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Intrasinusoidal bone marrow involvement is an infrequent histological pattern observed in a limited number of B and T cell lymphomas. Mantle cell lymphoma is a biologically and prognostically heterogeneous B cell lymphoma that frequently involves the bone marrow, with interstitial, nodular-paratrabecular, or diffuse patterns. Intrasinusoidal bone marrow involvement has been described only anecdotally in this lymphoma. Here, we describe the clinical, histopathological, and molecular features of four patients diagnosed with advanced-stage mantle cell lymphoma, showing intrasinusoidal bone marrow involvement, and other peculiar immunophenotypical features. As similar cases may represent a relevant issue in bone marrow diagnostic histopathology, we also reviewed the literature to discuss differential diagnoses of B and T cell lymphomas with intrasinusoidal bone marrow involvement.
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BACKGROUND/AIM: Extracellular vesicle DNA (EV-DNA) has emerged as a novel biomarker for tumor mutation detection using liquid biopsies, exhibiting biological advantages compared to cell-free DNA (cfDNA). This study assessed the feasibility of EV-DNA and cfDNA extraction and sequencing in old serum samples of patients with breast cancer (BC). PATIENTS AND METHODS: A total of 28 serum samples of 27 patients with corresponding clinical information were collected between 1983 and 1991. EV-DNA was extracted using Exo-GAG kit (Nasabiotech) and cfDNA using QIAsymphony DSP Virus/Pathogen Midi Kit (Qiagen), respectively. Subsequently, 10 matched samples (EV-DNA n=5, cfDNA n=5) of five patients were subjected to sequencing using the Oncomine™ Breast cfDNA Research Assay v2 (Thermo Fisher Scientific). RESULTS: Samples were collected on median 1.9 years after primary diagnosis [interquartile range (IQR)=0.2-7.2]. Median follow-up was 9.5 years (IQR=5.2-14.2). Median age of serum samples was 36.1 years (IQR=34.5-37.3). EV-DNA and cfDNA were extracted from 100% (28/28) of the included samples. Both, DNA quantity and concentration were comparable between EV-DNA and cfDNA. Sequencing was successfully performed in 100% (10/10) of the included samples. Two matched analyses yielded equivalent results in EV-DNA and cfDNA (no mutations, n=1; PIK3CA mutation, n=1), whilst in two analyses, PIK3CA mutation was only found in cfDNA, and in one analysis, a TP53 mutation was only found in EV-DNA. CONCLUSION: EV-DNA extraction and sequencing in old serum samples of patients with BC is feasible and has the potential to address clinically relevant questions in longitudinal studies.
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Biomarcadores Tumorais , Neoplasias da Mama , Vesículas Extracelulares , Humanos , Neoplasias da Mama/genética , Neoplasias da Mama/sangue , Feminino , Vesículas Extracelulares/genética , Adulto , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , Mutação , Pessoa de Meia-Idade , Ácidos Nucleicos Livres/sangue , Ácidos Nucleicos Livres/genética , Biópsia Líquida/métodos , Análise de Sequência de DNA/métodosRESUMO
Background: Richter transformation refers to the progression of an initially slow-growing small lymphocytic lymphoma/chronic lymphocytic leukemia (SLL/CLL) into an aggressive lymphoma, typically diffuse large B-cell lymphoma (DLBCL) or Hodgkin lymphoma. Case presentation: The patient presented with a rapid onset of localized cervical swelling, accompanied by monoclonal B-cell lymphocytosis displaying a CLL immunophenotype. The histopathological analysis identified a Burkitt lymphoma (BL) located in the submandibular gland and adjacent lymph node. The patient's bone marrow displayed a minor infiltration of monoclonal B-cells with a CLL immunophenotype (< 10%). Molecular analysis demonstrated the presence of the same monoclonal rearrangement in the framework region (FR3 region) of the immunoglobulin heavy chain (IGH) locus. High-throughput sequencing of the immunoglobulin heavy and light chains also confirmed the presence of the same rearrangement in SLL/CLL and in the Burkitt lymphoma sample, but also highlighted the presence of a second rearrangement in the Burkitt lymphoma cells, not shared with the SLL/CLL cells in the bone marrow. The patient was treated with DA-EPOCH-R, which lead to a complete metabolic response. Conclusion: This report provides an exceptionally rare description of a CLL-type monoclonal B-cell lymphocytosis transforming into a very aggressive Burkitt lymphoma in a treatment naïve patient.
