RESUMEN
Endometrial cancer (EC) is the most frequent gynecological cancer. The ESGO/ESTRO/ESP 2020 guidelines identify prognostic groups based on morpho-molecular characteristics. This study aims to evaluate the clinical applicability of NGS analysis to define an appropriate risk class and to improve the diagnostic and prognostic stratification of ECs. Cases of serous carcinoma (OHEC) and high- (HGEC) and low-grade (LGEC) endometrioid carcinoma diagnosed with the morphological and immunohistochemical (IHC) protocols were considered. After a standardized pre-analytical phase, tumor DNA was semi-automatically extracted and analyzed using NGS with a panel of 14 genes. A total of 63 cases were considered. NGS analysis was successful in 60 cases; all of these were classified according to the new diagnostic algorithm. The molecular risk classification showed a good correlation with the morphological (k = 0.8). The study showed that the protocols of the pre-analytical and analytical phases used are robust and can lead to molecular results that fall within the standards required, which can be used in clinical practice for more precise diagnostic-therapeutic management of patients. The implementation of the classification is particularly relevant for better prognostic stratification of HGECs. In addition, the identification of a suspicious VUS in POLE questions the classification of truncating variants.
RESUMEN
Liquid biopsy has the potential to drastically change clinical practice, paving the way to a novel non-invasive approach for cancer diagnosis and treatment. One of the limitations for the implementation of liquid biopsy in clinical practice is the lack of shared and reproducible standard operating procedures (SOPs) for sample collection, processing and storage. Here, we present a critical review of the literature focusing on the available SOPs to guide liquid biopsy management in research settings and describe SOPs that our laboratory developed and employed in the context of a prospective clinical-translational trial (RENOVATE, NCT04781062). The main aim of this manuscript is to address common issues, towards the implementation of interlaboratory shared protocols for optimized preanalytical handling of blood and urine samples. To our knowledge, this work is one of the few up-to-date, freely available comprehensive reports on trial-level procedures for the handling of liquid biopsy.
Asunto(s)
Manejo de Especímenes , Humanos , Estudios Prospectivos , Manejo de Especímenes/métodos , Biopsia Líquida , BiomarcadoresRESUMEN
Epithelial ovarian cancers (EOCs) harboring germline or somatic pathogenic variants in BRCA1 and BRCA2 genes show sensitivity to poly(ADP-ribose) polymerase inhibition. It has been suggested that BRCA1 promoter methylation is perhaps a better determinant of therapy response, because of its intrinsic dynamic feature, with respect to genomic scars or gene mutation. Conflicting evidence was reported so far, and the lack of a validated assay to measure promoter methylation was considered a main confounding factor in data interpretation. To contribute to the validation process of a pyrosequencing assay for BRCA1 promoter methylation, 109 EOCs from two Italian centers were reciprocally blindly investigated. By comparing two different pyrosequencing assays, addressing a partially overlapping region of BRCA1 promoter, an almost complete concordance of results was obtained. Moreover, the clinical relevance of this approach was also supported by the finding of BRCA1 transcript down-regulation in BRCA1-methylated EOCs. These findings could lead to the development of a simple and cheap pyrosequencing assay for diagnostics, easily applicable to formalin-fixed, paraffin-embedded tissues. This technique may be implemented in routine clinical practice in the near future to identify EOCs sensitive to poly(ADP-ribose) polymerase inhibitor therapy, thus increasing the subset of women affected by EOCs who could benefit from such treatment.
