High accuracy of a blood ctDNA-based multimodal test to detect colorectal cancer.

BACKGROUND: Detection of circulating tumor DNA (ctDNA) is a minimally invasive and convenient blood-based screening strategy that may increase effectiveness of colorectal cancer (CRC) screening. PATIENTS AND METHODS: A novel multimodal ctDNA-based blood assay that integrates genomics, epigenomics and fragmentomics, as well as proteomics in a refined version, was tested in blood samples from two cohorts: (i) consecutive fecal immunochemical test (FIT)-positive individuals from the CRC Barcelona stool-based screening program; (ii) patients diagnosed with CRC. Primary endpoint was the performance of the test to detect CRC at different tumor-node-metastasis (TNM) stages. Secondary endpoint was the ability of the test to detect advanced precancerous lesions (advanced adenoma or advanced serrated lesion). RESULTS: A total of 623 blood samples were analyzed in the primary analysis. Sensitivity and specificity of the assay to detect CRC was 93% and 90%, respectively. The sensitivity of CRC detection according to TNM stages was 84% for stage I, 94% for stage II and 96% for stage III (70/73) (P< 0.024). Sensitivity to detect advanced precancerous lesions was 23% with a refined version of the test (including protein and updating bioinformatic thresholding). CONCLUSION: A blood-based multimodal ctDNA assay detected CRC with high accuracy. This minimally invasive, accessible and convenient assay may help to increase the effectiveness of CRC screening.

miR-146a rs2431697 identifies myeloproliferative neoplasm patients with higher secondary myelofibrosis progression risk.

Myelofibrosis (MF) occurs as part of the natural history of polycythemia vera (PV) and essential thrombocythemia (ET), and remarkably shortens survival. Although JAK2V617F and CALR allele burden are the main transformation risk factors, inflammation plays a critical role by driving clonal expansion toward end-stage disease. NF-κB is a key mediator of inflammation-induced carcinogenesis. Here, we explored the involvement of miR-146a, a brake in NF-κB signaling, in MPN susceptibility and progression. rs2910164 and rs2431697, that affect miR-146a expression, were analyzed in 967 MPN (320 PV/333 ET/314 MF) patients and 600 controls. We found that rs2431697 TT genotype was associated with MF, particularly with post-PV/ET MF (HR = 1.5; p < 0.05). Among 232 PV/ET patients (follow-up time=8.5 years), 18 (7.8%) progressed to MF, being MF-free-survival shorter for rs2431697 TT than CC + CT patients (p = 0.01). Multivariate analysis identified TT genotype as independent predictor of MF progression. In addition, TT (vs. CC + CT) patients showed increased plasma inflammatory cytokines. Finally, miR-146a-/- mice showed significantly higher Stat3 activity with aging, parallel to the development of the MF-like phenotype. In conclusion, we demonstrated that rs2431697 TT genotype is an early predictor of MF progression independent of the JAK2V617F allele burden. Low levels of miR-146a contribute to the MF phenotype by increasing Stat3 signaling.

Correction to: Liquid biopsy in oncology: a consensus statement of the Spanish Society of Pathology and the Spanish Society of Medical Oncology.

The article Liquid biopsy in oncology.

Liquid biopsy in oncology: a consensus statement of the Spanish Society of Pathology and the Spanish Society of Medical Oncology.

The proportion of cancer patients with tumours that harbour a potentially targetable genomic alteration is growing considerably. The diagnosis of these genomic alterations can lead to tailored treatment at the onset of disease or on progression and to obtaining additional predictive information on immunotherapy efficacy. However, in up to 25% of cases, the initial tissue biopsy is inadequate for precision oncology and, in many cases, tumour genomic profiling at progression is not possible due to technical limitations of obtaining new tumour tissue specimens. Efficient diagnostic alternatives are therefore required for molecular stratification, which includes liquid biopsy. This technique enables the evaluation of the tumour genomic profile dynamically and captures intra-patient genomic heterogeneity as well. To date, there are several diagnostic techniques available for use in liquid biopsy, each one of them with different precision and performance levels. The objective of this consensus statement of the Spanish Society of Pathology and the Spanish Society of Medical Oncology is to evaluate the viability and effectiveness of the different methodological approaches in liquid biopsy in cancer patients and the potential application of this method to current clinical practice. The experts contributing to this consensus statement agree that, according to current evidence, liquid biopsy is an acceptable alternative to tumour tissue biopsy for the study of biomarkers in various clinical settings. It is therefore important to standardise pre-analytical and analytical procedures, to ensure reproducibility and generate structured and accessible clinical reports. It is essential to appoint multidisciplinary tumour molecular boards to oversee these processes and to enable the most suitable therapeutic decisions for each patient according to the genomic profile.

