B cell-helper neutrophils stimulate the diversification and production of immunoglobulin in the marginal zone of the spleen.

Neutrophils use immunoglobulins to clear antigen, but their role in immunoglobulin production is unknown. Here we identified neutrophils around the marginal zone (MZ) of the spleen, a B cell area specialized in T cell-independent immunoglobulin responses to circulating antigen. Neutrophils colonized peri-MZ areas after postnatal mucosal colonization by microbes and enhanced their B cell-helper function after receiving reprogramming signals, including interleukin 10 (IL-10), from splenic sinusoidal endothelial cells. Splenic neutrophils induced immunoglobulin class switching, somatic hypermutation and antibody production by activating MZ B cells through a mechanism that involved the cytokines BAFF, APRIL and IL-21. Neutropenic patients had fewer and hypomutated MZ B cells and a lower abundance of preimmune immunoglobulins to T cell-independent antigens, which indicates that neutrophils generate an innate layer of antimicrobial immunoglobulin defense by interacting with MZ B cells.

Prognostic value of stem cell markers in glioblastoma.

Background/Context: Glioblastoma (GB) is the most common primary brain tumour in adults and it is associated with a high mortality rate. According to the stem cell theory, the growth, relapse and treatment response of GB is determined by the stem cell subpopulation present in the tumour. Objective: Our aim is to study the prognostic value of stem cell markers (CD44, Nestin, Olig2 and SOX2) in a series of homogeneously treated GBs. Material and methods: We study 280 GBs treated with STUPP acheme with a histologican review of the cases and TMA with a máximum of 4 spots for each case. Each slide was immunohistochemically stained and Reading. We compared the immunohistochemical results with survival tme. Results: Only SOX2 immunoexpression (IE) excedding 10% of the tumour cells was found to be related to good survival (p= 0.037) in univariate analysis. However, amultivariate analysis indicate the age, surgery and MGMT promotes methylation but no SOX2 IE are prognostic factors. Conclusions: We conclude the immunohistochemical studies of stem cell markers in GB are not useful for predicting prognosis in daily practice.

Activated leukocyte cell adhesion molecule (ALCAM) is a marker of recurrence and promotes cell migration, invasion, and metastasis in early-stage endometrioid endometrial cancer.

Endometrial cancer is the most common gynaecological cancer in western countries, being the most common subtype of endometrioid tumours. Most patients are diagnosed at an early stage and present an excellent prognosis. However, a number of those continue to suffer recurrence, without means of identification by risk classification systems. Thus, finding a reliable marker to predict recurrence becomes an important unmet clinical issue. ALCAM is a cell-cell adhesion molecule and member of the immunoglobulin superfamily that has been associated with the genesis of many cancers. Here, we first determined the value of ALCAM as a marker of recurrence in endometrioid endometrial cancer by conducting a retrospective multicentre study of 174 primary tumours. In early-stage patients (N = 134), recurrence-free survival was poorer in patients with ALCAM-positive compared to ALCAM-negative tumours (HR 4.237; 95% CI 1.01-17.76). This difference was more significant in patients with early-stage moderately-poorly differentiated tumours (HR 9.259; 95% CI 2.12-53.47). In multivariate analysis, ALCAM positivity was an independent prognostic factor in early-stage disease (HR 6.027; 95% CI 1.41-25.74). Then we demonstrated in vitro a role for ALCAM in cell migration and invasion by using a loss-of-function model in two endometrial cancer cell lines. ALCAM depletion resulted in a reduced primary tumour size and reduced metastatic local spread in an orthotopic murine model. Gene expression analysis of ALCAM-depleted cell lines pointed to motility, invasiveness, cellular assembly, and organization as the most deregulated functions. Finally, we assessed some of the downstream effector genes that are involved in ALCAM-mediated cell migration; specifically FLNB, TXNRD1, and LAMC2 were validated at the mRNA and protein level. In conclusion, our results highlight the potential of ALCAM as a recurrent biomarker in early-stage endometrioid endometrial cancer and point to ALCAM as an important molecule in endometrial cancer dissemination by regulating cell migration, invasion, and metastasis. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Utility of Human Papillomavirus Genotyping in the Management of Low-Grade Squamous Intraepithelial Lesions.