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PURPOSE: The integration of immunotherapy in the perioperative setting of muscle-invasive urothelial carcinoma (MIUC) appears promising. SAKK 06/17 investigated the addition of neoadjuvant durvalumab to gemcitabine/cisplatin (GC) chemotherapy followed by radical surgery and adjuvant checkpoint inhibition with durvalumab. PATIENTS AND METHODS: SAKK 06/17 was an investigator-initiated, open-label, single-arm phase II study including cisplatin-fit patients with stage cT2-T4a cN0-1 operable MIUC. Four cycles of neoadjuvant GC in combination with four cycles of durvalumab (start with GC cycle 2) were administered, followed by radical surgery. Adjuvant durvalumab was given for 10 cycles. The primary end point was event-free survival (EFS) at 2 years. RESULTS: Sixty one patients were accrued at 12 sites. The full analysis set consisted of 57 patients, 54 (95%) had bladder cancer. Median follow-up was 40 months. The primary end point was met, with EFS at 2 years of 76% (one-sided 90% CI [lower bound], 67%; two-sided 95% CI, 62 to 85). EFS at 3 years was 73% (95% CI, 59 to 83). Complete pathologic response in resected patients (N = 52) was achieved in 17 patients (33%), and 31 (60%) had pathologic response Assuntos
Carcinoma de Células de Transição
, Neoplasias da Bexiga Urinária
, Humanos
, Neoplasias da Bexiga Urinária/tratamento farmacológico
, Neoplasias da Bexiga Urinária/cirurgia
, Carcinoma de Células de Transição/tratamento farmacológico
, Carcinoma de Células de Transição/cirurgia
, Cisplatino/efeitos adversos
, Desoxicitidina/efeitos adversos
, Músculos
, Imunoterapia
, Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos
, Terapia Neoadjuvante/efeitos adversos
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INTRODUCTION: The clinical course of prostate cancer (PCa) is highly variable, ranging from indolent behavior to rapid metastatic progression. The Gleason score is widely accepted as the primary histologic assessment tool with significant prognostic value. However, additional biomarkers are required to better stratify patients, particularly those at intermediate risk. METHODS: In this study, we analyzed the expression of 86 cancer hallmark genes in 171 patients with PCa who underwent radical prostatectomy and focused on the outcome of the 137 patients with postoperative R0-PSA0 status. RESULTS: Low expression of the IGF1 and SRD52A, and high expression of TIMP2, PLAUR, S100A2, and CANX genes were associated with biochemical recurrence (BR), defined as an increase of prostate-specific antigen above 0.2 ng/mL. Furthermore, the analysis of the expression of 462 noncoding RNAs (ncRNA) in a sub-cohort of 39 patients with Gleason score 7 tumors revealed that high levels of expression of the ncRNAs LINC00624, LINC00593, LINC00482, and cd27-AS1 were significantly associated with BR. Our findings provide further evidence for tumor-promoting roles of ncRNAs in PCa patients at intermediate risk. The strong correlation between expression of LINC00624 and KRT8 gene, encoding a well-known cell surface protein present in PCa, further supports a potential contribution of this ncRNA to PCa progression. CONCLUSION: While larger and further studies are needed to define the role of these genes/ncRNA in PCa, our findings pave the way toward the identification of a subgroup of patients at intermediate risk who may benefit from adjuvant treatments and new therapeutic agents.