Mutational Screening of BRCA1/2 Genes as a Predictive Factor for Therapeutic Response in Epithelial Ovarian Cancer: A Consensus Guide from the Spanish Society of Pathology (SEAP-IAP) and the Spanish Society of Human Genetics (AEGH).

Germline/somatic BRCA-mutated ovarian carcinomas (OC) are associated to have better response with platinum-based chemotherapy and long-term prognosis than non-BRCA-associated OCs. In addition, these mutations are predictive factors to response to Poly(ADP-ribose) polymerase (PARP) inhibitors. Different positioning papers have addressed the clinical recommendations for BRCA testing in OC. This consensus guide represents a collection of technical recommendations to address the detection of BRCA1/2 mutations in the molecular diagnostic testing strategy for OC. Under the coordination of Spanish Society of Pathology (SEAP-IAP) and the Spanish Society of Human Genetics (AEGH), these recommendations have been developed by pathologists and geneticists taking into account previously published recommendations and their experience in the molecular characterization of these genes. Since the implementation of BRCA testing as a predictive factor can initiate the workflow by testing germline mutations in the blood or by testing both germline and somatic mutations in tumor tissue, distinctive features of both strategies are discussed. Additionally, the recommendations included in this paper provide some references, quality parameters, and genomic tools aimed to standardize and facilitate the clinical genomic diagnosis of OC.

Ultra-selection of metastatic colorectal cancer patients using next-generation sequencing to improve clinical efficacy of anti-EGFR therapy.

BACKGROUND: Extended RAS analysis is mandatory in metastatic colorectal cancer (mCRC) patients. The optimal threshold of RAS mutated subclones to identify patients most likely to benefit from antiepidermal growth factor receptor (EGFR) therapy is controversial. Our aim was to assess the clinical impact of detecting mutations in RAS, BRAF, PIK3CA and EGFRS492R in basal tissue tumour samples by using a highly sensitive next-generation sequencing (NGS) technology in mCRC patients treated with chemotherapy plus anti-EGFR or anti-vascular endothelial growth factor. PATIENTS AND METHODS: Five hundred and eighty-one tumour samples from untreated mCRC patients from 7 clinical studies were collected. Mutational analysis was carried out by standard-of-care (therascreen pyro) with a sensitivity detection of 5% mutant allele fraction (MAF), and compared with NGS technology using 454GS Junior platform (Roche Applied Science, Germany) with a sensitivity of 1%. Molecular results were correlated with clinical outcomes. RESULTS: After quality assessment, 380 samples were evaluable for molecular analysis. Standard-of-care mutational analysis detected RAS, BRAFV600E or PIK3CA mutations in 56.05% of samples compared with 69.21% by NGS (P = 0.00018). NGS identified coexistence of multiple low-frequency mutant alleles in 96 of the 263 mutated cases (36.5%; range 2-7). Response rate (RR), progression-free survival (PFS) and overall survival (OS) were increasingly improved in patients with RAS wild-type, RAS/BRAF wild-type or quadruple (KRAS/NRAS/BRAF/PIK3CA) wild-type tumours treated with anti-EGFR, assessed by standard-of-care. No additional benefit in RR, PFS or OS was observed by increasing the detection threshold to 1% by NGS. An inverse correlation between the MAF of the most prevalent mutation detected by NGS and anti-EGFR response was observed (P = 0.039). EGFRS492Rmutation was not detected in untreated samples. CONCLUSIONS: No improvement in the selection of patients for anti-EGFR therapy was obtained by adjusting the mutation detection threshold in tissue samples from 5% to 1% MAF. Response to anti-EGFR was significantly better in patients with quadruple wild-type tumours.