OBJECTIVE: The aim of the study was to determine the usefulness of human papillomavirus (HPV) partial genotyping test in the triage of newly diagnosed low-grade squamous intraepithelial lesions (LSILs). MATERIALS AND METHODS: We analyzed 143 patients with LSIL diagnosed de novo. Lesions were classified as positive for HPV 16 or HPV 18, positive for HPV but not HPV 16 or HPV 18 (HPVno16no18) or no HPV detected (HPVneg). Patients were followed for a period of 2 years or until the lesion progressed. We calculated absolute and relative risks for progression and regression according to the HPV result. RESULTS: The mean (SD) age was 33.8 (11.1) years. A total of 19.6% were positive for HPV 16, 4.9% for HPV 18, and 63.6% for HPVno16no18. The absolute risk of HPV 16 for progression to cervical intraepithelial neoplasia grade 2 or more (CIN 2+) was 32.1%, 14.3% for HPV 18, and 5.8% for HPVno16no18. None of the HPVneg cases evolved to CIN 2+. The presence of HPV 16 conferred a 7.4 (95% CI = 2.7-20.3) times greater risk of developing CIN 2+ than its absence. The absolute risks for HPV 16, HPV 18, HPVno16no18, and HPVneg for regression were 53.6%, 57.1%, 75.4%, and 87.5%, respectively. Relative risks for regression were 0.7 (95% CI = 0.5-0.9) for HPV 16 and 1.3 (95% CI = 1.1-1.5) for HPVneg. CONCLUSIONS: The HPV 16 LSILs are more likely to progress to CIN 2+, so tight control and immediate colposcopy are crucial, whereas when HPV 16 is not present, follow-up could be less strict. Low-grade squamous intraepithelial lesions in which high-risk HPV is not detected do not progress to CIN 2+, so its control should be different from other LSIL, and conservative management could be an acceptable strategy.

Usefulness of p16INK4a staining for managing histological high-grade squamous intraepithelial cervical lesions.

p16INK4a (p16) tumor-suppressor protein is a biomarker of human papillomavirus (HPV) oncogenic activity that has revealed a high rate of positivity in histological high-gade squamous intraepithelial lesion/cervical intraepithelial neoplasia grade 2 (HSIL/CIN2) lesions. However, there is a paucity of data regarding p16 status as a surrogate marker of HSIL/CIN2 evolution. The aim of this study was to evaluate the outcome of HSIL/CIN2 patients followed up without treatment for 12 months according to p16 immunohistochemical staining. Patients diagnosed with HSIL/CIN2 colposcopy-directed biopsy, were recruited prospectively between December 2011 and October 2013. p16 staining was performed in all HSIL/CIN2 diagnostic biopsies. Follow-up was conducted every 4 months by cytology, colposcopy and biopsy if suspicion of progression and once the 12 months of follow-up completed. Complete regression, partial regression, persistence, and progression rates of HSIL/CIN2 were defined as a final outcome. A total of 96 patients were included in the analysis. The rate of spontaneous regression was 64%, while 28% had persistent disease, and 8% progressed at 12 months of follow-up. p16 was positive in 81 (84%) initial HSIL/CIN2 biopsies. Regression was observed in all 15 p16 negative cases and in 46 of 81 (57%) p16 positive cases (P=0.001). In conclusion, patients with p16 negative HSIL/CIN2 biopsy had a high rate of regression during first 12 months of follow-up. Status of p16 staining could be considered for HSIL/CIN2 management.

Fibrinogen nitrotyrosination after ischemic stroke impairs thrombolysis and promotes neuronal death.

