Miguel Martorell

Prevalence and Clinical Outcomes for Patients With ALK-Positive Resected Stage I to III Adenocarcinoma: Results From the European Thoracic Oncology Platform Lungscape Project

PURPOSE The prevalence of anaplastic lymphoma kinase (ALK) gene fusion (ALK positivity) in early-stage non-small-cell lung cancer (NSCLC) varies by population examined and detection method used. The Lungscape ALK project was designed to address the prevalence and prognostic impact of ALK positivity in resected lung adenocarcinoma in a primarily European population. METHODS Analysis of ALK status was performed by immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH) in tissue sections of 1,281 patients with adenocarcinoma in the European Thoracic Oncology Platform Lungscape iBiobank. Positive patients were matched with negative patients in a 1:2 ratio, both for IHC and for FISH testing. Testing was performed in 16 participating centers, using the same protocol after passing external quality assessment. RESULTS Positive ALK IHC staining was present in 80 patients (prevalence of 6.2%; 95% CI, 4.9% to 7.6%). Of these, 28 patients were ALK FISH positive, corresponding to a lower bound for the prevalence of FISH positivity of 2.2%. FISH specificity was 100%, and FISH sensitivity was 35.0% (95% CI, 24.7% to 46.5%), with a sensitivity value of 81.3% (95% CI, 63.6% to 92.8%) for IHC 2+/3+ patients. The hazard of death for FISH-positive patients was lower than for IHC-negative patients (P = .022). Multivariable models, adjusted for patient, tumor, and treatment characteristics, and matched cohort analysis confirmed that ALK FISH positivity is a predictor for better overall survival (OS). CONCLUSION In this large cohort of surgically resected lung adenocarcinomas, the prevalence of ALK positivity was 6.2% using IHC and at least 2.2% using FISH. A screening strategy based on IHC or H-score could be envisaged. ALK positivity (by either IHC or FISH) was related to better OS.

Lymphoepithelioma-like Carcinoma of the Uterine Cervix

CONTEXT It has been proposed that Epstein-Barr virus (EBV) plays a role in the etiology of lymphoepithelioma-like carcinoma (LELC) in diverse anatomic locations. In contrast to Asian women, Western women have a low prevalence of LELC of the uterine cervix, and EBV genomes have not been identified. OBJECTIVE To assess the presence of EBV in LELC of the uterine cervix in 4 white Western women. DESIGN We collected 4 cases of LELC of the uterine cervix between 1990 and 2000. We performed histologic and immunohistochemical analyses of formalin-fixed, paraffin-embedded tumor samples. We amplified tumor DNA with polymerase chain reaction to detect EBV, human papillomavirus, and simian virus 40 DNAs. RESULTS Immunohistochemically, tumor cells were positive for cytokeratins and showed strong expression of p53 and MIB-1. Staining for the oncoprotein c-Erb-B2 was focally positive, and staining for Bcl-2 and progesterone receptors was negative. Only one case showed focal nuclear staining for estrogen receptors. All cases had a dense infiltrate of mature lymphocytes expressing T-cell antigens CD45RO, CD3, and CD8. Polymerase chain reaction analysis did not detect EBV, human papillomavirus, or simian virus 40 DNA sequences in any of the 4 cases. One case had positive serologic results for anti-EBV antibodies, indicating a mild or chronic infection. CONCLUSIONS LELC of the uterine cervix shows the immunohistochemical profile of an aggressive tumor in spite of its good prognosis, in which CD8 cytotoxic suppressor lymphocytes could play an important role. Based on our results, the role of EBV, human papillomavirus, or simian virus 40 in the pathogenesis of LELC of the uterine cervix in Western women remains unclear.

