tumor specimens. The 29 lesions were subjected to mismatch repair status evaluation, using immunohistochemistry (IHC), and targeted genomic sequencing, after which they were analyzed using a panel of 90 cancer-related genes. IHC revealed that 45% (13/29) of the lesions exhibited deficient mismatch repair. The most common genomic variants in small-bowel cancers were in TP53 (48%, 13/27), followed by KRAS (44%, 12/27), ARID1A (33%, 9/27), PIK3CA (26%, 7/27), APC (26%, 7/27), and SMAD4, NOTCH3, CREBBP, PTCH1, and EP300 (22%, 6/27 each). Overall survival and disease-specific survival of patients with tumor mutational burden (TMB) ≥10 mutations/Mb (n = 17) were significantly better than those of patients with TMB <10 mutations/Mb (n = 6). Additionally, patients with a mutant SMAD4 had poorer recurrence-free survival than those with wild-type SMAD4. Our results suggested that TMB and SMAD4 mutations were associated with the prognosis of small-bowel cancer patients. Thus, cancer genomic analysis could be useful in the search for biomarkers of prognosis prediction in small-bowel cancers.
Publication Date: 2021-05-21
Journal: PloS one
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
Recommendations for Specimen and Therapy Selection in Colorectal Cancer.
Next-generation sequencing has emerged as a clinical tool for the identification of actionable mutations to triage advanced colorectal cancer patients for targeted therapies. The literature is conflicted as to whether primaries or their metastases should be selected for sequencing. Some authors suggest that either site may be sequenced, whereas others recommend sequencing the primary, the metastasis, or even both tumors. Here, we address this issue head on with a meta-analysis and provide for the first time a set of sensible recommendations to make this determination.
From our own series, we include 43 tumors from 13 patients including 14 primaries, 10 regional lymph node metastases, 17 distant metastases, and two anastomotic recurrences sequenced using the 50 gene Ion AmpliSeq cancer NGS panel v2.
Based on our new cohort and a meta-analysis, we found that ~ 77% of patient-matched primary-metastatic pairs have identical alterations in these 50 cancer-associated genes.
Low tumor cellularity, tumor heterogeneity, clonal evolution, treatment status, sample quality, and/or size of the sequencing panel accounted for a proportion of the differential detection of mutations at primary and metastatic sites. The therapeutic implications of the most frequently discordant alterations (TP53, APC, PIK3CA, and SMAD4) are discussed. Our meta-analysis indicates that a subset of patients who fail initial therapy may benefit from sequencing of additional sites to identify new actionable genomic abnormalities not present in the initial analysis. Evidence-based recommendations are proposed.
Publication Date: 2021-04-26
Journal: Oncology and therapy
Enhancing the landscape of colorectal cancer using targeted deep sequencing.
Targeted next-generation sequencing (NGS) technology detects specific mutations that can provide treatment opportunities for colorectal cancer (CRC) patients. We included 145 CRC patients who underwent surgery. We analyzed the mutation frequencies of common actionable genes and their association with clinicopathological characteristics and oncologic outcomes using targeted NGS. Approximately 97.9% (142) of patients showed somatic mutations. Frequent mutations were observed in TP53 (70%), APC (60%), and KRAS (49%). TP53 mutations were significantly linked to higher overall stage (p = 0.038) and lower disease-free survival (DFS) (p = 0.039). ATM mutation was significantly associated with higher tumor stage (p = 0.012) and shorter overall survival (OS) (p = 0.041). Stage 3 and 4 patients with ATM mutations (p = 0.023) had shorter OS, and FBXW7 mutation was significantly associated with shorter DFS (p = 0.002). However, the OS of patients with or without TP53, RAS, APC, PIK3CA, and SMAD4 mutations did not differ significantly (p = 0.59, 0.72, 0.059, 0.25, and 0.12, respectively). Similarly, the DFS between patients with RAS, APC, PIK3CA, and SMAD4 mutations and those with wild-type were not statistically different (p = 0.3, 0.79, 0.13, and 0.59, respectively). In multivariate Cox regression analysis, ATM mutation was an independent biomarker for poor prognosis of OS (p = 0.043). A comprehensive analysis of the molecular markers for CRC can provide insights into the mechanisms underlying disease progression and help optimize a personalized therapy.
Publication Date: 2021-04-16
Journal: Scientific reports
Accumulated genetic mutations leading to accelerated initiation and progression of colorectal cancer in a patient with Gardner syndrome: A case report.