Asunto(s)
Antineoplásicos , Neoplasias Ováricas , Femenino , Humanos , Mutación de Línea Germinal , Proteína BRCA1/genética , Mutación , Metilación de ADN/genética , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Antineoplásicos/uso terapéutico , Secuenciación de Nucleótidos de Alto Rendimiento , Neoplasias Ováricas/diagnóstico , Neoplasias Ováricas/genética , Neoplasias Ováricas/tratamiento farmacológico , Proteína BRCA2/genéticaRESUMEN
Knowledge of the mechanisms underpinning the development of protective immunity conferred by mRNA vaccines is fragmentary. Here, we investigated responses to coronavirus disease 2019 (COVID-19) mRNA vaccination via high-temporal resolution blood transcriptome profiling. The first vaccine dose elicited modest interferon and adaptive immune responses, which peaked on days 2 and 5, respectively. The second vaccine dose, in contrast, elicited sharp day 1 interferon, inflammation, and erythroid cell responses, followed by a day 5 plasmablast response. Both post-first and post-second dose interferon signatures were associated with the subsequent development of antibody responses. Yet, we observed distinct interferon response patterns after each of the doses that may reflect quantitative or qualitative differences in interferon induction. Distinct interferon response phenotypes were also observed in patients with COVID-19 and were associated with severity and differences in duration of intensive care. Together, this study also highlights the benefits of adopting high-frequency sampling protocols in profiling vaccine-elicited immune responses.
Asunto(s)
Vacunas contra la COVID-19 , COVID-19 , Humanos , COVID-19/prevención & control , ARN Mensajero/genética , Vacunas Sintéticas , Interferones , Vacunas de ARNmRESUMEN
BACKGROUND AND RATIONALE: Little is known about SARS-CoV-2 seroconversion in asymptomatic patients affected by solid cancer, and whether it is associated with specific transcriptomics changes in peripheral blood mononuclear cells (PBMC). METHODS: Patients affected by solid cancer treated in a top comprehensive cancer center in Italy during the first COVID-19 pandemic wave, and negative for COVID-19-symptoms since the first detection of COVID-19 in Italy, were prospectively evaluated by SARS-CoV-2 serology in the period between April 14th and June 23rd 2020. Follow-up serologies were performed, every 21-28 days, until August 23rd 2020. All SARS-CoV-2 IgM + patients underwent confirmatory nasopharyngeal swab (NPS). PBMCs from a subset of SARS-CoV-2 IgM + patients were collected at baseline, at 2 months, and at 7 months for transcriptome sequencing. RESULTS: SARS-CoV-2 serology was performed on 446 of the 466 recruited patients. A total of 14 patients (3.14%) tested positive for at least one SARS-CoV-2 immunoglobulin in the period between April 14th and August 23rd 2020. Incidence of SARS-CoV-2 IgM decreased from 1.48% in the first month of the accrual to 0% in the last month. Viral RNA could not be detected in any of the NPS. PBMC serial transcriptomic analysis showed progressive downregulation of interleukin 6 upregulated signatures, chemokine-mediated signaling and chemokine-chemokine receptor KEGG pathways. B- and T-cell receptor pathways (p-values = 0.0002 and 0.017 respectively) were progressively upregulated. CONCLUSIONS: SARS-CoV-2 seroconversion rate in asymptomatic patients affected by solid cancer is consistent with that of asymptomatic COVID-19 assessed in the general population through NPS at the peak of the first wave. Transcriptomic features over time in IgM + asymptomatic cases are suggestive of previous viral exposure.
