Performance of non-formalin fixed paraffin embedded samples in hybrid capture and amplicon next-generation sequencing panels.

BACKGROUND: Genomic profiling using next-generation sequencing (NGS) is fundamental for driving prognostic and therapy in cancer. Formalin-fixed paraffin embedded (FFPE) tissue is the widely used material, whereas non-FFPE may represent an alternative. However, studies comparing the NGS performance of non-FFPE materials to FFPE are still lacking in the literature. The objective of this study was to characterize in non-FFPE preparations the nucleic acid yield and NGS performance on both a capture-based and an amplicon-based NGS platform. NGS quality metrics obtained from non-FFPE preparations were compared to FFPE. METHODS: We analyzed the cellularity and nucleic acid yield in 111 tumors from non-FFPE preparations. In addition, comprehensive hybrid capture panel sequencing metrics obtained from DNA and RNA libraries were compared between independent non-FFPE and FFPE samples. A paired comparison between non-FFPE and FFPE samples was performed to analyze concordance in mutant allele detection using an amplicon panel. RESULTS: The mean target coverage from DNA libraries was 2× higher in non-FFPE samples than in FFPE. The detection of exogenous DNA was 2.5× higher in non-FFPE than in FFPE. Conversely, a lower performance was observed in non-FFPE RNA libraries in comparison to FFPE DNA libraries with no impact in minimum standard cutoffs. The variant allele detection in non-FFPE was found to be comparable to that of FFPE tumor samples in matched samples. CONCLUSIONS: Non-FFPE was demonstrated to be a suitable material for DNA and RNA library preparations using a comprehensive NGS panel. This is the first study reporting library quality metrics according to the TSO500 analysis pipeline.

A Prospective Cohort Study of Biomarkers in Squamous Cell Carcinoma of the Anal Canal (SCCAC) and their Influence on Treatment Outcomes.

Background: Although Chemoradiation (CRT) is the curative treatment for SCCAC, many patients present primary resistance. Since it is a rare tumor, response predictors remain unknown. Methods: We performed a prospective cohort study to evaluate biomarkers associated with CRT response, progression-free survival (PFS), and overall survival (OS). The primary endpoint was response at 6 months (m). Tumor DNA and HPV were analyzed by next-generation sequencing, while KI-67 and PD-L1 by immunohistochemistry in tumor tissue. Results: Seventy-eight patients were recruited between October/2011 and December/2015, and 75 were response evaluable. The median age was 57 years, 65% (n=49) were stage III and 12% (n=9) were HIV positive (HIV+). At 6m, 62.7% (n=47) presented CR. On multivariate analyses, stage II patients were 4.7 more likely to achieve response than stage III (OR, 4.70; 95%CI, 1.36-16.30; p=0.015). HIV+ was associated with a worse response (OR, 5.72; 95%CI, 2.5-13.0; p<0.001). 5-year PFS and OS rates were 63.3% and 76.4%, respectively, with a median follow up of 66m. On multivariate analyses, older age (HR 1.06, p=0.022, 95%IC 1.01-1.11) and absence of CR at 6m (HR 3.36, p=0.007, 95%IC 1.39-8.09) were associated with inferior OS. The 5-year OS rate was 62.5% in HIV+ group compared to 78% among HIV- pts, although this difference was not statistically significant (p=0.4). PIK3CA, MET and TP53 mutations, HPV, Ki-67 expression, and PD-L1 expression, were not associated with PFS and OS. Conclusions: Clinical stage III and HIV+ were associated with worse response to CRT at 6m. The absence of CR was the main factor associated with poor 5-year OS.

Identification of the TP53 p.R337H Variant in Tumor Genomic Profiling Should Prompt Consideration of Germline Testing for Li-Fraumeni Syndrome.