Ischemic stroke is an acute vascular event that compromises neuronal viability, and identification of the pathophysiological mechanisms is critical for its correct management. Ischemia produces increased nitric oxide synthesis to recover blood flow but also induces a free radical burst. Nitric oxide and superoxide anion react to generate peroxynitrite that nitrates tyrosines. We found that fibrinogen nitrotyrosination was detected in plasma after the initiation of ischemic stroke in human patients. Electron microscopy and protein intrinsic fluorescence showed that in vitro nitrotyrosination of fibrinogen affected its structure. Thromboelastography showed that initially fibrinogen nitrotyrosination retarded clot formation but later made the clot more resistant to fibrinolysis. This result was independent of any effect on thrombin production. Immunofluorescence analysis of affected human brain areas also showed that both fibrinogen and nitrotyrosinated fibrinogen spread into the brain parenchyma after ischemic stroke. Therefore, we assayed the toxicity of fibrinogen and nitrotyrosinated fibrinogen in a human neuroblastoma cell line. For that purpose we measured the activity of caspase-3, a key enzyme in the apoptotic pathway, and cell survival. We found that nitrotyrosinated fibrinogen induced higher activation of caspase 3. Accordingly, cell survival assays showed a more neurotoxic effect of nitrotyrosinated fibrinogen at all concentrations tested. In summary, nitrotyrosinated fibrinogen would be of pathophysiological interest in ischemic stroke due to both its impact on hemostasis - it impairs thrombolysis, the main target in stroke treatments - and its neurotoxicity that would contribute to the death of the brain tissue surrounding the infarcted area.

Expansion of the NKG2C+ natural killer-cell subset is associated with high-risk carotid atherosclerotic plaques in seropositive patients for human cytomegalovirus.

OBJECTIVE: Human cytomegalovirus (HCMV), a pathogen involved in the development and progression of atherosclerosis, promotes in some individuals a marked reconfiguration of the natural killer (NK)-cell compartment whose hallmark is a persistent expansion of a peripheral blood NK-cell subset expressing the CD94/NKG2C NK receptor. We aimed to evaluate whether the HCMV-associated NK-cell compartment reconfiguration is related to carotid atherosclerotic plaque (CAP) instability. APPROACH AND RESULTS: NK receptor expression (ie, LILRB1, NKG2A, NKG2C, and killer immunoglobulin-like receptors [KIR]) by peripheral NK and T cells was evaluated in 40 patients with HCMV+ with CAP, including nonatherosclerotic strokes (n=15) and healthy subjects (n=11) as controls. High-risk CAP (n=16), defined as carotid stenosis >50% with ipsilateral neurological symptomatology in the previous 180 days, compared with non-high-risk CAP had higher %NKG2C+ NK cells (29.5 ± 22.4% versus 16.3 ± 13.2%; P=0.026; odds ratio, 1.053; 95% confidence interval, 1.002-1.106; P=0.042), with a corresponding reduction in the NKG2A+ NK subset (31.7 ± 17.8% versus 41.8 ± 15.8%; P=0.072). The proportions of NKG2C+ NK cells in high-risk CAP were inversely correlated with the CD4+/CD8+ ratio (R(Spearman)=-0.629; P=0.009) and directly with high-sensitivity C-reactive protein levels (R(Pearson) = 0.591; P=0.012), consistent with higher subclinical systemic inflammation. The intraplaque inflammatory infiltrate, evaluated in 27 CAP obtained after endarterectomy, showed a higher presence of subintimal CD3+ lymphocytes in those patients with HCMV-induced changes in the peripheral NK- and T-cell compartments. CONCLUSIONS: The expansion of NKG2C+ NK cells in patients with CAP seems to be associated with an increased risk of plaque destabilization in some patients with chronic HCMV infection.

The C250T Mutation of TERTp Might Grant a Better Prognosis to Glioblastoma by Exerting Less Biological Effect on Telomeres and Chromosomes Than the C228T Mutation.