Plasma ctDNA RAS mutation analysis for the diagnosis and treatment monitoring of metastatic colorectal cancer patients.

BACKGROUND: RAS assessment is mandatory for therapy decision in metastatic colorectal cancer (mCRC) patients. This determination is based on tumor tissue, however, genotyping of circulating tumor (ct)DNA offers clear advantages as a minimally invasive method that represents tumor heterogeneity. Our study aims to evaluate the use of ctDNA as an alternative for determining baseline RAS status and subsequent monitoring of RAS mutations during therapy as a component of routine clinical practice. PATIENTS AND METHODS: RAS mutational status in plasma was evaluated in mCRC patients by OncoBEAM™ RAS CRC assay. Concordance of results in plasma and tissue was retrospectively evaluated. RAS mutations were also prospectively monitored in longitudinal plasma samples from selected patients. RESULTS: Analysis of RAS in tissue and plasma samples from 115 mCRC patients showed a 93% overall agreement. Plasma/tissue RAS discrepancies were mainly explained by spatial and temporal tumor heterogeneity. Analysis of clinico-pathological features showed that the site of metastasis (i.e. peritoneal, lung), the histology of the tumor (i.e. mucinous) and administration of treatment previous to blood collection negatively impacted the detection of RAS in ctDNA. In patients with baseline mutant RAS tumors treated with chemotherapy/antiangiogenic, longitudinal analysis of RAS ctDNA mirrored response to treatment, being an early predictor of response. In patients RAS wt, longitudinal monitoring of RAS ctDNA revealed that OncoBEAM was useful to detect emergence of RAS mutations during anti-EGFR treatment. CONCLUSION: The high overall agreement in RAS mutational assessment between plasma and tissue supports blood-based testing with OncoBEAM™ as a viable alternative for genotyping RAS of mCRC patients in routine clinical practice. Our study describes practical clinico-pathological specifications to optimize RAS ctDNA determination. Moreover, OncoBEAM™ is useful to monitor RAS in patients undergoing systemic therapy to detect resistance and evaluate the efficacy of particular treatments.

miR-203 and miR-221 regulate SOCS1 and SOCS3 in essential thrombocythemia.

The biological basis of essential thrombocythemia (ET) patients lacking known mutations is still unknown. MicroRNAs (miRNA) regulate hematopoietic differentiation and are deregulated in several hematopoietic malignancies. However, miRNA expression in ET patients has been poorly explored. We performed miRNA profiling in platelets from 19 ET patients and 10 healthy controls. Hierarchical cluster analysis showed two well-separated clusters between patients and controls, indicating that ET platelets had a characteristic 70-miRNA signature (P<0.0001), 68 of which were downregulated. According to the mutational status, three differentially expressed miRNAs, miR-15a (P=0.045), miR-150 (P=0.001) and miR-519a (P=0.036), were identified. A 40-miRNA signature was identified characterizing JAK2V617F-positive ET patients. Eight genes, whose interaction with the miRNAs could activate the JAK/STAT pathway were identified. An inverse correlation was observed between miRNAs expression and their target genes for SOCS1 and miR-221, SOCS3 and miR-221, SOCS3 and miR-203, and PTPN11 and miR-23a. All three miRNAs were upregulated in JAK2V617F-negative ET patients. SOCS1 and SOCS3 were validated as targets of miR-221 and miR-203, respectively. In summary, our study shows that platelets from JAK2V617F-negative ET patients harbor a specific miRNA signature that can participate in the modulation of the JAK/STAT pathway through regulation of key genes as SOCS1 and SOCS3.

Urine human papillomavirus prevalence in women with high-grade cervical lesions.