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Neoplasias da Próstata , RNA Longo não Codificante , Masculino , Humanos , RNA Longo não Codificante/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/cirurgia , Neoplasias da Próstata/patologia , Próstata/patologia , Antígeno Prostático Específico , Prostatectomia , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/cirurgia , Gradação de TumoresRESUMO
BACKGROUND & AIMS: Liver injury after COVID-19 vaccination is very rare and shows clinical and histomorphological similarities with autoimmune hepatitis (AIH). Little is known about the pathophysiology of COVID-19 vaccine-induced liver injury (VILI) and its relationship to AIH. Therefore, we compared VILI with AIH. METHODS: Formalin-fixed and paraffin-embedded liver biopsy samples from patients with VILI (n = 6) and from patients with an initial diagnosis of AIH (n = 9) were included. Both cohorts were compared by histomorphological evaluation, whole-transcriptome and spatial transcriptome sequencing, multiplex immunofluorescence, and immune repertoire sequencing. RESULTS: Histomorphology was similar in both cohorts but showed more pronounced centrilobular necrosis in VILI. Gene expression profiling showed that mitochondrial metabolism and oxidative stress-related pathways were more and interferon response pathways were less enriched in VILI. Multiplex analysis revealed that inflammation in VILI was dominated by CD8+ effector T cells, similar to drug-induced autoimmune-like hepatitis. In contrast, AIH showed a dominance of CD4+ effector T cells and CD79a+ B and plasma cells. T-cell receptor (TCR) and B-cell receptor sequencing showed that T and B cell clones were more dominant in VILI than in AIH. In addition, many T cell clones detected in the liver were also found in the blood. Interestingly, analysis of TCR beta chain and Ig heavy chain variable-joining gene usage further showed that TRBV6-1, TRBV5-1, TRBV7-6, and IgHV1-24 genes are used differently in VILI than in AIH. CONCLUSIONS: Our analyses support that SARS-CoV-2 VILI is related to AIH but also shows distinct differences from AIH in histomorphology, pathway activation, cellular immune infiltrates, and TCR usage. Therefore, VILI may be a separate entity, which is distinct from AIH and more closely related to drug-induced autoimmune-like hepatitis. IMPACT AND IMPLICATIONS: Little is known about the pathophysiology of COVID-19 vaccine-induced liver injury (VILI). Our analysis shows that COVID-19 VILI shares some similarities with autoimmune hepatitis, but also has distinct differences such as increased activation of metabolic pathways, a more prominent CD8+ T cell infiltrate, and an oligoclonal T and B cell response. Our findings suggest that VILI is a distinct disease entity. Therefore, there is a good chance that many patients with COVID-19 VILI will recover completely and will not develop long-term autoimmune hepatitis.
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COVID-19 , Doença Hepática Crônica Induzida por Substâncias e Drogas , Hepatite Autoimune , Humanos , Vacinas contra COVID-19/efeitos adversos , SARS-CoV-2 , COVID-19/prevenção & controle , Fígado/patologia , Receptores de Antígenos de Linfócitos T , VacinaçãoRESUMO
Vaccines against SARS-CoV-2 are the most effective measure against the COVID-19 pandemic. The safety profile of mRNA vaccines in patients with rare diseases has not been assessed systematically in the clinical trials, as these patients were typically excluded. This report describes the occurrence of agranulocytosis within days following the first dose of an mRNA-1273 vaccination against COVID-19 in a previously healthy older adult. The patient was diagnosed with a suspected STAT3 wild-type T-cell large granular lymphocytic leukaemia (T-LGL). Neutropenia was successfully treated with IVIG, glucocorticoids, and G-CSF. In vitro experiments aimed at elucidating the pathways potentially causing the mRNA vaccine-associated neutropenia indicated that the mRNA, but not the adenoviral Ad26.COV2.S vector vaccine, triggered strong IL-6/STAT3 activation in vitro, resulting in excessive T-cell activation and neutrophil degranulation in the patient but not in controls. mRNA-1273 activated TLR-3 suggesting TLR mediated IL-6/STAT3 pathway activation. To complete the primary series of COVID-19 immunization, we used a single dose of Ad26.COV2.S vector vaccine without reoccurrence of neutropenia. The T-LGL clone remained stable during the follow-up of more than 12 months without ongoing therapy. Our data suggest that switching the immunization platform may be a reasonable approach in subjects with rare associated hematologic side effects due to excess STAT3-mediated stimulation following mRNA vaccination. Using in vitro testing before re-administration of a (COVID) vaccine also has relevance for other rare immune events after (mRNA) vaccination.
