Heterogeneity of colorectal carcinoma (CRC) represents a major hurdle towards personalized medicine. Efforts based on whole tumor profiling demonstrated that the CRC molecular subtypes were associated with specific tumor morphological patterns representing tumor subregions. We hypothesize that whole-tumor molecular descriptors depend on the morphological heterogeneity with significant impact on current molecular predictors. We investigated intra-tumor heterogeneity by morphology-guided transcriptomics to better understand the links between gene expression and tumor morphology represented by six morphological patterns (morphotypes): complex tubular, desmoplastic, mucinous, papillary, serrated, and solid/trabecular. Whole-transcriptome profiling by microarrays of 202 tumor regions (morphotypes, tumor-adjacent normal tissue, supportive stroma, and matched whole tumors) from 111 stage II-IV CRCs identified morphotype-specific gene expression profiles and molecular programs and differences in their cellular buildup. The proportion of cell types (fibroblasts, epithelial and immune cells) and differentiation of epithelial cells were the main drivers of the observed disparities with activation of EMT and TNF-α signaling in contrast to MYC and E2F targets signaling, defining major gradients of changes at molecular level. Several gene expression-based (including single-cell) classifiers, prognostic and predictive signatures were examined to study their behavior across morphotypes. Most exhibited important morphotype-dependent variability within same tumor sections, with regional predictions often contradicting the whole-tumor classification. The results show that morphotype-based tumor sampling allows the detection of molecular features that would otherwise be distilled in whole tumor profile, while maintaining histopathology context for their interpretation. This represents a practical approach at improving the reproducibility of expression profiling and, by consequence, of gene-based classifiers.
Purpose Renal cell carcinoma belongs among the deadliest malignancies despite great progress in therapy and accessibility of primary care. One of the main unmet medical needs remains the possibility of early diagnosis before the tumor dissemination and prediction of early relapse and disease progression after a successful nephrectomy. In our study, we aimed to identify novel diagnostic and prognostic biomarkers using next-generation sequencing on a novel cohort of RCC patients. Methods Global expression profiles have been obtained using next-generation sequencing of paired tumor and non-tumor tissue of 48 RCC patients. Twenty candidate lncRNA have been selected for further validation on an independent cohort of paired tumor and non-tumor tissue of 198 RCC patients. Results Sequencing data analysis showed significant dysregulation of more than 2800 lncRNAs. Out of 20 candidate lncRNAs selected for validation, we confirmed that 14 of them are statistically significantly dysregulated. In order to yield better discriminatory results, we combined several best performing lncRNAs into diagnostic and prognostic models. A diagnostic model consisting of AZGP1P1, CDKN2B-AS1, COL18A1, and RMST achieved AUC 0.9808, sensitivity 95.96%, and specificity 90.4%. The model for prediction of early relapse after nephrectomy consists of COLCA1, RMST, SNHG3, and ZNF667-AS1 and achieved AUC 0.9241 with sensitivity 93.75% and specificity 71.07%. Notably, no combination has outperformed COLCA1 alone. Lastly, a model for stage consists of ZNF667-AS1, PVT1, RMST, LINC00955, and TCL6 and achieves AUC 0.812, sensitivity 85.71%, and specificity 69.41%. Conclusion In our work, we identified several lncRNAs as potential biomarkers and developed models for diagnosis and prognostication in relation to stage and early relapse after nephrectomy.