The aim of this study was to determine how TERTp mutations impact glioblastoma prognosis. MATERIALS AND METHODS: TERTp mutations were assessed in a retrospective cohort of 258 uniformly treated glioblastoma patients. RNA-sequencing and whole exome sequencing results were available in a subset of patients. RESULTS: Overall, there were no differences in outcomes between patients with mutated TERTp-wt or TERTp. However, we found significant differences according to the type of TERTp mutation. Progression-free survival (mPFS) was 9.1 months for those with the C250T mutation and 7 months for those with either the C228T mutation or TERTp-wt (p = 0.016). Overall survival (mOS) was 21.9 and 15 months, respectively (p = 0.026). This differential effect was more pronounced in patients with MGMTp methylation (mPFS: p = 0.008; mOS: p = 0.021). Multivariate analysis identified the C250T mutation as an independent prognostic factor for longer mOS (HR 0.69; p = 0.044). We found no differences according to TERTp mutation status in molecular alterations common in glioblastoma, nor in copy number variants in genes related to alternative lengthening of telomeres. Nevertheless, in the gene enrichment analysis adjusted for MGMTp methylation status, some Reactome gene sets were differentially enriched, suggesting that the C250T mutation may exert a lesser effect on telomeres or chromosomes. CONCLUSIONS: In our series, patients exhibiting the C250T mutation had a more favorable prognosis compared to those with either TERPp-wt or TERTp C228T mutations. Additionally, our findings suggest a reduced involvement of the C250T mutation in the underlying biological mechanisms related to telomeres.

Endometrial preparation with desogestrel before Essure hysteroscopic sterilization: preliminary study.

STUDY OBJECTIVE: To assess the effect of desogestrel on endometrial preparation for transcervical sterilization using the Essure device. DESIGN: Prospective nonrandomized clinical study (Canadian Task Force classification II-3). SETTING: Acute-care university-affiliated hospital in Barcelona, Spain. PATIENTS: Women undergoing sterilization using the Essure device between January 2010 and January 2011. INTERVENTIONS: Participants were offered desogestrel, 75 µg/d, for 6 weeks before the procedure. Sixteen who accepted were included in the desogestrel group, and 18 who refused were allocated to the no-treatment group. Endometrial biopsy samples were also obtained. MEASUREMENTS AND MAIN RESULTS: In women who received desogestrel, decidual transformation was observed in eight, glandular atrophy in three, and proliferative endometrium in five. In the no-treatment group, two women had menstruation, nine had proliferative endometrium, and seven had secretory endometrium. In the desogestrel group, the procedure was successful in all women. In the no-treatment group, the procedure was cancelled in two women because of menstruation and in four women with secretory endometrium in whom the tubal ostia were difficult to visualize because of endometrial thickness and bleeding. The median (interquartile range, 25th-75th percentile) duration of the procedure was shorter in the desogestrel group than in the no-treatment group (7 [6-7] minutes vs 8 [7-12] minutes; p = .002). CONCLUSION: Desogestrel, 75 µg/d, could be an alternative to combined hormonal contraception before placement of Essure inserts, facilitating the procedure and serving as a contraceptive method during the following 12 weeks until occlusion of the tubes.

High-grade squamous intraepithelial lesion could be managed conservatively in women up to 25 years: results from a retrospective cohort study.

OBJECTIVE: The aim of this study was to compare histologic findings and clinical outcomes of women up to 25 years with a high-grade squamous intraepithelial lesion (HSIL) compared to women older than 25 years. MATERIALS AND METHODS: Sixty-three women up to 25 years and 245 women older than 25 years with HSIL, diagnosed from June 1991 to September 2008, were examined and treated following the official Spanish guidelines. Colposcopic and histologic findings and needs for treatment were recorded, and patients were followed up for a minimum of 12 months. RESULTS: A total of 308 patients were evaluated; 63.49% of women up to 25 years and 77.10% of women older than 25 years with HSIL had cervical intraepithelial neoplasia (CIN) 2+ histology (p = .04). Also, 74.60% of women up to 25 years and 99.24% of women older than 25 years underwent an excisional procedure (p < .001). No significant or clinical differences were found in the 1-year follow-up outcomes (82.54% vs 78.37% had normal results; p = ns). CONCLUSIONS: Women up to 25 years have less CIN 2+ histologic findings and less need for conization compared to older women. Our findings support the feasibility to design an adequate protocol for these younger women, which would be less aggressive and would, consequently, minimize obstetric long-term secondary effects.