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COVID-19 , Leucemia Linfocítica Granular Grande , Neutropenia , Humanos , Idoso , Vacina de mRNA-1273 contra 2019-nCoV , Ad26COVS1 , Vacinas contra COVID-19 , Interleucina-6 , Pandemias , SARS-CoV-2 , Vacinação , Adenoviridae , Fator de Transcrição STAT3RESUMO
Cemento-osseous dysplasia (COD) belongs to the spectrum of benign fibro-osseous lesions occurring exclusively in the tooth-bearing areas of the jaws. Depending on site and extent of involvement, periapical, focal and florid subtypes can be distinguished that share an identical histomorphology. Most cases are asymptomatic and follow a self-limited course requiring no specific treatment. Over time, lesions progressively mineralise while the cellularity decreases. However, the molecular pathogenesis of COD, has not yet been explored. We analysed a series of 31 COD samples by targeted sequencing and detected pathogenic hotspot mutations involving the RAS-MAPK signalling pathway in 5/18 evaluable cases (28%). The mutations were found in the BRAF, HRAS, KRAS, NRAS, and FGFR3 genes. Our findings suggest that COD is driven by RAS-MAPK activation; however, the mechanism underlying the spontaneous growth arrest typically occuring in most of the lesions remains elusive.
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Tumores Odontogênicos , Humanos , Mutação , Transdução de Sinais , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas ras/metabolismoRESUMO
INTRODUCTION: Colorectal carcinoma (CRC) is among the most common carcinomas in women and men. In the advanced stage, patients are treated based on the RAS status. Recent studies indicate that in the future, in addition to KRAS and NRAS, alterations in other genes, such as PIK3CA or TP53, will be considered for therapy. Therefore, it is important to know the mutational landscape of routinely diagnosed CRC. METHOD: We report the molecular profile of 512 Swiss CRC patients analyzed by targeted next-generation sequencing as part of routine diagnostics at our institute. RESULTS: Pathogenic and likely pathogenic variants were found in 462 (90%) CRC patients. Variants were detected in TP53 (54.3%), KRAS (48.2%), PIK3CA (15.6%), BRAF (13.5%), SMAD4 (10.5%), FBXW7 (7.8%), NRAS (3.5%), PTEN (2.7%), ERBB2 (1.6%), AKT1 (1.5%), and CTNNB1 (0.9%). The remaining pathogenic alterations were found in the genes ATM(n= 1), MAP2K1(n= 1), and IDH2(n= 1). DISCUSSION/CONCLUSIONS: Our analysis revealed the prevalence of potential predictive markers in a large cohort of CRC patients obtained during routine diagnostic analysis. Furthermore, our study is the first of this size to uncover the molecular landscape of CRC in Switzerland.
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Neoplasias Colorretais , Proteínas Proto-Oncogênicas p21(ras) , Masculino , Humanos , Feminino , Prevalência , Proteínas Proto-Oncogênicas p21(ras)/genética , Suíça/epidemiologia , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Mutação , Classe I de Fosfatidilinositol 3-Quinases/genética , Sequenciamento de Nucleotídeos em Larga EscalaRESUMO
BACKGROUND: Patients with advanced squamous-cell lung cancer (SQCLC) frequently (46%) exhibit tumor overexpression of fibroblast growth factor receptor (FGFR) messenger ribonucleic acid (mRNA). Rogaratinib is a novel oral pan-FGFR inhibitor with a good safety profile and anti-tumor activity in early clinical trials as a single agent in FGFR pathway-addicted tumors. SAKK 19/18 determined clinical activity of rogaratinib in patients with advanced SQCLC overexpressing FGFR1-3 mRNA. METHODS: Patients with advanced SQCLC failing standard systemic treatment and with FGFR1-3 mRNA tumor overexpression as defined in the protocol received rogaratinib 600 mg BID until disease progression or intolerable toxicity. A 6-months progression-free survival rate (6mPFS) ≤15 % was considered uninteresting (H0), whereas a 6mPFS ≥38 % was considered promising (H1). According to a Simon 2-stage design, 2 out of 10 patients of the first stage were required to be progression-free at 6 months. Comprehensive Genomic Profiling was performedusing the Oncomine Comprehensive Assay Plus (Thermo Fisher Scientific). RESULTS: Between July 2019 and November 2020, 49 patients were screened and 20 were classified FGFR-positive. Among a total of 15 patients, 6mPFS was reached in 1 patient (6.7 %), resulting in trial closure for futility after the first stage. There were 7 (46.7 %) patients with stable disease and 5 (33.3 %) patients with progressive disease. Median PFS was 1.6 (95 % CI 0.9-3.5) months and median overall survival (OS) 3.5 (95 % CI 1.0-5.9) months. Most frequent treatment-related adverse events (TRAEs) included hyperphosphatemia in 8 (53 %), diarrhea in 5 (33 %), stomatitis in 3 (20 %) and nail changes in 3 (20 %) patients. Grade ≥3 TRAEs occurred in 6 (40 %) patients. No associations between mutational profile and treatment outcome were observed. CONCLUSION: Despite preliminary signals of activity, rogaratinib failed to improve PFS in patients with advanced SQCLC overexpressing FGFR mRNA. FGFR inhibitors in SQCLC remain a challenging field, and more in-depth understanding of pathway crosstalks may lead to the development of drug combinations with FGFR inhibitors resulting in improved outcomes.
