Next-Generation DNA Sequencing-Based Gene Panel for Diagnosis and Genetic Risk Stratification in Onco-Hematology.

A suitable diagnostic classification of myeloid neoplasms and acute leukemias requires testing for a large number of molecular biomarkers. Next-generation sequencing is a technology able to integrate identification of the vast majority of them in a single test. This manuscript includes the design, analytical validation and clinical feasibility evaluation of a molecular diagnostic kit for onco-hematological diseases. It is based on sequencing of the coding regions of 76 genes (seeking single-nucleotide variants, small insertions or deletions and CNVs), as well as the search for fusions in 27 target genes. The kit has also been designed to detect large CNVs throughout the genome by including specific probes and employing a custom bioinformatics approach. The analytical and clinical feasibility validation of the Haematology OncoKitDx panel has been carried out from the sequencing of 170 patient samples from 6 hospitals (in addition to the use of commercial reference samples). The analytical validation showed sensitivity and specificity close to 100% for all the parameters evaluated, with a detection limit of 2% for SNVs and SVs, and 20% for CNVs. Clinically relevant mutations were detected in 94% of all patients. An analysis of the correlation between the genetic risk classification of AML (according to ELN 2017) established by the hospitals and that obtained by the Haematology OncoKitDx panel showed an almost perfect correlation (K = 0.94). Among the AML samples with a molecular diagnosis, established by the centers according to the WHO, the Haematology OncoKitDx analysis showed the same result in 97% of them. The panel was able to adequately differentiate between MPN subtypes and also detected alterations that modified the diagnosis (FIP1L1-PDGFRA). Likewise, the cytogenetic risk derived from the CNV plot generated by the NGS panel correlated substantially with the results of the conventional karyotype (K = 0.71) among MDS samples. In addition, the panel detected the main biomarkers of prognostic value among patients with ALL. This validated solution enables a reliable analysis of a large number of molecular biomarkers from a DNA sample in a single assay.

Unmasking a new prognostic marker and therapeutic target from the GDNF-RET/PIT1/p14ARF/p53 pathway in acromegaly.

BACKGROUND: Acromegaly is produced by excess growth hormone secreted by a pituitary adenoma of somatotroph cells (ACRO). First-line therapy, surgery and adjuvant therapy with somatostatin analogs, fails in 25% of patients. There is no predictive factor of resistance to therapy. New therapies are investigated using few dispersed tumor cells in acute primary cultures in standard conditions where the cells do not grow, or using rat pituitary cell lines that do not maintain the full somatotroph phenotype. The RET/PIT1/p14ARF/p53 pathway regulates apoptosis in normal pituitary somatotrophs whereas the RET/GDNF pathway regulates survival, controlling PIT1 levels and blocking p14ARF (ARF) and p53 expression. METHODS: We investigated these two RET pathways in a prospective series of 32 ACRO and 63 non-functioning pituitary adenomas (NFPA), studying quantitative RNA and protein gene expression for molecular-clinical correlations and how the RET pathway might be implicated in therapeutic success. Clinical data was collected during post-surgical follow-up. We also established new'humanized' pituitary cultures, allowing 20 repeated passages and maintaining the pituitary secretory phenotype, and tested five multikinase inhibitors (TKI: Vandetanib, Lenvatinib, Sunitinib, Cabozantinib and Sorafenib) potentially able to act on the GDNF-induced RET dimerization/survival pathway. Antibody arrays investigated intracellular molecular pathways. FINDINGS: In ACRO, there was specific enrichment of all genes in both RET pathways, especially GDNF. ARF and GFRA4 gene expression were found to be opposing predictors of response to first-line therapy. ARF cut-off levels, calculated categorizing by GNAS mutation, were predictive of good response (above) or resistance (below) to therapy months later. Sorafenib, through AMPK, blocked the GDNF/AKT survival action without altering the RET apoptotic pathway. INTERPRETATION: Tumor ARF mRNA expression measured at the time of the surgery is a prognosis factor in acromegaly. The RET inhibitor, Sorafenib, is proposed as a potential treatment for resistant ACRO. FUND: This project was supported by national grants from Agencia Estatal de Investigación (AEI) and Instituto Investigación Carlos III, with participation of European FEDER funds, to IB (PI150056) and CVA (BFU2016-76973-R). It was also supported initially by a grant from the Investigator Initiated Research (IIR) Program (WI177773) and by a non-restricted Research Grant from Pfizer Foundation to IB. Some of the pituitary acromegaly samples were collected in the framework of the Spanish National Registry of Acromegaly (REMAH), partially supported by an unrestricted grant from Novartis to the Spanish Endocrine Association (SEEN). CVA is also supported from a grant of Medical Research Council UK MR/M018539/1.

Early oral cancer diagnosis: The Aarhus statement perspective. A systematic review and meta-analysis.