Asunto(s)
COVID-19 , Neoplasias , Anticuerpos Antivirales , Quimiocinas , Humanos , Inmunoglobulina M , Incidencia , Leucocitos Mononucleares , Neoplasias/complicaciones , Neoplasias/epidemiología , Pandemias , Estudios Prospectivos , SARS-CoV-2RESUMEN
PURPOSE: The study of plasma cell-free DNA integrity (cfDI) has shown potential for providing useful information in neoplastic patients. The aim of this study is to estimate the accuracy of an electrophoresis-based method for cfDI evaluation in the assessment of pathologic complete response (pCR) in patients with breast cancer (BC) undergoing neoadjuvant chemotherapy (NACT). PATIENTS AND METHODS: Fifty-one patients with BC undergoing anthracycline-/taxane-based NACT were recruited. Plasma samples were collected from each patient at diagnosis (t0), after anthracycline administration (t1), and after NACT completion (t2). The concentration of differently sized cell-free DNA fragments was assessed by automated electrophoresis. cfDI, expressed as cfDI index, was calculated as the ratio of 321-1,000 bp sized fragment concentration to 150-220 bp sized fragment concentration assessed at t2. cfDI index was then used to build an exploratory classifier for BC response to NACT, directly comparing its sensitivity and specificity with magnetic resonance imaging (MRI), through bootstrapped logistic regression. RESULTS: cfDI index was assessed on 38 plasma samples collected from as many patients at t2, maintaining a 30/70 ratio between pCR and non-pCR patients. cfDI index showed an area under the receiver operating characteristic curve in predicting the achievement of pCR of 81.6, with a cutoff above 2.71 showing sensitivity = 81.8 and specificity = 81.5. The combination of cfDI index and MRI showed, in case of concordance, an area under the receiver operating characteristic curve of 92.6 with a predictive value of complete response of 87.5 and a predictive value of absence of complete response of 94.7. CONCLUSION: cfDI index measured after NACT completion shows great potential in the assessment of pCR in patients with BC. The evaluation of its use in combination with MRI is strongly warranted in prospective studies.
Asunto(s)
Neoplasias de la Mama , Ácidos Nucleicos Libres de Células , Neoplasias de la Mama/tratamiento farmacológico , Electroforesis , Femenino , Humanos , Terapia Neoadyuvante/métodos , Estudios ProspectivosRESUMEN
Next-generation sequencing (NGS) is the technology of choice for the routine screening of tumor samples in clinical practice. In this setting, the targeted sequencing of a restricted number of clinically relevant genes represents the most practical option when looking for genetic variants associated with cancer, as well as for the choice of targeted treatments. In this review, we analyze available NGS platforms and clinical applications of multi-gene testing in breast cancer, with a focus on metastatic triple-negative breast cancer (mTNBC). We make an overview of the clinical utility of multi-gene testing in mTNBC, and then, as immunotherapy is emerging as a possible targeted therapy for mTNBC, we also briefly report on the results of the latest clinical trials involving immune checkpoint inhibitors (ICIs) and TNBC, where NGS could play a role for the potential predictive utility of homologous recombination repair deficiency (HRD) and tumor mutational burden (TMB).
Asunto(s)
Pruebas Genéticas/métodos , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/metabolismo , Biomarcadores de Tumor/genética , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/tendencias , Humanos , Mutación/genética , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
Current approaches for cancer detection and characterization are based on radiological procedures coupled with tissue biopsies, despite relevant limitations in terms of overall accuracy and feasibility, including relevant patients' discomfort. Liquid biopsies enable the minimally invasive collection and analysis of circulating biomarkers released from cancer cells and stroma, representing therefore a promising candidate for the substitution or integration in the current standard of care. Despite the potential, the current clinical applications of liquid biopsies are limited to a few specific purposes. The lack of standardized procedures for the pre-analytical management of body fluids samples and the detection of circulating biomarkers is one of the main factors impacting the effective advancement in the applicability of liquid biopsies to clinical practice. The aim of this work, besides depicting current methods for samples collection, storage, quality check and biomarker extraction, is to review the current techniques aimed at analyzing one of the main circulating biomarkers assessed through liquid biopsy, namely cell-free nucleic acids, with particular regard to circulating tumor DNA (ctDNA). ctDNA current and potential applications are reviewed as well.