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Carcinoma Pulmonar de Células não Pequenas , Carcinoma de Células Escamosas , Neoplasias Pulmonares , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma de Células Escamosas/tratamento farmacológico , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Piperazinas , Inibidores de Proteínas Quinases/uso terapêutico , Pirróis , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , TiofenosRESUMO
Brown tumors are rare and generally self-limiting mass lesions of bone occurring in the context of hyperparathyroidism. Although commonly regarded as endocrine-driven tumor-like lesions, we detected pathogenic hotspot KRAS mutations in 10/16 brown tumors (62%) with similar frequencies found in cases affecting the peripheral and axial skeleton. Pathogenic mutations in other driver genes of the RAS-MAPK pathway were not identified. Our findings suggest brown tumors to represent true neoplasms driven by the activation of the RAS-MAPK signaling pathway. The frequent regression of brown tumors after normalization of hyperparathyroidism points to a second hit mediated by endocrine stimulation to be required for tumor development. Our findings underline the pathogenic relation of brown tumors to nonossifying fibroma and giant cell granuloma of the jaws which both appear histologically similar to brown tumors and are also driven by RAS-MAPK signaling pathway activation.
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Fibroma , Granuloma de Células Gigantes , Hiperparatireoidismo , Granuloma de Células Gigantes/genética , Granuloma de Células Gigantes/patologia , Humanos , Mutação , Proteínas Proto-Oncogênicas p21(ras)/genéticaRESUMO
Broad molecular profiling by next-generation sequencing of solid tumors has become a critical tool for clinical decision-making in the era of precision oncology. In addition to many already approved targeted therapies, more than half of ongoing oncology-related clinical trials are biomarker-driven. Therefore, accurate and reliable assays are needed to assess the genetic make-up of tumor cells and guide clinicians in the therapy decision process. In order to obtain high-quality NGS data for variant detection, certain preanalytical steps and quality metrics should be followed. These include assessment of sample types, choice of extraction method, library preparation technology, sequencing platform, and finally sequencing quality control. Each of these steps has certain challenges and pitfalls that need to be addressed and overcome, respectively. In this chapter, we address the preanalytical quality control and how each of the involved steps may influence the final result. Following these guidelines and QC metrics may help in obtaining optimal results that will allow the precise and robust assessment of genetic variants in a clinical setting.
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Neoplasias , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Neoplasias/diagnóstico , Neoplasias/genética , Medicina de Precisão , Controle de QualidadeRESUMO
Anti-PD-1 therapy can provide long, durable benefit to a fraction of patients. The on-label PD-L1 test, however, does not accurately predict response. To build a better biomarker, we created a method called T Cell Subtype Profiling (TCSP) that characterizes the abundance of T cell subtypes (TCSs) in FFPE specimens using five RNA models. These TCS RNA models are created using functional methods, and robustly discriminate between naïve, activated, exhausted, effector memory, and central memory TCSs, without the reliance on non-specific, classical markers. TCSP is analytically valid and corroborates associations between TCSs and clinical outcomes. Multianalyte biomarkers based on TCS estimates predicted response to anti-PD-1 therapy in three different cancers and outperformed the indicated PD-L1 test, as well as Tumor Mutational Burden. Given the utility of TCSP, we investigated the abundance of TCSs in TCGA cancers and created a portal to enable researchers to discover other TCSP-based biomarkers.