BACKGROUND: Mortality is linked to diagnostic intervals in certain cancers. As symptom perception is conditioned by tumor site, a specific study on oral cancer is needed. METHODS: This study's inclusion criteria were original data, symptomatic primary oral squamous cell carcinoma, and exposure of interest, diagnostic interval, or diagnostic delay. The outcome of interest was survival and disease stage. A meta-analysis was undertaken to investigate the relationship between intervals to diagnosis, TNM classification, and survival in oral cancer. RESULTS: Regarding referral delay, the results present no heterogeneity and showed a risk increase in mortality of 2.48 (range = 1.39-4.42). The larger the diagnostic delay, the more advanced the stage at diagnosis. High quality studies reveal a higher risk increase than low quality studies (odds ratio [OR] = 2.44; 95% confidence interval [CI] = 1.36-4.36 vs OR = 1.53; 95% CI = 1.26-1.86). CONCLUSION: A longer time interval from first symptom to referral for diagnosis is a risk factor for advanced stage and mortality of oral cancer. © 2015 Wiley Periodicals, Inc. Head Neck 38: E2182-E2189, 2016.

Proliferative activity and diagnostic delay in oral cancer.

BACKGROUND: Tumor stage may relate to the chronology of neoplasm growth and has been used as an outcome variable when studying diagnostic delay in oral cancer. However, tumor growth rate may act as a confounding factor. METHODS: We reviewed a total of 63 incident cases of oral cancer. The variables considered for the study included age, sex, smoking history, tumor site, TNM stage, Ki-67 score, and diagnostic delay. RESULTS: Significant differences between survivors and exitus were found in terms of tumor stage at diagnosis (I-II vs III-IV), sex, and Ki-67 scores. When the analysis was adjusted for tumor stage at diagnosis (I-II vs III-IV), proliferative activity resulted to be an independent prognostic factor for survival, whereas diagnostic delay did not influence survival. CONCLUSION: These results seem to suggest that survival from oral cancer is affected more by the biology of the cancer (rapid tumor growth) than by diagnostic delay.

Is diagnostic delay related to advanced-stage oral cancer? A meta-analysis.

Diagnostic delay in oropharyngeal cancer may be associated with poor prognosis. As controversy exists on this topic because of contradictory results, the aim of this study was to perform a systematic review of the relationship between total diagnostic delay and advanced disease stages. A systematic search of MEDLINE, EMBASE, and ISI proceedings was made to identify observational studies that provided relative risks (RRs) and 95% confidence intervals (CIs) for patients with confirmed pathological diagnosis. The outcome of interest was disease stage (TNM classification), while the exposure of interest was the total diagnostic delay. The study-specific adjusted log RRs for cohort studies were weighted by the inverse of their variance to compute a pooled RR and its 95% CI. The fixed-effects pooled RR of advanced stages of oropharyngeal cancer when diagnostic delay is present was 1.32 (95% CI: 1.07-1.62). This association was stronger when the analysis was restricted to oral cancer (pooled RR: 1.47; 95% CI: 1.09-1.99) and when delay was longer than 1 month (pooled RR: 1.69; 95% CI: 1.26-2.77). The probability for patients with delayed diagnosis to present an advanced-stage tumour at diagnosis was significantly higher than that of patients with no delay in diagnosis. However, new prospective studies with strict methodology are needed to shed more light on this association.

Yellowish lesions of the oral cavity. Suggestion for a classification.

The colour of a lesion is due to its nature and to its histological substratum. In order to ease diagnosis, oral cavity lesions have been classified according to their colour in: white, red, white and red, bluish and/or purple, brown, grey and/or black lesions. To the best of our knowledge, there is no such a classification for yellow lesions. So, a suggestion for a classification of yellowish lesions according to their semiology is made with the following headings: diffuse macular lesions, papular, hypertrophic, or pustular lesions, together with cysts and nodes. This interpretation of the lesions by its colour is the first step to diagnosis. It should be taken into account that, as happens with any other classification, the yellowish group of lesions includes items with different prognosis as well as possible markers of systemic disorders.

Yellowish lesions of the oral cavity. Suggestion for a classification

Para facilitar el diagnóstico de las lesiones de la cavidad oral estas han sido clasificadas atendiendo a su color en: blancas, rojas, blancas y rojas, azules y/o púrpura, marrones, grises y/o negras. El color de la lesión se debe a su naturaleza y al sustrato histológico. Debido a que no existe ninguna clasificación de las lesiones, según nuestro conocimiento, atendiendo a su color amarillo, proponemos aquí esta clasificación desde una perspectiva semiológica, agrupándolas en lesiones maculares difusas, lesiones papulares, lesiones hipertróficas, lesiones pustulosas, quistes y nódulos. Esta lectura de las lesiones según su color constituye el primer paso para ayudar a su diagnóstico. Finalmente, debemos considerar que al igual que ocurre con las lesiones que se clasifican en cualquier grupo de color, el grupo de lesiones amarillas incluirá lesiones con diferente pronóstico y además lesiones que pueden indicar una patología sistémica de base

The colour of a lesion is due to its nature and to its histological substratum. In order to ease diagnosis, oral cavity lesions have been classified according to their colour in: white, red, white and red, bluish and/or purple, brown, grey and/or black lesions. To the best of our knowledge, there is no such a classification for yellow lesions. So, a suggestion for a classification of yellowish lesions according to their semiology is made with the following headings: diffuse macular lesions, papular, hypertrophic, or pustular lesions, together with cysts and nodes. This interpretation of the lesions by its colour is the first step to diagnosis. It should be taken into account that, as happens with any other classification, the yellowish group of lesions includes items with different prognosis as well as possible markers of systemic disorders