PURPOSE: Li-Fraumeni syndrome (LFS) is rare in the worldwide population, but it is highly prevalent in the Brazilian population because of a founder mutation, TP53 p.R337H, accounting for 0.3% of south and southeastern population. Clinical criteria for LFS may not identify all individuals at risk of carrying the Brazilian founder mutation because of its lower penetrance and variable expressivity. This variant is rarely described in databases of somatic mutations. Somatic findings in tumor molecular profiling may give insight to identify individuals who might be carriers of LFS and allow the adoption of risk reduction strategies for cancer. MATERIALS AND METHODS: We determined the frequency of the TP53 p.R337H variant in tumor genomic profiling from 755 consecutive Brazilian patients with pan-cancer. This is a retrospective cohort from January 2013 to March 2020 at a tertiary care center in Brazil. RESULTS: The TP53 p.R337H variant was found in 2% (15 of 755) of the samples. The mutation allele frequency ranged from 30% to 91.7%. A total of seven patients were referred for genetic counseling and germline testing after tumor genomic profiling results were disclosed. All the patients who proceeded with germline testing (6 of 6) confirmed the diagnosis of LFS. Family history was available in 12 cases. Nine patients (9 of 12) did not meet LFS clinical criteria. CONCLUSION: The identification of the TP53 p.R337H variant in tumor genomic profiling should be a predictive finding of LFS in the Brazilian population and should prompt testing for germline status confirmation.

Effects of overinflation on procollagen type III expression in experimental acute lung injury.

INTRODUCTION: In acute lung injury (ALI), elevation of procollagen type III (PC III) occurs early and has an adverse impact on outcome. We examined whether different high-inflation strategies of mechanical ventilation (MV) in oleic acid (OA) ALI alter regional expression of PC III. METHODS: We designed an experimental, randomized, and controlled protocol in which rats were allocated to two control groups (no injury, recruited [alveolar recruitment maneuver after tracheotomy without MV; n = 4 rats] and control [n = 5 rats]) or four injured groups (one exposed to OA only [n = 10 rats] and three OA-injured and ventilated). The three OA-injured groups were ventilated for 1 hour according to the following strategies: LVHP-S (low volume-high positive end-expiratory pressure [PEEP], supine; n = 10 rats, tidal volume [VT] = 8 ml/kg, PEEP = 12 cm H2O), HVLP-S (high volume-low PEEP, supine; n = 10 rats, VT = 20 ml/kg, PEEP = 5 cm H2O), and HVLP-P (high volume-low PEEP, prone; n = 10 rats). Northern blot analysis for PC III and interleukin-1-beta (IL-1beta) and polymorphonuclear infiltration index (PMI) counting were performed in nondependent and dependent regions. Regional differences between groups were assessed by two-way analysis of variance after logarithmic transformation and post hoc tests. RESULTS: A significant interaction for group and region effects was observed for PC III (p = 0.012) with higher expression in the nondependent region for HVLP-S and LVHP-S, intermediate for OA and HVLP-P, and lower for control (group effect, p < 0.00001, partial eta2 = 0.767; region effect, p = 0.0007, partial eta2 = 0.091). We found high expression of IL-1beta (group effect, p < 0.00001, partial eta2 = 0.944) in the OA, HVLP-S, and HVLP-P groups without regional differences (p = 0.16). PMI behaved similarly (group effect, p < 0.00001, partial eta2 = 0.832). CONCLUSION: PC III expression is higher in nondependent regions and in ventilatory strategies that caused overdistension. This response was partially attenuated by prone positioning.

Low tidal volume ventilation induces proinflammatory and profibrogenic response in lungs of rats.

OBJECTIVE: We examined whether mechanical ventilation with low tidal volume induces polymorphonuclear infiltration and proinflammatory and profibrogenic responses in rat lungs compared dependent and nondependent lung region to expression of interleukin-1beta (IL-1beta) and alpha-1 procollagen III (PC III) mRNA. DESIGN: An experimental, randomized and controlled protocol with previously normal rats. INTERVENTIONS: Three groups of ten animals were studied. Two groups were ventilated (FIO2=0.3) in supine position for 1 h without positive end expiratory pressure, one group with a low tidal volume (6 ml/kg), and the other with a high tidal volume (24 ml/kg). In the third group animals were kept in spontaneous ventilation for 1 h. MEASUREMENTS AND RESULTS: After ventilation the right lung was used to quantify polymorphonuclear infiltration. The left lung was divided into dependent and nondependent regions, and expression of IL-1beta and PC III mRNA was quantified by northern blot analysis. The group ventilated with low tidal volume had greater polymorphonuclear infiltration IL-1beta and PC III mRNA expression than the nonventilated group. Similar results were observed with high tidal volumes. There was no difference between low and high tidal volume ventilation. Expression levels of IL-1beta and PC III mRNA were higher in the nondependent region of ventilated groups and equal in the nonventilated group. CONCLUSIONS: Even a low tidal volume mode of mechanical ventilation induces proinflammatory and profibrogenic response, with a nondependent predominance for IL-1beta and PC III mRNA expression in supine, ventilated, previously normal rats.