Presence of Human Papillomavirus DNA in Testicular Biopsies From Nonobstructive Azoospermic Men

CONTEXT Human papillomavirus (HPV) plays a major role in the etiology of many malignancies of diverse localization, such as uterine cervical carcinoma and its precursors. Human papillomavirus sequences have been detected throughout the male lower genitourinary tract, but the role of men as transmitters remains unclear. OBJECTIVE To investigate the relationship between azoospermia and the presence of HPV DNA in testicular cells. DESIGN One hundred eighty-five patients with azoospermia undergoing testicular biopsy were studied. Histologic study was done on formalin-fixed, paraffin-embedded samples from testicular biopsies, stained with hematoxylin-eosin. Molecular study to detect HPV sequences was performed on genomic DNA isolated from paraffin sections by standard protocols. Seven cases containing HPV sequences were studied after microdissection with PALM microlaser technology in order to determine the presence of HPV DNA sequences in different cells, as well as from seminal tubules or stromal (Leydig) cells. RESULTS Human papillomavirus DNA sequences were detected in testicular biopsies of 12 patients (6.48%). Human papillomavirus type 16 was the most common genotype encountered. Among the 92 patients who underwent bilateral testicular biopsy, HPV sequences were detected in 9 patients (9.78%), all of whom showed only unilateral testicular affection, more often in the left testicle (ratio, 2: 1). After microdissection, HPV DNA sequences were seen in Leydig and Sertoli cells; the presence of HPV in germinal cells could not be ruled out. CONCLUSIONS Leydig cells, Sertoli cells, and probably germinal cells (cases 2, 3, and 4) harbored HPV DNA sequences. Such findings have not been previously described in testicular tissue.

Solitary fibrous tumor of the thigh with epithelioid features: a case report

BackgroundExtrapleural Solitary Fibrous tumors (SFTs) have been increasingly reported. The retroperitoneum, deep soft tissues of proximal extremities, abdominal cavity, trunk, head and neck are the most common extraserosal locations reported. Microscopically they show a wide range of morphological features, and so the differential diagnosis is extensive. Immunohistochemically, they commonly express CD34, vimentin, bcl-2 and CD99. Epithelial membrane antigen (EMA) and smooth muscle actin (SMA) may occasionally be expressed. Epithelioid morphology in extrapleural SFT has only very occasionally been described (five cases reported), some of them with biphasic pattern and others with malignant characteristics.Case presentationA SFT of the thigh with epithelioid areas in a 63 year old woman is reported. Microscopically the tumor showed areas hypo and hipercellular. At the periphery of the hipercellular areas there were nodules composed of epithelioid cells. Immunohistochemically both the spindle and epithelioid cells were positive for CD34, vimentin, bcl-2 and CD99. Epithelial, neural and muscular markers were negative. Molecular study was done and ruled out a synovial sarcoma.ConclusionTen cases of SFT of the thigh have been reported but to our knowledge this is the first case with epithelioid morphology affecting the extremities. Identification of this pattern of SFT is of importance, to avoid misdiagnosis with other more aggressive conditions in soft tissue.

Effects of small interfering RNAs targeting fascin on human esophageal squamous cell carcinoma cell lines

BackgroundFascin induces membrane protrusions and cell motility. Fascin overexpression was associated with poor prognosis, and its downregulation reduces cell motility and invasiveness in esophageal squamous cell carcinoma (ESCC). Using a stable knockdown cell line, we revealed the effect of fascin on cell growth, cell adhesion and tumor formation.MethodsWe examined whether fascin is a potential target in ESCC using in vitro and in vivo studies utilizing a specific siRNA. We established a stable transfectant with downregulated fascin from KYSE170 cell line.ResultsThe fascin downregulated cell lines showed a slower growth pattern by 40.3% (p < 0.01) and detachment from collagen-coated plates by 53.6% (p < 0.01), compared to mock cells, suggesting that fascin plays a role in cell growth by maintaining cell adhesion to the extracellular matrix. In vivo, the tumor size was significantly smaller in the tumor with fascin knockdown cells than in mock cells by 95% at 30 days after inoculation.ConclusionsThese findings suggest that fascin overexpression plays a role in tumor growth and progression in ESCC and that cell death caused by its downregulation might be induced by cell adhesion loss. This indicates that targeting fascin pathway could be a novel therapeutic strategy for the human ESCC.