RESUMEN
INTRODUCTION: Standard procedures aimed at the early diagnosis of breast cancer (BC) present suboptimal accuracy and imply the execution of invasive and sometimes unnecessary tissue biopsies. The assessment of circulating biomarkers for diagnostic purposes, together with radiomics, is of great potential in BC management. METHODS AND ANALYSIS: This is a prospective translational study investigating the accuracy of the combined assessment of multiple circulating analytes together with radiomic variables for early BC diagnosis. Up to 750 patients will be recruited at their presentation at the Diagnostic Senology Unit of Ospedale Policlinico San Martino (Genoa, IT) for the execution of a diagnostic biopsy after the detection of a suspect breast lesion (t0). Each recruited patient will be asked to donate peripheral blood and urine before undergoing breast biopsy. Blood and urine samples will also be collected from a cohort of 100 patients with negative mammography. For cases with histological diagnosis of invasive BC, a second sample of blood and urine will be collected after breast surgery. Circulating tumour DNA, cell-free methylated DNA and circulating proteins will be assessed in samples collected at t0 from patients with stage I-IIA BC at surgery together with those collected from patients with histologically confirmed benign lesions of similar size and from healthy controls with negative mammography. These analyses will be combined with radiomic variables extracted with freeware algorithms applied to cases and matched controls for which digital mammography is available. The overall goal of the present study is to develop a horizontal data integration classifier for the early diagnosis of BC. ETHICS AND DISSEMINATION: This research protocol has been approved by Regione Liguria Ethics Committee (reference number: 2019/75, study ID: 4452). Patients will be required to provide written informed consent. Results will be published in international peer-reviewed scientific journals. TRIAL REGISTRATION NUMBER: NCT04781062.
Asunto(s)
Neoplasias de la Mama , Mama/diagnóstico por imagen , Mama/patología , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/patología , Detección Precoz del Cáncer/métodos , Femenino , Humanos , Mamografía/métodos , Estudios ProspectivosRESUMEN
In breast cancer patients undergoing neoadjuvant chemotherapy before surgery, there is an unmet need for noninvasive predictive biomarkers of response. The analysis of circulating tumor DNA (ctDNA) in particular has been the object of several reports, but few of them have studied the applicability of tagged targeted deep sequencing (tTDS) to clinical practice and its performance compared with droplet digital PCR (ddPCR). Here, we present the first results from an ongoing study involving a prospectively accrued, monocentric cohort of patients affected by invasive breast cancer, undergoing neoadjuvant chemotherapy followed by surgery with curative intent as per clinical practice. A pretreatment tumor biopsy and plasma samples were collected before and during treatment, after surgery, and every six months henceforth or until relapse, whichever came first. Pretreatment biopsies were sequenced with a 409-gene massive parallel sequencing (MPS) panel, allowing the identification of target mutations and their research in plasma by tTDS and ddPCR as a complementary approach. Using tTDS, we demonstrated the presence of at least one deleterious mutation in all the relapsed cases we studied (n = 4), with an average lead time of six months before clinical relapse. The association with ddPCR was suboptimal, and only one relapsed patient could be identified with such method. tTDS shows potential as an early noninvasive method for the detection of MRD in BC patients.
RESUMEN
Standard treatment for locally advanced rectal adenocarcinoma (LARC) includes a combination of chemotherapy with pyrimidine analogues, such as capecitabine, and radiation therapy, followed by surgery. Currently no clinically useful genomic predictors of benefit from neoadjuvant chemoradiotherapy (nCRT) exist for LARC. In this study we assessed the expression of 8,127 long noncoding RNAs (lncRNAs), poorly studied in LARC, to infer their ability in classifying patients' pathological complete response (pCR). We collected and analyzed, using lncRNA-specific Agilent microarrays a consecutive series of 61 LARC cases undergoing nCRT. Potential lncRNA predictors in responders and non-responders to nCRT were identified with LASSO regression, and a model was optimized using k-fold cross-validation after selection of the three most informative lncRNA. 11 lncRNAs were differentially expressed with false discovery rate < 0.01 between responders and non-responders to NACT. We identified lnc-KLF7-1, lnc-MAB21L2-1, and LINC00324 as the most promising variable subset for classification building. Overall sensitivity and specificity were 0.91 and 0.94 respectively, with an AUC of our ROC curve = 0.93. Our study shows for the first time that lncRNAs can accurately predict response in LARC undergoing nCRT. Our three-lncRNA based signature must be independently validated and further analyses must be conducted to fully understand the biological role of the identified signature, but our results suggest lncRNAs may be an ideal biomarker for response prediction in the studied setting.