Despite the great achievements in treating pediatric cancer patients in the last several decades, approximately one fifth of patients remains uncurable using standard therapeutic modalities and require search for innovative therapeutic approaches. Advances in sequencing techniques and bioinformatic data processing enabled identification of wide spectrum of molecular alterations including single nucleotide variants, copy number aberrations, fusion genes or changes in expression and methylation patterns, which could serve as therapeutic targets. Translation of comprehensive molecular profiling into clinical practice is still limited, however, multiple precision oncology initiatives have already explored feasibility of this approach. From September 2016 to December 2020, a total of 160 patients with high-risk solid tumors that were treated or consulted at Department of Pediatric Oncology of University Hospital Brno were subjected to molecular analysis of tumor tissue using whole-exome sequencing, targeted RNA sequencing, whole-transcriptome profiling and array-CGH. In 18 patients, 2 or more biopsies were analyzed due to relapse or progression of the disease. In the cohort, CNS tumors were the most prevalent (41%), followed by sarcomas (33%) and neuroblastoma (9%). All patients were presented at multidisciplinary molecular tumor board, where treatment recommendations were discussed. In 37% of patients (n = 59), therapeutic targets were identified. Most commonly identified targets included BRAF (n = 9), FGFR1 (n = 7), NF1 (n = 6), NRAS (n = 5) and PIK3CA (n = 4), making RAS/MAPK signaling most frequently altered pathway in the subgroup. Single nucleotide variants or small indels accounted for 65% of actionable findings, followed by fusion genes (12%), copy number aberrations (9%), CD274 expression (7%, confirmed by IHC staining for PD-L1 protein), and high tumor mutational burden (7%). Clinically relevant fusions were found in 25% of patients and 20% of identified fusions were targetable. 8 patients were eligible for immunotherapy based on either PD-L1 expression, or high tumor mutational burden (>10 mut/Mb). Using molecular-based approach in treating high-risk patients represents a promising strategy and helps to understand the complexity of pediatric malignancies though examining tumor biology at multiple levels. Implementing the concept of precision oncology into clinical practice could not only be beneficial in the context of chances for improved survival of high-risk patients, but might also be convenient for other patients, whose successful treatment comes at cost of various secondary complications due to intensive chemotherapy/radiotherapy approaches. Supported by Ministry of Health of the Czech Republic, grant nr. NV19-03-00562, NV19-03-00501, NV19-03-00559 and NU20-03-00240. All rights reserved. Citation Format: Petra Pokorna, Hana Palova, Tina Catela Ivkovic, Sona Adamcova, Michal Kyr, Vojtech Bystry, Robin Jugas, Karolina Trachtova, Dagmar Al Tukmachi, Tomas Merta, Jaroslav Juracek, Jiri Sana, Sona Mejstrikova, Marta Jezova, Peter Mudry, Zdenek Pavelka, Jaroslav Sterba, Ondrej Slaby. Comprehensive genomic profiling as an approach to guide therapeutic planning in pediatric patients with high-risk solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 76.
Brain metastases (BMs) comprise a heterogeneous group of the most frequent intracranial tumors in adults, most originating in lung, breast, renal cell, and colorectal carcinomas and melanomas. Despite the recent improvements in imaging methodology resulting in earlier BM identification and advancements in treatment strategies, BMs are still a significant cause of patient morbidity. Furthermore, BMs frequency increases due to more prolonged survival of cancer patients and population aging. Since the most widely used prognostic scoring systems for BMs require prior knowledge of the primary origin and up to 14% of BMs are classified as BMs of unknown primary, there is an urgent unmet need for accurate biomarkers for identification of BM origin. MiRNAs are non-coding RNAs with an approximate length of 22 nucleotides, functioning as post-transcriptional regulators of gene expression. Dysregulated miRNA expression profile has been observed in many pathological processes, including the complex and not fully understood metastatic cascade. These molecules are very stable and present not only in tissues but also in human body fluids, including blood plasma and cerebrospinal fluid (CSF). Based on these facts, both tissue and circulating miRNAs are extensively studied as potential diagnostic biomarkers. Specific miRNA signatures of