Distribution of Human Papillomavirus Types in Women from Valencia, Spain, with Abnormal Cytology

OBJECTIVE To determine human papillomavirus (HPV) types among cervical smears using polymerase chain reaction (PCR) and to contribute to the knowledge of human papillomavirus genotype distribution and prevalence of oncogenic types in cervical lesions in Spain. STUDY DESIGN Consensus PCR and direct s quencing of PCR products (DNA HPV typing) were used in a retrospective study to determinate the type or types of HPVon 974 cytology smears of women with abnormal cytology results. RESULTS Of 974 smears, 79.8% were high-risk (H-R) HPVs, 19.7% low-risk (L-R) HPVs, 4.6% indeterminate-risk (I-R) HPVs, considering both single and multiple infections. Multiple infections were detected in 4.7% of the cytologies. We detected 40 different HPV types: 17 H-R (HPV26 not detected), 10 L-R (HPVs 40 and HPV 61 not detected) and 13 I-R. The highest percentage of H-R HPV was found in those women with a cytologic high grade squamous intraepithelial lesion (HSIL) (87.4%). HPV 16 was the most frequent genotype. CONCLUSION There was a significantly her prevalence rate of H-R HPV in HSIL than in low grade squamous intraepithelial lesion (LSIL) and atypical squamous cells of undetermined significance (ASC-US) (p < 0.01). HPV 16 (39.5%) was the most frequent genotype, with a significantly higher prevalence rate of this type in HSIL than in LSIL and ASC-US (p < 0.05 and p < 0.001, respectively). The study of the distribution of HPV and the presence of oncogenic HPV types in our population is important to assess the cost effectiveness of the current vaccines.

Angiolymphoid hyperplasia with eosinophilia involving the cubital nerve

A tumor involving cubital nerve was resected and studied; it was classified as an angiolymphoid hyperplasia with eosinophilia (ALHE). Immunohistochemical and molecular study was done both to confirm the reactive nature of the process and rule out the presence of clonal T or B cell rearrangement. This lesion has been designated as epitheloid hemangioma [Coindre (1994) Ann Pathol 14:426]. Typically, ALHE occurs in the skin and the subcutaneous tissue, and extracutaneous involvement is rare. No cases of ALHE affecting a nerve have been described, but a case of Kimura’s disease, the lesions of which have repeatedly been confused with ALHE, has been reported involving median nerve.

MicroRNA profiling associated with non-small cell lung cancer: next generation sequencing detection, experimental validation, and prognostic value

BACKGROUND The average five-year survival index for non-small cell lung cancer (NSCLC) patients is approximately 15%. Emerging evidence indicates that microRNAs constitute a new class of gene regulators in humans that may play an important role in tumorigenesis. Hence, there is growing interest in studying their role as possible new biomarkers whose expression is aberrant in cancer. Therefore, in this study we identified deregulated miRNAs by next generation sequencing (NGS) and analyzed their prognostic value. METHODS Sequencing by oligo ligation detection technology was used to identify dysregulated miRNAs in a training cohort comprising paired tumor/normal tissue samples (N = 32). We validated 22 randomly selected differentially-expressed miRNAs by quantitative real time PCR in tumor and adjacent normal tissue samples (N = 178). Kaplan-Meier survival analysis and Cox regression were used in multivariate analysis to identify independent prognostic biomarkers. RESULTS NGS analysis revealed that 39 miRNAs were dysregulated in NSCLC: 28 were upregulated and 11 were downregulated. Twenty-two miRNAs were validated in an independent cohort. Interestingly, the group of patients with high expression of both miRNAs (miR-21high and miR-188high) showed shorter relapse-free survival (RFS) and overall survival (OS) times. Multivariate analysis showed that this miRNA combination is an independent prognostic marker for RFS and OS (p = 0.001 and p < 0.0001, respectively). CONCLUSIONS NGS technology can specifically identify dysregulated miRNA profiles in rsectable NSCLC samples. MiR-21 or miR-188 overexpression correlated with a negative prognosis, and their combined signature may represent a new independent prognostic biomarker for RFS and OS.Background The average five-year survival for non-small cell lung cancer (NSCLC) patients is approximately 15%. Emerging evidence indicates that microRNAs (miRNAs) constitute a new class of gene regulators in humans that may play an important role in tumorigenesis. Hence, there is growing interest in studying their role as possible new biomarkers whose expression is aberrant in cancer. Therefore, in this study we identified dysregulated miRNAs by next generation sequencing (NGS) and analyzed their prognostic value. Methods Sequencing by oligo ligation detection technology was used to identify dysregulated miRNAs in a training cohort comprising paired tumor/normal tissue samples (N = 32). We validated 22 randomly selected differentially-expressed miRNAs by quantitative real time PCR in tumor and adjacent normal tissue samples (N = 178). Kaplan-Meier survival analysis and Cox regression were used in multivariate analysis to identify independent prognostic biomarkers. Results NGS analysis revealed that 39 miRNAs were dysregulated in NSCLC: 28 were upregulated and 11 were downregulated. Twenty-two miRNAs were validated in an independent cohort. Interestingly, the group of patients with high expression of both miRNAs (miR-21high and miR-188high) showed shorter relapse-free survival (RFS) and overall survival (OS) times. Multivariate analysis confirmed that this combined signature is an independent prognostic marker for RFS and OS (p = 0.001 and p < 0.0001, respectively). Conclusions NGS technology can specifically identify dysregulated miRNA profiles in resectable NSCLC samples. MiR-21 or miR-188 overexpression correlated with a negative prognosis, and their combined signature may represent a new independent prognostic biomarker for RFS and OS.