Desenvolvimento de um método de diagnóstico molecular para o câncer gástrico baseado na análise do perfil da expressão gência através da metodologia de cDNA array / Development of a molecular diagnosis tool for gastric cancer based on gene expression profile using the cDNA array methodology

O estudo do Câncer gástrico é de grande importância tendo em vista a sua alta taxa de incidência e mortalidade em todo o mundo. As estimativas do Instituto nacional do Câncer (INCA) para 1999 mostram que o câncer de estômago provocou o maior número de óbitos por câncer no Brasil. Hoje o maior avanço no entendimento do câncer gástrico advêm de estados relacionando o processo de oncogêneses com a infecção por H. pilori. O tratamento da infecção com antibióticos levou a uma redução significativa na incidência de câncer gástrico em países desenvolvidos, como o Japão e os EUA. Em países em desenvolvimento, como o Brasil, essa queda foi menos significativa devido a uma grande incidência de casos de re-infecção. A identificação de genes diferencialmente expressos no tecido gástrico normal e tumoral permitirá a descoberta de marcadores genéticos indicadores da evolução do processo de transformação celular. Considerando que as alterações morfológicas, hoje a base do diagnóstico, são consequentes de alterações moleculares, os métodos de diagnóstico baseados no perfil de expressão gênica poderão ser capazes de identificar lesões pré-malignas. Além disso, a determinação do perfil molecular de um tumor poderá determinar um regime de tratamento de forma tumor-específica... Identificamos, duas proteínas ribossomais denominadas de L26 e L27. A expressão de L26 foi encontrada em amostra tumoral e é um importante marcador para a diferenciação entre um adenocarcinoma pouco diferenciado e um linfoma gástrico. Encontramos uma sequência homóloga ao gene da Mucina 4 (MUC4). Sabe-se que há um aumento na expressão de MUC4 em Câncer de estômago e as mucinas sintetizadas pelas células malignas podem contribuir para a capacidade de invasão das células tumorais, favorecendo o surgimento de metástases... A partir deste estudo, poderemos identificar os genes relacionados com o desenvolvimento do câncer gástrico e viabilizar um diagnóstico molecular...(AU)

Gastric cancer is one of the leading causes of cancer-related death in Brazil and in the world. High frequency of gastric cancer-related death is mainly due to late-stage diagnosis. Hence, new tools aimed to early diagnostic would have a positive impact in the outcome of the disease. Using cDNA arrays, we analised the expression proflle of normal gastric mucosa (N), gastritis (G), intestinal metaplasia (M) and gastric tumor (I). Based on the differentially expressed genes, we developed diagnosis tools for identification of lesions in gastric mucosa. In a ftrst step, we used cDNA arrays having around 4,500 elements to compare the expression proflle of six samples of normal gastric mucosa and six samples of tumor gastric mucosa. Eighty differentially expressed cDNAs were identified and, using Self Organizing Map (SOM), their expression proflle allowed the precise separation of the normal from the tumor sample groups. In a second step, the expression pro file o f 3 7 6 distinct genes, derived from the ftrst analysis and plus a set of known altered genes in human cancer according to the literature, were analised in 99 gastric fragments represented by: N (n=28), G (n=21), M (n=22) and T (n=28). Pair wise comparisons between these samples allowed the identification of 42 differentially expressed genes with p<0.0009 in a Wilcoxon test. Using the clustering algorithm k-means, the expression profile of 18 genes allowed the clustering of the majority of N and G samples in a distinct group, the majority of M and T samples in two aditional groups and fourth heterogeneous group. We then applied Fisher's linear discriminat to identify trios of genes that could be used to build classifiers for class distinction. A lager number of classifiers could distinguish between NxT whereas, for the distinction of GxT and MxT, fewer classifiers were identified.Importandy, it was possible identify samples of intestinal metaplasia whose expression pattem resembled that of an adenocarcinoma and can now be used for follow-up of patients in order to determine their potencial as prognostic test for malignant transformation (AU)