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Linfócitos T CD8-Positivos/metabolismo , Neoplasias/tratamento farmacológico , Receptor de Morte Celular Programada 1/metabolismo , Biomarcadores Tumorais/metabolismo , Linfócitos T CD8-Positivos/patologia , Células Cultivadas , Humanos , Leucócitos MononuclearesRESUMO
Diffuse intrinsic pontine glioma (DIPG) is an incurable paediatric malignancy. Identifying the molecular drivers of DIPG progression is of the utmost importance. Long non-coding RNAs (lncRNAs) represent a large family of disease- and tissue-specific transcripts, whose functions have not yet been elucidated in DIPG. Herein, we studied the oncogenic role of the development-associated H19 lncRNA in DIPG. Bioinformatic analyses of clinical datasets were used to measure the expression of H19 lncRNA in paediatric high-grade gliomas (pedHGGs). The expression and sub-cellular location of H19 lncRNA were validated in DIPG cell lines. Locked nucleic acid antisense oligonucleotides were designed to test the function of H19 in DIPG cells. We found that H19 expression was higher in DIPG vs. normal brain tissue and other pedHGGs. H19 knockdown resulted in decreased cell proliferation and survival in DIPG cells. Mechanistically, H19 buffers let-7 microRNAs, resulting in the up-regulation of oncogenic let-7 target (e.g., SULF2 and OSMR). H19 is the first functionally characterized lncRNA in DIPG and a promising therapeutic candidate for treating this incurable cancer.
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Neoplasias do Tronco Encefálico/genética , Proliferação de Células , Glioma/genética , RNA Longo não Codificante/metabolismo , Neoplasias do Tronco Encefálico/metabolismo , Neoplasias do Tronco Encefálico/patologia , Células Cultivadas , Regulação Neoplásica da Expressão Gênica , Glioma/metabolismo , Glioma/patologia , Histonas/metabolismo , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Mutação , RNA Longo não Codificante/genéticaRESUMO
Tumor mutation burden (TMB) is evaluated as a biomarker of response to immunotherapy. We present the efforts of the Onconetwork Immuno-Oncology Consortium to validate a commercial targeted sequencing test for TMB calculation. A three-phase study was designed to validate the Oncomine Tumor Mutational Load (OTML) assay at nine European laboratories. Phase 1 evaluated reproducibility and accuracy on seven control samples. In phase 2, six formalin-fixed, paraffin-embedded samples tested with FoundationOne were reanalyzed with the OTML panel to evaluate concordance and reproducibility. Phase 3 involved analysis of 90 colorectal cancer samples with known microsatellite instability (MSI) status to evaluate TMB and MSI association. High reproducibility of TMB was demonstrated among the sites in the first and second phases. Strong correlation was also detected between mean and expected TMB in phase 1 (r2 = 0.998) and phase 2 (r2 = 0.96). Detection of actionable mutations was also confirmed. In colorectal cancer samples, the expected pattern of MSI-high/high-TMB and microsatellite stability/low-TMB was present, and gene signatures produced by the panel suggested the presence of a POLE mutation in two samples. The OTML panel demonstrated robustness and reproducibility for TMB evaluation. Results also suggest the possibility of using the panel for mutational signatures and variant detection. Collaborative efforts between academia and companies are crucial to accelerate the translation of new biomarkers into clinical research.
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Neoplasias Colorretais/genética , DNA/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Instabilidade de Microssatélites , Carga Tumoral/genética , Células A549 , Biomarcadores Tumorais/genética , Neoplasias Colorretais/patologia , DNA/isolamento & purificação , Reparo de Erro de Pareamento de DNA/genética , Análise Mutacional de DNA/métodos , Confiabilidade dos Dados , Humanos , Células MCF-7 , Reprodutibilidade dos TestesRESUMO
Metastatic neuroendocrine prostate cancer (NEPC) is a highly aggressive disease, whose incidence is rising. Long noncoding RNAs (lncRNAs) represent a large family of disease- and tissue-specific transcripts, most of which are still functionally uncharacterized. Thus, we set out to identify the highly conserved lncRNAs that play a central role in NEPC pathogenesis. To this end, we performed transcriptomic analyses of donor-matched patient-derived xenograft models (PDXs) with immunohistologic features of prostate adenocarcinoma (AR+ /PSA+ ) or NEPC (AR- /SYN+ /CHGA+ ) and through differential expression analyses identified lncRNAs that were upregulated upon neuroendocrine transdifferentiation. These genes were prioritized for functional assessment based on the level of conservation in vertebrates. Here, LINC00261 emerged as the top gene with over 3229-fold upregulation in NEPC. Consistently, LINC00261 expression was significantly upregulated in NEPC specimens in multiple patient cohorts. Knockdown of LINC00261 in PC-3 cells dramatically attenuated its proliferative and metastatic abilities, which are explained by parallel downregulation of CBX2 and FOXA2 through distinct molecular mechanisms. In the cell cytoplasm, LINC00261 binds to and sequesters miR-8485 from targeting the CBX2 mRNA, while inside the nucleus, LINC00261 functions as a transcriptional scaffold to induce SMAD-driven expression of the FOXA2 gene. For the first time, these results demonstrate hyperactivation of the LINC00261-CBX2-FOXA2 axes in NEPC to drive proliferation and metastasis, and that LINC00261 may be utilized as a therapeutic target and a biomarker for this incurable disease.