A Gene Signature Combining the Tissue Expression of Three Angiogenic Factors is a Prognostic Marker in Early-stage Non-small Cell Lung Cancer

AbstractBackground Angiogenesis and lymphangiogenesis are key mechanisms for tumor growth and dissemination. They are mainly regulated by the vascular endothelial growth factor (VEGF) family of ligands and receptors. The aim of this study was to analyze relative expression levels of angiogenic markers in resectable non-small cell lung cancer patients in order to asses a prognostic signature that could improve characterization of patients with worse clinical outcomes. Methods RNA was obtained from tumor and normal lung specimens from 175 patients. Quantitative polymerase chain reaction was performed to analyze the relative expression of HIF1A, PlGF, VEGFA, VEGFA165b, VEGFB, VEGFC, VEGFD, VEGFR1, VEGFR2, VEGFR3, NRP1 and NRP2.ResultsUnivariate analysis showed that tumor size and ECOG-PS are prognostic factors for time to progression (TTP) and overall survival (OS). This analysis in the case of angiogenic factors also revealed that PlGF, VEGFA, VEGFB and VEGFD distinguish patients with different outcomes. Taking into account the complex interplay between the different ligands of the VEGF family and to more precisely predict the outcome of the patients, we considered a new analysis combining several VEGF ligands. In order to find independent prognostic variables, we performed a multivariate Cox analysis, which showed that the subgroup of patients with higher relative expression of VEGFA plus lower VEGFB and VEGFD presented the poorest outcome for both TTP and OS.ConclusionsThe relative expression of these three genes can be considered as an angiogenic gene signature whose applicability for the selection of candidates for targeted therapies needs to be further validated.

Analysis of the immune microenvironment in resected non-small cell lung cancer: the prognostic value of different T lymphocyte markers

The prognosis of non-small cell lung cancer (NSCLC) remains poor and heterogeneous and new biomarkers are needed. As the immune system plays a pivotal role in cancer, the study of immune-related markers may provide valuable prognostic information of NSCLC. In 122 formalin-fixed, paraffin-embedded tumor tissue samples from early-stage NSCLC, tumor and tumor-near stromal areas were microdissected and gene expression levels of conventional and regulatory T cell markers were assessed by quantitative polymerase chain reaction. Also, the presence of infiltrating CD4+, CD8+, and FOXP3+ cells in tumor samples was assessed by immunohistochemistry. The relative proportion of conventional and regulatory T cells present in the tumor environment was assessed and found to be key to understand the importance that the immune system analysis has in the prognostics of NSCLC patients. The presence of CD8+ cells in the tumor compartment was associated with better outcome, whereas the presence of FOXP3+ cells was associated with worse overall survival. The negative prognostic value of combined biomarkers, indicating high levels of FOXP3 in the stroma and low levels of CD4 or CD8 in tumors, was observed at mRNA level and was validated by immunohistochemistry.In conclusion, the proportion of T helper and cytotoxic cells vs. regulatory T cells in different locations of the tumor microenvironment have opposite prognostic impacts in resected NSCLC.