OBJECTIVE: To determine the prevalence of human papillomavirus (HPV) in urine samples from women with high-grade cervical lesions. Secondary objectives are to identify the influence of socio-demographic factors and the different genotypes with urinary HPV positivity. STUDY DESIGN: 75 women with a positive biopsy for CIN2+ were included in the study from October 2010 to July 2011. A sample of urine was collected immediately before conization at the outpatient clinic. We analyzed the presence of HPV using a PCR technique. RESULTS: The mean age of the patients was 34.8 years (range 24 to 61). All patients had histological CIN2+, of whom 54.67% had CIN3. The prevalence of HPV in urine test was 58.82% in CIN2 population versus 78.05% in CIN3 patients (p 0.072). 31 different genotypes were found. The most frequent HPV genotype was 16-HPV, which was identified in 58% of women with positive HPV-DNA in urine samples. No demographic characteristics were significantly associated to urinary HPV prevalence. CONCLUSION: Most of the patients with CIN2+ showed positive results for urine HPV test. The prevalence of positive urinary HPV test was higher for patients with CIN3. HPV urine detection could be considered as an acceptable option for high-risk population who skip regular screening programs.

Interlaboratory reproducibility and proficiency testing within the human papillomavirus cervical cancer screening program in Catalonia, Spain.

In Catalonia, a screening protocol for cervical cancer, including human papillomavirus (HPV) DNA testing using the Digene Hybrid Capture 2 (HC2) assay, was implemented in 2006. In order to monitor interlaboratory reproducibility, a proficiency testing (PT) survey of the HPV samples was launched in 2008. The aim of this study was to explore the repeatability of the HC2 assay's performance. Participating laboratories provided 20 samples annually, 5 randomly chosen samples from each of the following relative light unit (RLU) intervals: <0.5, 0.5 to 0.99, 1 to 9.99, and ≥10. Kappa statistics were used to determine the agreement levels between the original and the PT readings. The nature and origin of the discrepant results were calculated by bootstrapping. A total of 946 specimens were retested. The kappa values were 0.91 for positive/negative categorical classification and 0.79 for the four RLU intervals studied. Sample retesting yielded systematically lower RLU values than the original test (P<0.005), independently of the time elapsed between the two determinations (median, 53 days), possibly due to freeze-thaw cycles. The probability for a sample to show clinically discrepant results upon retesting was a function of the RLU value; samples with RLU values in the 0.5 to 5 interval showed 10.80% probability to yield discrepant results (95% confidence interval [CI], 7.86 to 14.33) compared to 0.85% probability for samples outside this interval (95% CI, 0.17 to 1.69). Globally, the HC2 assay shows high interlaboratory concordance. We have identified differential confidence thresholds and suggested the guidelines for interlaboratory PT in the future, as analytical quality assessment of HPV DNA detection remains a central component of the screening program for cervical cancer prevention.

Nuclear PARP-1 protein overexpression is associated with poor overall survival in early breast cancer.

BACKGROUND: Poly(ADP-ribose)polymerase-1 (PARP-1) is a highly promising novel target in breast cancer. However, the expression of PARP-1 protein in breast cancer and its associations with outcome are yet poorly characterized. PATIENTS AND METHODS: Quantitative expression of PARP-1 protein was assayed by a specific immunohistochemical signal intensity scanning assay in a range of normal to malignant breast lesions, including a series of patients (N = 330) with operable breast cancer to correlate with clinicopathological factors and long-term outcome. RESULTS: PARP-1 was overexpressed in about a third of ductal carcinoma in situ and infiltrating breast carcinomas. PARP-1 protein overexpression was associated to higher tumor grade (P = 0.01), estrogen-negative tumors (P < 0.001) and triple-negative phenotype (P < 0.001). The hazard ratio (HR) for death in patients with PARP-1 overexpressing tumors was 7.24 (95% CI; 3.56-14.75). In a multivariate analysis, PARP-1 overexpression was an independent prognostic factor for both disease-free (HR 10.05; 95% CI 5.42-10.66) and overall survival (HR 1.82; 95% CI 1.32-2.52). CONCLUSIONS: Nuclear PARP-1 is overexpressed during the malignant transformation of the breast, particularly in triple-negative tumors, and independently predicts poor prognosis in operable invasive breast cancer.