Gal-1 Expression Analysis in the GLIOCAT Multicenter Study: Role as a Prognostic Factor and an Immune-Suppressive Biomarker.

Glioblastoma (GBM) is the most frequent primary malignant brain tumor and has a dismal prognosis. Unfortunately, despite the recent revolution of immune checkpoint inhibitors in many solid tumors, these have not shown a benefit in overall survival in GBM patients. Therefore, new potential treatment targets as well as diagnostic, prognostic, and/or predictive biomarkers are needed to improve outcomes in this population. The ß-galactoside binding protein Galectin-1 (Gal-1) is a protein with a wide range of pro-tumor functions such as proliferation, invasion, angiogenesis, and immune suppression. Here, we evaluated Gal-1 expression by immunohistochemistry in a homogenously treated cohort of GBM (the GLIOCAT project) and correlated its expression with clinical and molecular data. We observed that Gal-1 is a negative prognostic factor in GBM. Interestingly, we observed higher levels of Gal-1 expression in the mesenchymal/classical subtypes compared to the less aggressive proneural subtype. We also observed a Gal-1 expression correlation with immune suppressive signatures of CD4 T-cells and macrophages, as well as with several GBM established biomarkers, including SHC1, PD-L1, PAX2, MEOX2, YKL-40, TCIRG1, YWHAG, OLIG2, SOX2, Ki-67, and SOX11. Moreover, Gal-1 levels were significantly lower in grade 4 IDH-1 mutant astrocytomas, which have a better prognosis. Our results confirm the role of Gal-1 as a prognostic factor and also suggest its value as an immune-suppressive biomarker in GBM.

Prediction of cervical intraepithelial neoplasia grade 2+ (CIN2+) using HPV DNA testing after a diagnosis of atypical squamous cell of undetermined significance (ASC-US) in Catalonia, Spain.

BACKGROUND: A protocol for cervical cancer screening among sexually active women 25 to 65 years of age was introduced in 2006 in Catalonia, Spain to increase coverage and to recommend a 3-year-interval between screening cytology. In addition, Human Papillomavirus (HPV) was offered as a triage test for women with a diagnosis of atypical squamous cells of undetermined significance (ASC-US). HPV testing was recommended within 3 months of ASC-US diagnosis. According to protocol, HPV negative women were referred to regular screening including a cytological exam every 3 years while HPV positive women were referred to colposcopy and closer follow-up. We evaluated the implementation of the protocol and the prediction of HPV testing as a triage tool for cervical intraepithelial lesions grade two or worse (CIN2+) in women with a cytological diagnosis of ASC-US. METHODS: During 2007-08 a total of 611 women from five reference laboratories in Catalonia with a novel diagnosis of ASC-US were referred for high risk HPV (hrHPV) triage using high risk Hybrid Capture version 2. Using routine record linkage data, women were followed for 3 years to evaluate hrHPV testing efficacy for predicting CIN2+ cases. Logistic regression analysis was used to estimate the odds ratio for CIN2 +. RESULTS: Among the 611 women diagnosed with ASC-US, 493 (80.7%) had at least one follow-up visit during the study period. hrHPV was detected in 48.3% of the women at study entry (mean age 35.2 years). hrHPV positivity decreased with increasing age from 72.6% among women younger than 25 years to 31.6% in women older than 54 years (p < 0.01). At the end of the 3 years follow-up period, 37 women with a diagnosis of CIN2+ (18 CIN2, 16 CIN3, 2 cancers, and 1 with high squamous intraepithelial lesions--HSIL) were identified and all but one had a hrHPV positive test at study entry. Sensitivity to detect CIN2+ of hrHPV was 97.2% (95%confidence interval (CI) = 85.5-99.9) and specificity was 68.3% (95%CI = 63.1-73.2). The odds ratio for CIN2+ was 45.3 (95% CI: 6.2-333.0), when among ASC-US hrHPV positive women were compared to ASC-US hrHPV negative women. CONCLUSIONS: Triage of ASC-US with hrHPV testing showed a high sensitivity for the detection of CIN2+ and a high negative predictive value after 3 years of follow-up. The results of this study are in line with the current guidelines for triage of women with ASC-US in the target age range of 25-65. Non adherence to guidelines will lead to unnecessary medical interventions. Further investigation is needed to improve specificity of ASC-US triage.