Asunto(s)
Adenocarcinoma/genética , Adenocarcinoma/terapia , Quimioradioterapia , Terapia Neoadyuvante , Recurrencia Local de Neoplasia/genética , ARN Largo no Codificante/genética , Neoplasias del Recto/genética , Neoplasias del Recto/terapia , Adenocarcinoma/patología , Anciano , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia/patología , Recurrencia Local de Neoplasia/terapia , Análisis de Componente Principal , ARN Largo no Codificante/metabolismo , Neoplasias del Recto/patología , Máquina de Vectores de SoporteRESUMEN
BACKGROUND: Over the past few years, next-generation sequencing (NGS) has become reliable and cost-effective, and its use in clinical practice has become a reality. A relevant role for NGS is the prediction of response to anti-EGFR agents in metastatic colorectal cancer (mCRC), where multiple exons from KRAS, NRAS, and BRAF must be sequenced simultaneously. METHODS: We optimized a 14-amplicon NGS panel to assess, in a consecutive cohort of 219 patients affected by mCRC, the presence and clinico-pathological associations of mutations in the KRAS, NRAS, BRAF, and PIK3CA genes from formalin-fixed, paraffin-embedded specimens collected for diagnostics and research at the time of diagnosis. RESULTS: We observed a statistically significant association of RAS mutations with sex, young age, and tumor site. We demonstrated that concomitant mutations in the RAS/RAF pathway are not infrequent in mCRC, and as anticipated by whole-genome studies, RAS and PIK3CA tend to be concurrently mutated. We corroborated the association of BRAF mutations in right mCRC tumors with microsatellite instability. We established tumor side as prognostic parameter independently of mutational status. CONCLUSIONS: To our knowledge, this is the first monocentric, consecutively accrued clinical mCRC cancer cohort tested by NGS in a real-world context for KRAS, NRAS, BRAF, and PIK3CA. Our study has highlighted in clinical practice findings such as the concomitance of mutations in the RAS/RAF pathway, the presence of multiple mutations in single gene, the co-occurrence of RAS and PIK3CA mutations, the prognostic value of tumor side and possible associations of sex with specific mutations.
Asunto(s)
Neoplasias Colorrectales/genética , Neoplasias Colorrectales/secundario , Mutación/genética , Proteínas Proto-Oncogénicas B-raf/genética , Transducción de Señal , Proteínas ras/genética , Anciano , Neoplasias Colorrectales/patología , Femenino , Genes Relacionados con las Neoplasias , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Transducción de Señal/genéticaRESUMEN
Oncogenic alteration of the cholesterol synthesis pathway is a recognized mechanism of metabolic adaptation. In the present review, we focus on squalene epoxidase (SE), one of the two rate-limiting enzymes in cholesterol synthesis, retracing its history since its discovery as an antimycotic target to its description as an emerging metabolic oncogene by amplification with clinical relevance in cancer. We review the published literature assessing the association between SE over-expression and poor prognosis in this disease. We assess the works demonstrating how SE promotes tumor cell proliferation and migration, and displaying evidence of cancer cell demise in presence of human SE inhibitors in in vitro and in vivo models. Taken together, robust scientific evidence has by now accumulated pointing out SE as a promising novel therapeutic target in cancer treatment.