BMs were obtained using high-throughput miRNA profiling (Illumina small RNA sequencing) on 3 types of samples (metastatic tissue, blood plasma, CSF) from a cohort of 30 patients with BMs originating in the 5 tumor types – lung, breast, renal cell and colorectal carcinomas and melanomas (6 patients per group, 87 samples in total, only 3 CSF samples from RCC patients available). We identified significantly differentially expressed miRNAs in BM tissues with the ability to differentiate between primary origins. Tissue miRNAs could identify BMs originating from breast, colorectal and renal cell carcinomas and melanomas with high specificity and sensitivity. Interestingly, the heterogeneity of lung carcinomas was also characteristic for the corresponding BMs, making it challenging to distinguish accurately from other BMs. Even though the tissue-specific miRNA signature was the most precise, our results suggest a significant diagnostic potential of circulating miRNAs from CSF for BM patients. Therefore, these short and stable molecules could potentially help identify the origin of BMs of unknown primary. The research is supported by project National Institute for Cancer Research (Programme EXCELES, ID Project No. LX22NPO5102) - Funded by the European Union - Next Generation EU. Citation Format: Dagmar Al Tukmachi, Michaela Ruckova, Marek Vecera, Tana Machackova, Petra Pokorna, Marketa Hermanova, Michal Hendrych, Leos Kren, Ivana Roskova, Vaclav Vybihal, Hana Valekova, Radim Jancalek, Jiri Sana, Martin Smrcka, Ondrej Slaby. Tumor tissue and cerebrospinal fluid microRNA profiles enable the classification of brain metastasis accordingly to their origin. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3758.
Background: Despite major improvements in the survival of pediatric cancer patients that were achieved through the intensification of chemotherapy and the perfection of supportive care in the past decades, treatment outcomes for high-risk, relapsed, and refractory solid cancers remain unsatisfactory. Accelerating the progress of pediatric oncology requires both therapeutic advances and attention to reducing the long-term cytotoxic treatment-related side effects. This could be achieved by targeting specific molecular changes that drive pediatric malignancies. Material and Methods: From September 2016 to August 2020, a total of 192 patients with pediatric high-risk solid tumors successfully underwent comprehensive genomic profiling. Since more than thirty patients had two or more biopsies from recurrent relapses, the total number of samples examined was 295. In the cohort, there were 78 cases of central nervous system tumors, 68 sarcomas, 14 neuroblastomas, 10 lymphomas, and 22 tumors of other histology. Whole-exome sequencing was performed in all patients, fusion gene analysis in 96% of patients, whole-transcriptome profiling in 84% of patients, and CNV analysis in 63% of patients. Results: The diagnostic yield of therapeutically actionable findings was 40%, with single-nucleotide variants and small insertions/deletions being the most common actionable alteration types. In 23% of patients, a clinically relevant gene fusion was identified. The majority of the identified fusions were of diagnostic significance, and 18% of those were therapeutically targetable gene fusions involving BRAF, RAF1, ALK, FGFR1, or NTRK2. Four patients were eligible for immunotherapy based on high tumor mutational burden (>10 mut/Mb). Lymphomas and CNS tumors showed the highest rate of patients with therapeutically actionable findings (60% and 56%, respectively), followed by neuroblastomas (36%), sarcomas (25%), and other solid tumors (23%). All results and individual treatment plans were discussed at multidisciplinary molecular tumor boards. Conclusion: Precision medicine in pediatric oncology has rapidly developed over the last decade and resulted in new therapeutic options based on molecular biomarkers and increased our understanding of the complexity of pediatric malignancies. Supported by the Ministry of Health of the Czech Republic, grant nr. NU20-03-00240 and the project National Institute for Cancer Research (Programme EXCELES, ID Project No. LX22NPO5102) - Funded by the European Union - Next Generation EU. Citation Format: Petra Pokorna, Hana Palova, Sona Adamcova, Vojtech Bystry, Michal Kyr, Dagmar Al Tukmachi, Sona Mejstrikova, Peter Mudry, Jaroslav Sterba, Ondrej Slaby. Impact of the comprehensive genomic profiling on the individual therapeutic planning in high-risk/refractory tumors: real-world precision medicine in pediatric oncology. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4511.
