A Standardized Liquid Biopsy Preanalytical Protocol for Downstream Circulating-Free DNA Applications.

The term liquid biopsy (LB) refers to molecules such as proteins, DNA, RNA, cells, or extracellular vesicles in blood and other bodily fluids that originate from the primary and/or metastatic tumor. LB has emerged as a mainstay in translational research and has started to become part of clinical oncology practice, providing a minimally invasive alternative to solid biopsy. The LB allows real-time monitoring of a tumor via a minimally invasive sample extraction, such as blood. The applications include early cancer detection, patient follow-up for the detection of disease progression, assessment of minimal residual disease, and potential identification of molecular progression and mechanism of resistance. In order to achieve a reliable analysis of these samples that can be reported in the clinic, the preanalytical procedures should be carefully considered and strictly followed. Sample collection, quality, and storage are crucial steps that determine their usefulness in downstream applications. Here, we present standardized protocols from our liquid biopsy working module for collecting, processing, and storing plasma and serum samples for downstream liquid biopsy analysis based on circulating-free DNA. The protocols presented here require standard equipment and are sufficiently flexible to be applied in most laboratories focused on biological procedures.

Patients with triple-negative, JAK2V617F- and CALR-mutated essential thrombocythemia share a unique gene expression signature.

Approximately 10% to 15% of patients with essential thrombocythemia (ET) lack the common driver mutations, so-called "triple-negative" (TN) disease. We undertook a systematic approach to investigate for somatic mutations and delineate gene expression signatures in 46 TN patients and compared the results to those with known driver mutations and healthy volunteers. Deep, error-corrected, next-generation sequencing of peripheral blood mononuclear cells using the HaloPlexHS platform and whole-exome sequencing was performed. Using this platform, 10 (22%) of 46 patients had detectable mutations (MPL, n = 6; JAK2V617F, n = 4) with 3 of 10 cases harboring germline MPL mutations. RNA-sequencing and DNA methylation analysis were also performed by using peripheral blood mononuclear cells. Pathway analysis comparing healthy volunteers and ET patients (regardless of mutational status) identified significant enrichment for genes in the tumor necrosis factor, NFκB, and MAPK pathways and upregulation of platelet proliferative drivers such as ITGA2B and ITGB3. Correlation with DNA methylation showed a consistent pattern of hypomethylation at upregulated gene promoters. Interrogation of these promoter regions highlighted enrichment of transcriptional regulators, which were significantly upregulated in patients with ET regardless of mutation status, including CEBPß and NFκB. For "true" TN ET, patterns of gene expression and DNA methylation were similar to those in ET patients with known driver mutations. These observations suggest that the resultant ET phenotype may, at least in part and regardless of mutation type, be driven by transcriptional misregulation and may propagate downstream via the MAPK, tumor necrosis factor, and NFκB pathways with resultant JAK-STAT activation. These findings identify potential novel mechanisms of disease initiation that require further evaluation.

The risk of thrombosis in essential thrombocythemia is associated with the type of CALR mutation: A multicentre collaborative study.

OBJECTIVES: In patients with essential thrombocythemia (ET), after the JAK2V617F driver mutation, mutations in CALR are common (classified as type 1, 52-bp deletion or type 2, 5-bp insertion). CALR mutations have generally been associated with a lower risk of thrombosis. This study aimed to confirm the impact of CALR mutation type on thrombotic risk. METHODS: We retrospectively investigated 983 ET patients diagnosed in Spanish and Polish hospitals. RESULTS: With 7.5 years of median follow-up from diagnosis, 155 patients (15.8%) had one or more thrombotic event. The 5-year thrombosis-free survival (TFS) rate was 83.8%, 91.6% and 93.9% for the JAK2V617F, CALR-type 1 and CALR-type 2 groups, respectively (P = .002). Comparing CALR-type 1 and CALR-type 2 groups, TFS for venous thrombosis was lower in CALR-type 1 (P = .046), with no difference in TFS for arterial thrombosis observed. The cumulative incidence of thrombosis was significantly different comparing JAK2V617F vs CALR-type 2 groups but not JAK2V617F vs CALR-type 1 groups. Moreover, CALR-type 2 mutation was a statistically significant protective factor for thrombosis with respect to JAK2V617F in multivariate logistic regression (OR: 0.45, P = .04) adjusted by age. CONCLUSIONS: Our results suggest that CALR mutation type has prognostic value for the stratification of thrombotic risk in ET patients.