Asunto(s)
Neoplasias/metabolismo , Escualeno-Monooxigenasa/genética , Escualeno-Monooxigenasa/metabolismo , Regulación hacia Arriba , Animales , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Progresión de la Enfermedad , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Amplificación de Genes , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Terapia Molecular Dirigida , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Transducción de Señal/efectos de los fármacos , Escualeno-Monooxigenasa/antagonistas & inhibidores , Regulación hacia Arriba/efectos de los fármacosRESUMEN
BACKGROUND: Immunohistochemistry (IHC) and fluorescent-in situ hybridization (FISH) are standard methods to assess human epidermal growth factor receptor 2 (HER2) status in breast cancer (BC) patients. Real-time quantitative polymerase-chain-reaction (qRT-PCR) is able to detect HER2 overexpression. Here we compared FISH, IHC, quantitative PCR (qPCR), and qRT-PCR to determine the concordance rates and evaluate their relative roles in HER2 determination. PATIENTS AND METHODS: We determined HER2 status in 153 BC patients, using IHC, FISH, Q-PCR and qRT-PCR. In discordant cases, we directly measured HER2 protein levels using Western blotting. RESULTS: The overall agreement (OA) between FISH and Q-PCR was 94.1, with a k value of 0.87. Assuming FISH as the standard reference, Q-PCR showed an 86.1% sensitivity and a 99.0% specificity with a global accuracy of 91.6%. OA between FISH and qRT-PCR was 90.8% with a k value of 0.81. Of interest, the disagreement between FISH and qRT-PCR was mostly restricted to equivocal cases. HER2 protein analysis suggested that qRT-PCR correlates better than FISH with HER2 protein levels, particularly where FISH fails to provide conclusive results. SIGNIFICANCE: qRT-PCR may outperform FISH in identifying patients overexpressing HER2 protein. Q-PCR cannot be used for HER2 status assessment, due to its suboptimal level of agreement with FISH. Both FISH and Q-PCR may be less accurate than qRT-PCR as surrogates of HER2 protein determination.
Asunto(s)
Neoplasias de la Mama/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptor ErbB-2/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Femenino , Humanos , Inmunohistoquímica , Hibridación Fluorescente in Situ , Curva ROC , Receptor ErbB-2/metabolismoRESUMEN
SQLE encodes squalene epoxidase, a key enzyme in cholesterol synthesis. SQLE has sporadically been reported among copy-number driven transcripts in multi-omics cancer projects. Yet, its functional relevance has never been subjected to systematic analyses. Here, we assessed the correlation of SQLE copy number (CN) and gene expression (GE) across multiple cancer types, focusing on the clinico-pathological associations in breast cancer (BC). We then investigated whether any biological effect of SQLE inhibition could be observed in BC cell line models. Breast, ovarian, and colorectal cancers showed the highest CN driven GE among 8,783 cases from 22 cancer types, with BC presenting the strongest one. SQLE overexpression was more prevalent in aggressive BC, and was an independent prognostic factor of unfavorable outcome. Through SQLE pharmacological inhibition and silencing in a panel of BC cell lines portraying the diversity of SQLE CN and GE, we demonstrated that SQLE inhibition resulted in a copy-dosage correlated decrease in cell viability, and in a noticeable increase in replication time, only in lines with detectable SQLE transcript. Altogether, our results pinpoint SQLE as a bona fide metabolic oncogene by amplification, and as a therapeutic target in BC. These findings could have implications in other cancer types.
Asunto(s)
Neoplasias de la Mama/genética , Oncogenes , Escualeno-Monooxigenasa/genética , Biomarcadores de Tumor , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/patología , Línea Celular Tumoral , Supervivencia Celular/genética , Transformación Celular Neoplásica/genética , Cromosomas Humanos Par 8 , Variaciones en el Número de Copia de ADN , Progresión de la Enfermedad , Femenino , Dosificación de Gen , Expresión Génica , Silenciador del Gen , Genes myc , Sitios Genéticos , Humanos , Clasificación del Tumor , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Evaluación del Resultado de la Atención al Paciente , Pronóstico , Modelos de Riesgos Proporcionales , Receptor ErbB-2/metabolismo , Escualeno-Monooxigenasa/metabolismo , Transcripción GenéticaRESUMEN
Oral potentially malignant disorders (OPMDs) characterized by the presence of dysplasia and DNA copy number aberrations (CNAs), may reflect chromosomal instability (CIN) and predispose to oral squamous cell carcinoma (OSCC). Early detection of OPMDs with such characteristics may play a crucial role in OSCC prevention. The aim of this study was to explore the relationship between CNAs, histological diagnosis, oral subsite and aneuploidy in OPMDs/OSCCs. Samples from OPMDs and OSCCs were processed by high-resolution DNA flow cytometry (hr DNA-FCM) to determine the relative nuclear DNA content. Additionally, CNAs were obtained for a subset of these samples by genome-wide array comparative genomic hybridization (aCGH) using DNA extracted from either diploid or aneuploid nuclei suspension sorted by FCM. Our study shows that: i) aneuploidy, global genomic imbalance (measured as the total number of CNAs) and specific focal CNAs occur early in the development of oral cancer and become more frequent at later stages; ii) OPMDs limited to tongue (TNG) mucosa display a higher frequency of aneuploidy compared to OPMDs confined to buccal mucosa (BM) as measured by DNA-FCM; iii) TNG OPMDs/OSCCs show peculiar features of CIN compared to BM OPMDs/OSCCs given the preferential association with total broad and specific focal CNA gains. Follow-up studies are warranted to establish whether the presence of DNA aneuploidy and specific focal or broad CNAs may predict cancer development in non-dysplastic OPMDs.