Heterogeneity of colorectal carcinoma (CRC) represents a major hurdle towards personalized medicine. Efforts based on whole tumor profiling demonstrated that the CRC molecular subtypes were associated with specific tumor morphological patterns representing tumor subregions. We hypothesize that molecular descriptors depend on morphological heterogeneity of the tumor, a dependency that negatively impacts the efficiency of current molecular predictors. We investigated intra-tumor heterogeneity by morphology-guided transcriptomics to better understand the links between gene expression and tumor morphology. The morphological patterns (morphotypes) considered were complex tubular, desmoplastic, mucinous, papillary, serrated, and solid/trabecular. Whole-transcriptomic profiling by microarrays of 202 tumor regions (representing the six morphotypes, tumor-adjacent normal tissue, supportive stroma, and matched whole tumors) from 111 stage II-IV CRCs identified morphotype-specific gene expression profiles and molecular programs and differences in their cellular buildup. The proportion of cell types (fibroblasts, epithelial and immune cells) and differentiation of epithelial cells were the main drivers of the observed disparities with activation of EMT and TNF-a signaling in contrast to MYC and E2F targets signaling, defining major gradients of changes at molecular level. Several types of gene expression-based (including single-cell) classifiers, prognostic and predictive signatures were examined to study their behavior across morphotypes. Most exhibited important morphotype-dependent variability within same tumor sections, with regional predictions often contradicting the whole-tumor classification. The results of our study show that morphotype-based tumor sampling allows for detection of molecular features that would otherwise be distilled in whole tumor profile, while maintaining histopathology context for their interpretation. This represents a practical approach at improving the reproducibility of expression profiling and, by consequence, of gene-based classifiers.
Introduction: Currently used molecular diagnostic tests for colorectal cancer (CRC) are underperforming and more sensitive, non-invasive biomarkers are needed. Long non-coding RNAs (lncRNA) and microRNA (miRNA) have shown potential as diagnostic biomarkers. Unfortunately, the identification of non-coding RNA circulating biomarkers in blood serum is significantly burdened by abundant RNA specimens from disrupted blood cells. Recently, small extracellular vesicles (sEVs) emerged as potential reservoirs of clinically relevant biomarkers, including lncRNAs and miRNAs. In theory, sEVs protect RNAs from degradation and might serve as a source of intact RNA for further analyses. However, there is a lack of evidence supporting the superior quality of RNA extracted from sEVs over RNA from whole blood serum. This study aimed to analyze the RNA content of blood serum and the sEVs derived from the blood serum of CRC patients and healthy controls using RNA sequencing. Moreover, small RNA sequencing was used to evaluate the difference in miRNA profiles of sEVs and corresponding blood sera of CRC patients and healthy controls. Methods: Spin-column chromatography (Exiqon), precipitation-based method (Norgen), and size-exclusion chromatography (iZON) were used to extract sEVs from blood sera. The concentration of sEVs was measured by dynamic light scattering (DLS), the size was evaluated by electron microscopy (EM), and sEV-specific content was analyzed by western blot and qRT-PCR. RNA was extracted using the column-based method. Next-generation sequencing (NGS) analyses of blood serum and sEVs extracted from blood serum included samples from 10 CRC patients and 10 healthy controls for RNAseq, and 5 CRC patients and 5 healthy controls for small RNAseq. Differential expression analysis was carried out in R using DESeq2 package. Results: DLS and EM showed that size-exclusion chromatography yielded the purest population of sEVs characterized according to ISEV recommendations. Extraction of sEVs and subsequent RNA extraction and sequencing library preparation from ultra-low input samples were optimized. Over 30k different RNAs were identified in the sEVs derived from blood sera of CRC patients and healthy controls, including lncRNAs, miRNAs, and protein-coding RNAs. A detailed comparison of the transcriptome of blood sera and corresponding sEVs is a part of the poster. Conclusion: sEVs could serve as a source of RNA biomarkers; however, proper characterization and optimal methodology are necessary. This work was supported by the Ministry of Health of the Czech Republic grant No. NU20-03-00127, by The project National Institute for Cancer Research (Programme EXCELES, ID Project No. LX22NPO5102) - Funded by the European Union - Next Generation EU, by the project BBMRI-CZ, nr. LM2018125, and in co-operation with CEMCOF, CEITEC MU (CIISB) supported by MEYS CR, LM2018127. Citation Format: Tana Machackova, Petra Vychytilova-Faltejskova, Marie Madrzyk, Karolina Trachtova, Marketa Pavlikova, Jan Kotoucek, Jana Halamkova, Dagmar Al Tukmachi, Jiri Sana, Petra Pokorna, Milana Sachlova, Ondrej Slaby. Utility of RNA sequencing for transcriptome analysis of small extracellular vesicles derived from blood sera of colorectal cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6709.