Impacto del virus papiloma humano en pacientes afectos de carcinoma escamoso de cavidad oral y orofaringe / Impact of human papilloma virus in patients with oral and oropharyngeal squamous cell carcinomas

Antecedentes y objetivos: Aunque existe evidencia científica que demuestra la relación de causalidad del virus papiloma humano (VPH) sobre el carcinoma escamoso de cabeza y cuello, su porcentaje de causalidad en las distintas regiones anatómicas permanece todavía en controversia. El presente estudio tiene como objetivos evaluar la relación del VPH con el carcinoma escamoso de cavidad oral y orofaringe (CECOO) en nuestra población de referencia, y estudiar la correlación entre diferentes pruebas de detección del VPH basadas en métodos de PCR e inmunohistoquímica. Material y método: Estudio retrospectivo en pacientes tratados de CECOO durante el año 2011, con un seguimiento de 6 años. La muestra se dividió en 2 grupos según la positividad a VPH, detectado mediante 2 técnicas: p16 por inmunohistoquímica y PCR. Se analizaron variables demográficas y clínicas mediante SPSS(R) 22.0, considerando una significación estadística con p<0,05. Resultados: Se analizaron 155 pacientes afectos de CECOO (edad media de 62,7 años y un 69% varones). Veintiséis casos resultaron p16+ (16,8%) y 19 casos PCR+ (12,3%). Los tumores VPH+ se localizaron predominantemente en orofaringe (42,1%; p=0,017) y mostraron una tendencia a ser más frecuentes en el sexo masculino, mayor incidencia en pacientes más jóvenes, menos en fumadores y bebedores, y mayor afectación ganglionar cervical en el momento diagnóstico. Los pacientes PCR+ presentaron mayor supervivencia (p=0,024), al igual que los p16+ (p=0,005). Conclusiones: La incidencia de VPH en CECOO en nuestro entorno actualmente es baja (12,3%), pero la presentación clínica y el pronóstico del paciente VPH+ difiere del clásico paciente fumador y/o bebedor, lo que implica valorar el manejo de estos pacientes de forma independiente. La tinción inmunohistoquímica para p16 tiene una gran capacidad diagnóstica para predecir el VPH (95,5%), aunque la herramienta de referencia sigue siendo la detección de secuencias del genoma del VPH

Background and objectives: Although there is scientific evidence demonstrating causation of human papilloma virus (HPV) on squamous cell carcinoma of head and neck, its percentage of causality on the anatomic region remains in dispute. This study was developed with the objectives of evaluating the relationship between HPV and oral and oropharyngeal squamous cell carcinomas (OOSCC), and of studying the correlation between HPV detection tests (PCR and p16). Material and method: Retrospective study of patients treated for OOSCC during 2011, with a follow-up of 6 years. The sample was divided into 2 groups according to HPV positivity, detected by 2 techniques: p16 by immunohistochemistry and PCR. Demographic and clinical variables were analysed using SPSS(R) 22.0, considering P<.05 to be statistically significant. Results: We analysed 155 patients affected by OOSCC (mean age of 62.7, where 69% were males). Twenty six cases were p16+ (16.8%) and 19 cases PCR+ (12.3%), The HPV+ tumours were located predominantly in the oropharynx (42.1%, P=.017) and demonstrated the tendency to be more frequent in males, with higher incidence in younger patients, lower in smokers and drinkers, and higher when patients have a greater cervical lymph node involvement at the time of diagnosis. The PCR+ patients had higher survival (P=.024), as did the p16+ (P=.005). Conclusions: The incidence of HPV in OOSCC is low (12.3%), but the clinical presentation and prognosis of the HPV+ patient differs from the classic smoker and/or drinker, which implies assessing the management of these patients independently. The p16 staining has a great diagnostic capacity to predict HPV (95.5%), although the detection of the HPV genome is still the gold standard technique

Impact of human papilloma virus in patients with oral and oropharyngeal squamous cell carcinomas. / Impacto del virus papiloma humano en pacientes afectos de carcinoma escamoso de cavidad oral y orofaringe.

BACKGROUND AND OBJECTIVES: Although there is scientific evidence demonstrating causation of human papilloma virus (HPV) on squamous cell carcinoma of head and neck, its percentage of causality on the anatomic region remains in dispute. This study was developed with the objectives of evaluating the relationship between HPV and oral and oropharyngeal squamous cell carcinomas (OOSCC), and of studying the correlation between HPV detection tests (PCR and p16). MATERIAL AND METHOD: Retrospective study of patients treated for OOSCC during 2011, with a follow-up of 6 years. The sample was divided into 2 groups according to HPV positivity, detected by 2 techniques: p16 by immunohistochemistry and PCR. Demographic and clinical variables were analysed using SPSS® 22.0, considering P<.05 to be statistically significant. RESULTS: We analysed 155 patients affected by OOSCC (mean age of 62.7, where 69% were males). Twenty six cases were p16+ (16.8%) and 19 cases PCR+ (12.3%), The HPV+ tumours were located predominantly in the oropharynx (42.1%, P=.017) and demonstrated the tendency to be more frequent in males, with higher incidence in younger patients, lower in smokers and drinkers, and higher when patients have a greater cervical lymph node involvement at the time of diagnosis. The PCR+ patients had higher survival (P=.024), as did the p16+ (P=.005). CONCLUSIONS: The incidence of HPV in OOSCC is low (12.3%), but the clinical presentation and prognosis of the HPV+ patient differs from the classic smoker and/or drinker, which implies assessing the management of these patients independently. The p16 staining has a great diagnostic capacity to predict HPV (95.5%), although the detection of the HPV genome is still the gold standard technique.