Gardner syndrome is a rare autosomal dominant disorder with a high degree of penetrance, which is characterized by intestinal polyposis, osteomas, and dental abnormalities. Majority of patients with Gardner syndrome will develop colorectal cancer by the age of 40 to 50 years. Mutations in the adenomatous polyposis coli gene are supposed to be responsible for the initiation of Gardner syndrome.
A 22-year-old Chinese female was admitted to our hospital due to abdominal pain and bloody stool.
The patient presented with multiple intestinal polyposis, desmoid tumors, and dental abnormalities was diagnosed as Gardner syndrome and further examination revealed a colon tumor.
Patients were implanted with stents to alleviate bowel obstruction, and were treated with oxaliplatin combined with 5-Fu for 4 cycles, but the efficacy was not good. We performed next generation sequencing of 390 genes for the tumor specimens. We detected adenomatous polyposis coli E1538Ifs∗5, KRAS G12D, NF1 R652C, loss of SMAD4, TP53 R175H, IRF2 p.R82S, TCF7L2 p.A418Tfs∗14, and SMAD4 p.L43F in this patient.
We reported serial mutations in key genes responsible for initiation and progression of colorectal cancer from a patient with Gardner syndrome.
Publication Date: 2021-04-01
Prognostic and Therapeutic Implications of Tumor Biology, Including Gene Alterations, in Colorectal Liver Metastases.
For patients with colorectal liver metastases (CLM), the combination of surgical resection with other therapeutic options is essential. This article shows how recent advances in knowledge of tumor biology, including genetic alterations, affect the choice of therapeutic approach for patients with CLM.
We reviewed the literature on recent advances in knowledge about CLM tumor biology including genetic profiles, clinical risk score models for CLM, preoperative therapy for CLM, and liver-directed therapy for CLM.
Studies showed that RAS alteration is a negative prognostic factor in addition to traditional clinical risk factors (e.g., larger diameter and higher number of CLM, spread of the primary tumor to regional lymph nodes). Although the response to preoperative chemotherapy is an important predictor of survival, poor response is not a contraindication to surgical resection. The combination of surgical therapy and percutaneous ablation can be considered in marginally resectable cases; however, a wider ablation margin is required for RAS-mutant CLM. More recently, genetic analysis using next-generation sequencing showed the negative prognostic impact of alterations in TP53, SMAD4, FBXW7, and RAS/BRAF in patients with CLM. In RAS-mutant CLM, intensive follow-up is required in patients who remain recurrence free 2 years after surgery.
In patients with CLM, RAS mutation status is important in predicting postoperative survival, selecting the treatment approach, and tailoring postoperative follow-up. In addition, more recent genetic analyses of CLM have identified additional predictors of survival.
Publication Date: 2021-03-21
Journal: Journal of gastrointestinal surgery : official journal of the Society for Surgery of the Alimentary Tract
Crosstalk between miRNAs and signaling pathways involved in pancreatic cancer and pancreatic ductal adenocarcinoma.
Pancreatic cancer (PC) is the seventh leading cause of cancer-related deaths worldwide with 5-year survival rates below 8%. Most patients with PC and pancreatic ductal adenocarcinoma (PDAC) die after relapse and cancer progression as well as resistance to treatment. Pancreatic tumors contain a high desmoplastic stroma that forms a rigid mass and has a potential role in tumor growth and metastasis. PC initiates from intraepithelial neoplasia lesions leading to invasive cancer through various pathways. These lesions harbor particular changes in signaling pathways involved in the tumorigenesis process. These events affect both the epithelial cells, including the tumor and the surrounding stroma, and eventually lead to the formation of complex signaling networks. Genetic studies of PC have revealed common molecular features such as the presence of mutations in KRAS gene in more than 90% of patients, as well as the inactivation or deletion mutations of some tumor suppressor genes including TP53, CDKN2A, and SMAD4. In recent years, studies have also identified different roles of microRNAs in PC pathogenesis as well as their importance in PC diagnosis and treatment, and their involvement in various signaling pathways. In this study, we discussed the most common pathways involved in PC and PDAC as well as their role in tumorigenesis and progression. Furthermore, the miRNAs participating in the regulation of these signaling pathways in PC progression are summarized in this study. Therefore, understanding more about pathways involved in PC can help with the development of new and effective therapies in the future.
Publication Date: 2021-03-13
Journal: European journal of pharmacology
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 β-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.
Publication Date: 2021-03-13
Journal: Scientific reports