Asunto(s)
Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Variaciones en el Número de Copia de ADN/genética , Neoplasias de la Boca/genética , Neoplasias de la Boca/patología , Aneuploidia , Inestabilidad Cromosómica/genética , Aberraciones Cromosómicas , ADN de Neoplasias/genética , Diploidia , Femenino , Citometría de Flujo/métodos , Estudios de Seguimiento , Genómica , Humanos , Masculino , Mucosa Bucal/patología , Lesiones Precancerosas/genética , Lesiones Precancerosas/patología , Lengua/patologíaRESUMEN
OBJECTIVES: Reliable assessment of estrogen, progesterone (ER and PR), and HER2 receptor status are essential in breast cancer (BC) treatment. Immunohistochemical methods are limited by intra- and inter-laboratory variability. Furthermore, current methods are not the ideal approach for reproducing the biological continuum of ER, PR, and HER2 receptor levels, due to their intrinsic, semi-quantitative nature, relying in part on subjective interpretation. METHODS: In the present study, we tested a molecular approach to define ER, PR, and HER2 status in fine-needle-aspirate (FNA) samples from patients with early BC. We performed flow cytometry analysis on 88 FNA specimens from suspect BC patients to determine cellularity. We used quantitative Real Time PCR (QRT-PCR) to assess ER, PR, HER2 status, and qPCR for HER2 gene copy number (GCN). RESULTS: ER and PR mRNA levels showed a highly significant correlation with IHC data on surgical samples. qPCR showed greater accuracy than IHC in defining HER2 status. QRT-PCR defined better than IHC the continuous spectrum of the expression of the assessed receptors. Moreover, PCR analysis demonstrated a strict correlation between HER2 status and higher levels of its transcript, correctly stratifying HER2+ and HER2- patients. Finally, there was a strongly significant agreement between HER2 GCN assessed on FNA specimens by qPCR and FISH data obtained on pathological tissue specimens. CONCLUSIONS: The present results support a comprehensive approach to determine ER, PR, and HER2 status by PCR (QRT-PCR and qPCR) in FNA specimens, with high relevance for therapeutic strategies like neoadjuvant treatment.
Asunto(s)
Neoplasias de la Mama/metabolismo , Receptor ErbB-2/metabolismo , Receptores de Estrógenos/metabolismo , Receptores de Progesterona/metabolismo , Biomarcadores de Tumor/metabolismo , Biopsia con Aguja Fina , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Estudios de Cohortes , Femenino , Citometría de Flujo , Humanos , Inmunohistoquímica , Estudios Prospectivos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptor ErbB-2/genética , Receptores de Estrógenos/genética , Receptores de Progesterona/genéticaRESUMEN
Most chronic myeloid leukemia (CML) patients show the Philadelphia chromosome (Ph) arising from the reciprocal t(9;22), but 5-10% present variants of this translocation involving different breakpoints besides 9q34 and 22q11. WE REPORT THE NON SIMULTANEOUS OCCURRENCE OF TWO DIFFERENT TYPES OF PH TRANSLOCATION IN A CML PATIENT: a t(9;22)(q34;q11) standard and a three-way variant t(9;11;22)(q34;p15;q11). Bone marrow cells with standard translocation did not have BCR/ABL kinase domain (KD) mutations and were sensitive to imatinib therapy. In contrast, bone marrow cells with the variant translocation showed two BCR/ABL KD mutations and were resistant to imatinib, thus inducing transformation to the blast phase and karyotype evolution.