Background:Acute myeloid leukemia (AML) is a malignancy with a high prevalence of NRAS mutations. Transforming point mutations, located dominantly at hotspot codons 12, 13 and 61 of NRAS gene, occur in 11 to 22% of patients. Mutations in NRAS gene stimulate signalling activity of RAS pathway, resulting in an increased cellular proliferation and reduced apoptosis. Prognostic significance of NRAS gene mutations in AML patients remains controversial.Aims:The aim of our work was to analyze the association of NRAS gene mutations in AML with patient characteristics and outcomes.Methods:A total of 297 consecutive AML (non‐acute promyelocyte leukemia) patients (median age 53 years; range 19–70) with curative therapy were analyzed by next generation sequencing (NGS) using ClearSeq AML panel (Agilent Technologies) on MiSeq and NextSeq (Illumina). The NRAS mutations detected by NGS with variant allele frequency (VAF) ≥1.0% were verified by NRAS StripAssay (ViennaLab). Only mutations scored as positive by both methods were included in further analyses. Overall survival (OS) was analyzed by log‐rank (Mantel‐Cox) test. The comparative statistical analyses were evaluated by t‐test and Fisher's exact test.Results:In total, 84 mutations were identified in 66/297 (22.2%) analyzed AML patients. Fifteen patients (22.7%) were carriers of multiple gene mutations (range 1–3 mutations per patient). The mutations were most frequently found in the codon G12 (44/84 mutations, 52.4%), the most frequent gene mutations were: G12D (26/84; 31.0% mutations), G13D (15/84; 17.9%) and G12S (14/84; 16.7%). Furthermore, rare mutation G60E was identified in one patient. The median VAF of NRAS mutations was 7.0% (range 1.0–53.4%). Frequency of co‐mutation with FLT3‐TKD reached statistical significance (p = 0.025) and a trend for co‐mutation with NPM1 (p = 0.082) and DNMT3A (p = 0.091). A trend for mutual exclusivity was observed for FLT3‐ITD mutations with allelic ratio above 0.5 (p = 0.063). Occurrence of NRAS mutations was associated with younger age (median 48 vs. 54 years, respectively, p = 0.012), and a trend towards higher white blood cell count (median 41.0 vs. 14.2, respectively, p = 0.069). There was no association with OS, blast percentage in BM or sex. The presence of multiple NRAS mutations was not associated with analyzed characteristics nor patient outcomes.Summary/Conclusion:Our study showed NRAS gene mutations detected by two independent methods in more than one fifth of AML patients with curative therapy. Most of them have low VAF, therefore suggesting their later occurrence during leukemogenesis and limited proliferative potential. Although approximately 5% of AML patients harbor multiple NRAS mutations, we were not able to identify the difference between patients with one or more NRAS mutations.Supported by Ministry of Health of the Czech Republic, grant nr. 15–25809A, project FNBr. 65269705 and project MUNI/A/1105/2018. All rights reserved.
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