Análise comparativa da expressão global de genes em mucosa gástrica normal e tumoraL / Comparative analysis of global gene expression in normal and tumoral gastric mucosa

O estudo do câncer gástrico é de grande importância tendo em vista a sua alta taxa de incidência e mortalidade em todo o mundo. As estimativas do Instituto Nacional do Câncer (INCA) para 1999 mostram que o câncer de estômago provocou o maior número de óbitos por câncer no Brasil. Hoje, o maior avanço no entendimento do câncer gástrico advêm de estudos relacionando o processo de oncogênese com a infecção por H. pylorí. O tratamento da infecção com antibióticos levou a uma redução significativa na incidência de câncer gástrico em países desenvolvidos, como o Japão e os EUA. Em países em desenvolvimento, como o Brasil, essa queda foi menos significativa devido a uma grande incidência de casos de re-infecção. A identificação de genes diferencialmente expressos no tecido gástrico normal e tumoral permitirá a descoberta de marcadores genéticos indicadores da evolução do processo de transformação celular. Considerando que as alterações morfológicas, hoje a base do diagnóstico, são consequentes de alterações moleculares, os métodos de diagnóstico baseados no perfil de expressão gênica poderão ser capazes de identificar lesões pré-malignas. Além disso, a determinação do perfil molecular de um tumor poderá determinar um regime de tratamento de forma tumorespecífica. Com isso, os estudos de análise de expressão gênica podem contribuir significativamente para a descoberta de marcadores moleculares de câncer. Neste trabalho, comparamos a expressão global de genes entre amostras de tecido gástrico normal e tu moral. Através do DDRT -PCR, recuperamos cerca de 50 bandas com perfil diferencial. A busca de homologia foi feita no banco de dados e cerca de 45°/o dos clones apresentaram homologia com sequências nucleotídicas de genes conhecidos e 31,6% apresentaram homologia com ESTs (Etiquetas de Análise comparativa da expressão global de genes em mucosa gástrico normal e tumoral Sequências Expressas). Cerca de 23, 7°/o não apresentaram nenhuma homologia significativa e portanto, podem representar novos genes. Identificamos, duas proteínas ribossomais denominadas de L26 e L27. A expressão de L26 foi encontrada em amostra tumoral e é um importante marcador para a diferenciação entre um adenocarcinoma pouco diferenciado e um linfoma gástrico. Encontramos uma sequência homóloga ao gene da Mucina 4 (MUC4). Sabe-se que há um aumento na expressão de MUC4 em câncer de estômago e as mucinas sintetizadas pelas células malignas podem contribuir para a capacidade de invasão das células tumorais, favorecendo o surgimento de metástases. Identificamos também, através do DDRT -PCR, o RNA mensageiro para a enzima Aflatoxina-p 1 Aldeído Redutase (AFAR) sendo expressa em uma amostra tumoral. Esta enzima está envolvida no mecanismo de detoxificação da aflatoxina. O que justifica um estudo das suas funções biológicas na mucosa gástrica. Este é o primeiro relato na literatura mostrando a expressão de AFAR na mucosa gástrica. O perfil de expressão deste gene entre mucosa gástrica normal e tumoral se mostrou variado. Será necessário uma amostragem maior para concluírmos sobre o perfil de expressão de AFAR e a sua utilização como marcador molecular. Entre os 18.376 clones analisados através do cDNA microarray, 150 clones apresentaram diferenças de expressão de, no mínimo, cinco vezes entre mucosa gástrica normal e tumoral. Um dos clones encontrados corresponde a uma sequência de cDNA denominada de SMSO. Através de uma análise por RT -PCR, SMSO apresentou maior nível de expressão em mucosa gástrica tumoral em relação à mucosa normal em quatro de sete pacientes analisados. Todas as seqüências identificadas irão compor um "microarray" e o seu nível de expressão será avaliado em diferentes amostras de mucosa gástrica normal e tumoral. A partir deste estudo, poderemos identificar os genes relacionados com o desenvolvimento do câncer gástrico e viabilizar um diagnóstico molecular