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Neoplasias da Próstata , RNA Longo não Codificante , Animais , Linhagem Celular Tumoral , Proliferação de Células/genética , Citoplasma/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Próstata/metabolismo , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismoRESUMO
OBJECTIVES: Tumor mutational burden (TMB) has emerged as a promising predictive biomarker for immune checkpoint inhibitor therapy. While the feasibility of TMB analysis on formalin-fixed paraffin-embedded (FFPE) samples has been thoroughly evaluated, only limited analyses have been performed on cytological samples, and no dedicated study has investigated concordance of TMB between different sample types. Here, we assessed TMB on matched histological and cytological samples from lung cancer patients and evaluated the accuracy of TMB estimation in these sample types. MATERIALS AND METHODS: We analyzed mutations and resulting TMB in FFPE samples and matched ethanol-fixed cytological smears (nâ¯=â¯12 matched pairs) by using a targeted next-generation sequencing assay (Oncomine™ Tumor Mutational Load). Two different variant allele frequency (VAF) thresholds were used to estimate TMB (VAFâ¯=â¯5% or 10%). RESULTS: At 5% VAF threshold, 73% (107/147) of mutations were concordantly detected in matched histological and cytological samples. Discordant variants were mainly unique to FFPE samples (34/40 discordant variants) and mostly C:Gâ¯>â¯T:A transitions with low allelic frequency, likely indicating formalin fixation artifacts. Increasing the VAF threshold to 10% clearly increased the number of concordantly detected mutations in matched histological and cytological samples to 96% (100/106 mutations), and drastically reduced the number of FFPE-only mutations (from 34 to 4 mutations). In contrast, cytological samples showed consistent mutation count and TMB values at both VAF thresholds. Using FFPE samples, 2 out of 12 patients were classified as TMB-high at VAF cutoff of 5% but TMB-low at 10%, whereas cytological specimens allowed consistent patient classification independently from VAF cutoff. CONCLUSION: Our results show that cytological smears provide more consistent TMB values due to high DNA quality and lack of formalin-fixation induced artifacts. Therefore, cytological samples should be the preferred sample type for robust TMB estimation.
Assuntos
Neoplasias Pulmonares , Biomarcadores Tumorais , Análise Mutacional de DNA , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Neoplasias Pulmonares/genética , Mutação , Carga TumoralRESUMO
Background: Castration-resistant prostate cancer (CRPC) is an incurable malignancy. Long noncoding RNAs (lncRNAs) play key roles in drug resistance. Materials & methods: LncRNA HORAS5 role in cabazitaxel resistance (i.e., cell-count, IC50 and caspase activity) was studied via lentiviral-mediated overexpression and siRNA-based knockdown. Genes expression was analyzed with RNA-sequencing, reverse transcription quantitative PCR (RT-qPCR) and western blot. HORAS5 expression was queried in clinical database. Results: Cabazitaxel increased HORAS5 expression that upregulated BCL2A1, thereby protecting CRPC cells from cabazitaxel-induced apoptosis. BCL2A1 knockdown decreased cell-count and increased apoptosis in CRPC cells. HORAS5-targeting antisense oligonucleotide decreased cabazitaxel IC50. In CRPC clinical samples, HORAS5 expression increased upon taxane treatment. Conclusion:HORAS5 stimulates the expression of BCL2A1 thereby decreasing apoptosis and enhancing cabazitaxel resistance in CRPC cells.