RESUMEN
Chronic myeloid leukemia (CML) is a hematological disease accounting for about 15-20% of all adult leukemias. The clinical and biologic advances achieved in such a malignancy, represent one of the best successes obtained by translational medicine. Indeed, identification of the fusion oncogene BCR-ABL has allowed using of small molecule inhibitors of its tyrosine kinase activity which, in turn, have literally revolutionized the treatment of CML. Importantly the successfully clinical management was also realized on appropriate diagnosis, disease monitoring as well as early identification of such mutations causing drug resistance. Notably the recent availability of refined laboratory equipments represented by the Next Generation Sequencing (NGS) and genomic analyses has further contributed to gain ground towards the cure of this tumor. These issues are discussed here together with an overview on how patients treated with tyrosine kinase inhibitors should be monitored.
Asunto(s)
Antineoplásicos/uso terapéutico , Proteínas de Fusión bcr-abl/antagonistas & inhibidores , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Animales , Análisis Mutacional de ADN , Monitoreo de Drogas , Proteínas de Fusión bcr-abl/genética , Proteínas de Fusión bcr-abl/metabolismo , Pruebas Genéticas , Genómica/métodos , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/enzimología , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Técnicas de Diagnóstico Molecular , Terapia Molecular Dirigida , Mutación , Selección de Paciente , Medicina de Precisión , Valor Predictivo de las Pruebas , Transducción de Señal/efectos de los fármacos , Resultado del TratamientoRESUMEN
Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder characterized by the expansion of a leukemic stem cell (LSC) clone, carrying a Philadelphia translocation, able to overcome the non-malignant hematopoietic stem cells. The tyrosine kinase inhibitors (TKIs) imatinib, nilotinib and dasatinib are gold-standard for CML treatment. Each shows an impressive rate of complete cytogenetic response in chronic phase (CP)-CML. However, relapse and treatment failure are major problems with long-term use of TKIs. A polymerase chain reaction (PCR) assay to detect the mRNA expression of BCR-ABL1 represents the main molecular approach to monitoring response to treatment. However, using this analysis it is currently not possible to prospectively identify patients whose disease will relapse due to LSC reappearance. The aim of our study was to investigate whether the mRNA expression analysis of two Hedgehog (Hh) stemness signaling molecules, Smoothened (SMO) and Patched-1 (PTCH1), could predict upcoming molecular relapse. At the time of diagnosis, patients with high Sokal risk (n = 12) showed higher and lower levels of SMO and PTCH1, respectively (p = 0.0132), compared with patients with different Sokal scores (p = 0.0316 for intermediate risk and p = 0.0340 for low risk). These data suggest that Hh signaling was functionally more active in this risk group at the time of diagnosis. Furthermore, the kinetics of Hh signaling activity during the individual medical history correlated with BCR-ABL1 mRNA level and with upcoming molecular relapse. Also, mutation analysis of BCR-ABL1 revealed that activation of Hh signaling precedes molecular relapse by several months, mostly in patients carrying the gatekeeper mutation T315I. Importantly, in vitro data showed a synergistic effect of chemical inhibitors of Hh signaling and TKIs in both wild-type and resistant (T315I) CML cell lines. Collectively our data show that monitoring Hh pathway activity contemporaneously with BCR-ABL1 mRNA level may improve the chance of early detection of patients who will experience a relapse (mainly in the high Sokal risk group), paving the way for an innovative management of this hematologic malignancy.