MET phosphorylation predicts poor outcome in small cell lung carcinoma and its inhibition blocks HGF-induced effects in MET mutant cell lines.

BACKGROUND: Small cell lung carcinoma (SCLC) has poor prognosis and remains orphan from targeted therapy. MET is activated in several tumour types and may be a promising therapeutic target. METHODS: To evaluate the role of MET in SCLC, MET gene status and protein expression were evaluated in a panel of SCLC cell lines. The MET inhibitor PHA-665752 was used to study effects of pathway inhibition in basal and hepatocyte growth factor (HGF)-stimulated conditions. Immunohistochemistry for MET and p-MET was performed in human SCLC samples and association with outcome was assessed. RESULTS: In MET mutant SCLC cells, HGF induced MET phosphorylation, increased proliferation, invasiveness and clonogenic growth. PHA-665752 blocked MET phosphorylation and counteracted HGF-induced effects. In clinical samples, total MET and p-MET overexpression were detected in 54% and 43% SCLC tumours (n = 77), respectively. MET phosphorylation was associated with poor median overall survival (132 days) vs p-MET negative cases (287 days) (P < 0.001). Phospho-MET retained its prognostic value in a multivariate analysis. CONCLUSIONS: MET activation resulted in a more aggressive phenotype in MET mutant SCLC cells and its inhibition by PHA-665752 reversed this phenotype. In patients with SCLC, MET activation was associated with worse prognosis, suggesting a role in the adverse clinical behaviour in this disease.

Mitogen-activated protein kinase phosphatase-1 (MKP-1) impairs the response to anti-epidermal growth factor receptor (EGFR) antibody cetuximab in metastatic colorectal cancer patients.

BACKGROUND: The validation of KRAS mutations as a negative marker of response to anti-epidermal growth factor receptor (EGFR) antibodies has meant a seminal advance towards treatment individualisation of colorectal cancer (CRC) patients. However, as a KRAS wild-type status does not guarantee a response to anti-EGFR antibodies, a current challenge is the identification of other biomarkers of response. On the basis of pre-clinical evidence, we hypothesised that mitogen-activated protein kinase phosphatase-1 (MKP-1), a phosphatase that inactivates MAPKs, could be a mediator of resistance to anti-EGFR antibodies. METHODS: Tumour specimens from 48 metastatic CRC patients treated with cetuximab-based chemotherapy were evaluated for KRAS and BRAF mutational status and MKP-1 expression as assessed by immunohistochemistry. RESULTS: As expected, clinical benefit was confined to wild-type KRAS and BRAF patients. Mitogen-activated protein kinase phosphatase-1 was overexpressed in 16 patients (33%) and was not associated with patient baseline clinicopathological characteristics and KRAS mutational status. All patients with BRAF mutations (n=3) had MKP-1 overexpression. Among KRAS wild-type patients, MKP-1 overexpressors had a 7% response rate (RR), whereas patients not overexpressing MKP-1 had a 44% RR (P=0.03). Moreover, median time to progression was significantly longer in MKP-1 non-overexpressing patients (32 vs 13 weeks, P=0.009). CONCLUSION: These results support the concept of MKP-1 as a promising negative marker of response to cetuximab-based treatment in CRC patients with wild-type KRAS.

Diarrheic syndrome as a clinical sign of intestinal infiltration in progressive B-cell chronic lymphocytic leukemia.

Gastrointestinal involvement is a rare event in patients with B-cell chronic lymphocytic leukemia (B-CLL) and is usually associated to lymphomatous transformation. However, in autopsy studies the reported incidence of microscopic infiltration can reach up to 50% of cases. Seven B-CLL patients in advanced stage/progressive disease were evaluated by colonoscopy because of continuous diarrhea. Five out of seven patients (71%) presented histological evidence of colonic infiltration. Persistent diarrhea in patients with progressive/advanced B-CLL can be a clinical sign of intestinal infiltration and justifies endoscopic examinations.

[Effector function of CLA(+) T lymphocytes on autologous keratinocytes in psoriasis]. / Función efectora de linfocitos T CLA1 sobre queratinocitos autólogos en psoriasis.