Assuntos
Antineoplásicos/farmacologia , Regulação Neoplásica da Expressão Gênica , Antígenos de Histocompatibilidade Menor/genética , Neoplasias de Próstata Resistentes à Castração/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA Longo não Codificante/metabolismo , Taxoides/farmacologia , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Masculino , Antígenos de Histocompatibilidade Menor/biossíntese , Antígenos de Histocompatibilidade Menor/fisiologia , Oligonucleotídeos Antissenso , Neoplasias de Próstata Resistentes à Castração/metabolismo , Neoplasias de Próstata Resistentes à Castração/patologia , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , RNA Longo não Codificante/antagonistas & inibidores , RNA Longo não Codificante/biossínteseRESUMO
In non-small cell lung cancer (NSCLC), immune checkpoint inhibitors (ICIs) significantly improve overall survival (OS). Tumor mutational burden (TMB) has emerged as a predictive biomarker for patients treated with ICIs. Here, we evaluated the predictive power of TMB measured by the Oncomine™ Tumor Mutational Load targeted sequencing assay in 76 NSCLC patients treated with ICIs. TMB was assessed retrospectively in 76 NSCLC patients receiving ICI therapy. Clinical data (RECIST 1.1) were collected and patients were classified as having either durable clinical benefit (DCB) or no durable benefit (NDB). Additionally, genetic alterations and PD-L1 expression were assessed and compared with TMB and response rate. TMB was significantly higher in patients with DCB than in patients with NDB (median TMB = 8.5 versus 6.0 mutations/Mb, Mann-Whitney p = 0.0244). 64% of patients with high TMB (cut-off = third tertile, TMB ≥ 9) were responders (DCB) compared to 33% and 29% of patients with intermediate and low TMB, respectively (cut-off = second and first tertile, TMB = 5-9 and TMB ≤ 4, respectively). TMB-high patients showed significantly longer progression-free survival (PFS) and OS (log-rank test p = 0.0014 for PFS and 0.0197 for OS). While identifying different subgroups of patients, combining PD-L1 expression and TMB increased the predictive power (from AUC 0.63 to AUC 0.65). Our results show that the TML panel is an effective tool to stratify patients for ICI treatment. A combination of biomarkers might maximize the predictive precision for patient stratification. Our study supports TMB evaluation through targeted NGS in NSCLC patient samples as a tool to predict response to ICI therapy. We offer recommendations for a reliable and cost-effective assessment of TMB in a routine diagnostic setting. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
Assuntos
Biomarcadores Tumorais/genética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Análise Mutacional de DNA/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Neoplasias Pulmonares/genética , Mutação , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos Imunológicos/uso terapêutico , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/imunologia , Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Tomada de Decisão Clínica , Feminino , Predisposição Genética para Doença , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Seleção de Pacientes , Fenótipo , Medicina de Precisão , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , SuíçaRESUMO
Pre-symptomatic screening of genetic alterations might help identify subpopulations of individuals that could enter into early access prevention programs. Since liquid biopsy is minimally invasive it can be used for longitudinal studies in healthy volunteers to monitor events of progression from normal tissue to pre-cancerous and cancerous condition. Yet, cell-free DNA (cfDNA) analysis in healthy individuals comes with substantial challenges such as the lack of large cohort studies addressing the impact of mutations in healthy individuals or the low abundance of cfDNA in plasma. In this study, we aimed to investigate the technical feasibility of cfDNA analysis in a collection of 114 clinically healthy individuals. We first addressed the impact of pre-analytical factors such as cfDNA yield and quality on sequencing performance and compared healthy to cancer donor samples. We then confirmed the validity of our testing strategy by evaluating the mutational status concordance in matched tissue and plasma specimens collected from cancer patients. Finally, we screened our group of healthy donors for genetic alterations, comparing individuals who did not develop any tumor to patients who developed either a benign neoplasm or cancer during 1-10 years of follow-up time. To conclude, we have established a rapid and reliable liquid biopsy workflow that allowed us to study genomic alterations with a limit of detection as low as 0.08% of variant allelic frequency in healthy individuals. We detected pathogenic cancer mutations in four healthy donors that later developed a benign neoplasm or invasive breast cancer up to 10 years after blood collection. Even though larger prospective studies are needed to address the specificity and sensitivity of liquid biopsy as a clinical tool for early cancer detection, systematic screening of healthy individuals will help understanding early events of tumor formation.