BACKGROUND: Cutaneous lymphocyte antigen (CLA) is expressed by a subgroup of memory T cells that exhibit skin homing and are implicated in cutaneous T-cell-mediated diseases. MATERIAL AND METHODS: Expression of genes associated with psoriasis was analyzed in keratinocytes taken from patients and healthy individuals and cultured under different conditions, including activation using supernatants from CLA(+) T lymphocytes activated with anti-CD3 and anti-CD28 antibodies. RESULTS: Keratinocytes from psoriasis patients activated by CLA(+)T lymphocytes expressed higher levels of interferon-inducible protein 10, HLA-DR, intercellular cell adhesion molecule 1, and inducible nitric oxide synthase. CONCLUSIONS: Our results suggest that we have developed an in vitro model that will allow analysis of the effector role of CLA(+) T lymphocytes on keratinocytes in psoriasis. This model may allow the identification of genes involved in the pathology of psoriasis through induction by CLA(+) T lymphocytes.

[Genotypic analysis in primary cutaneous lymphomas using the standardized BIOMED-2 polymerase chain reaction protocols]. / Aplicación de los protocolos de PCR BIOMED-2 en el análisis genotípico de los linfomas cutáneos primarios.

The European Biomedicine and Health (BIOMED-2) Concerted Action Project BMH4-CT98-3936 has defined standardized protocols for polymerase chain reaction (PCR) amplification of different loci of the T-cell receptor (TCR) and immunoglobulin (Ig) genes with a view to achieving greater sensitivity and specificity in the assessment of clonality of lymphoid neoplasms. To assess T-cell clonality, analysis of TCRbeta gene and TCRdelta rearrangements (useful in cases of Tgammadelta + cell neoplasms) is proposed alongside that of TCRgamma. For analysis of B-cell clonality, along with the framework (FR) III segment of the IgH gene, other segments are studied (FRI, FRII) in addition to Igl and Igk genes or incomplete DJ rearrangements of the IgH gene and the k deleting element. The results of the amplification are read using automatic reading systems (GeneScan) or using a heteroduplex system.

Función efectora de linfocitos T CLA+ sobre queratinocitos autólogos en psoriasis / Effector function of CLA+ T lymphocytes on autologous keratinocytes in psoriasis

Introducción. Los linfocitos T CLA+ representan un subgrupo de linfocitos T de memoria con tropismo cutáneo que están implicados en diferentes enfermedades cutáneas mediadas por células T. Material y métodos. Se estudió la expresión de algunos genes asociados a la psoriasis en queratinocitos de enfermos y sanos cultivados en diferentes condiciones, entre ellas la activación por sobrenadantes de linfocitos T CLA+ activados mediante anti-CD3/anti-CD28.Resultados. Los queratinocitos psoriásicos activados por linfocitos CLA+ expresan más IP-10, HLA-DR, ICAM-1 e iNOS. Discusión. Estos resultados sugieren que hemos establecido un modelo in vitro que permite estudiar la función efectora de los linfocitos T CLA+ sobre los queratinocitos en la psoriasis. Este modelo puede permitir la identificación de genes relevantes en la patogenia de la psoriasis al ser inducidos por linfocitos T CLA+ (AU)

Background. Cutaneous lymphocyte antigen (CLA) is expressed by a subgroup of memory T cells that exhibit skin homing and are implicated in cutaneous T-cell-mediated diseases. Material and methods. Expression of genes associated with psoriasis was analyzed in keratinocytes taken from patients and healthy individuals and cultured under different conditions, including activation using supernatants from CLA+ T lymphocytes activated with anti-CD3 and anti-CD28 antibodies. Results. Keratinocytes from psoriasis patients activated by CLA+ T lymphocytes expressed higher levels of interferon-inducible protein 10, HLA-DR, intercellular cell adhesion molecule 1, and inducible nitric oxide synthase. Conclusions. Our results suggest that we have developed an in vitro model that will allow analysis of the effector role of CLA+ T lymphocytes on keratinocytes in psoriasis. This model may allow the identification of genes involved in the pathology of psoriasis through induction by CLA+ T lymphocytes (AU)