Incidence of human papillomavirus infection in male sexual partners of women diagnosed with CIN II-III.

INTRODUCTION: Human papillomavirus (HPV) infection is a very common sexually transmitted disease which has been strongly related to cervical cancer and cervical intraepithelial neoplasia (CIN), penile cancer and intraepithelial and infiltrating anal squamous cell carcinoma. OBJECTIVES: To describe the HPV status of male sexual partners of women diagnosed with CIN II/III and to evaluate the practical usefulness of the HPV detection in urine as a reliable marker of genital high-risk HPV infection in men. METHODS: Ninety-one heterosexual male partners (mean age 32.7) were included in the study. A panel of epidemiological data was recorded. Peniscopy was performed at the first visit and after 6 months. Urine samples and anal and penile scrapings were obtained and Hybrid Capture II test for high-risk HPV was performed. Physical examination disclosed clinically or peniscopic lesions in only 5.4% patients. HPV was isolated in 12.9% and 6.2% of penile and anal scrapings respectively whereas HPV detection was positive in 28% of urine samples. Overall, 41% of the evaluated patients presented at least one finding diagnostic of HPV infection. CONCLUSION: HPV detection in male partners of women with CIN is a frequent event, and urine HPV detection by Hybrid Capture test is a sensitive method for its detection. The determination of HPV in urine samples seems to be a simple method to investigate the subrogated genital HPV infection in men.

Safety and Efficacy of Crizotinib in Combination with Temozolomide and Radiotherapy in Patients with Newly Diagnosed Glioblastoma: Phase Ib GEINO 1402 Trial.

BACKGROUND: MET-signaling and midkine (ALK ligand) promote glioma cell maintenance and resistance against anticancer therapies. ALK and c-MET inhibition with crizotinib have a preclinical therapeutic rationale to be tested in newly diagnosed GBM. METHODS: Eligible patients received crizotinib with standard radiotherapy (RT)/temozolomide (TMZ) followed by maintenance with crizotinib. The primary objective was to determine the recommended phase 2 dose (RP2D) in a 3 + 3 dose escalation (DE) strategy and safety evaluation in the expansion cohort (EC). Secondary objectives included progression-free (PFS) and overall survival (OS) and exploratory biomarker analysis. RESULTS: The study enrolled 38 patients. The median age was 52 years (33-76), 44% were male, 44% were MGMT methylated, and three patients had IDH1/2 mutation. In DE, DLTs were reported in 1/6 in the second cohort (250 mg/QD), declaring 250 mg/QD of crizotinib as the RP2D for the EC. In the EC, 9/25 patients (32%) presented grade ≥3 adverse events. The median follow up was 18.7 months (m) and the median PFS was 10.7 m (95% CI, 7.7-13.8), with a 6 m PFS and 12 m PFS of 71.5% and 38.8%, respectively. At the time of this analysis, 1 died without progression and 24 had progressed. The median OS was 22.6 m (95% CI, 14.1-31.1) with a 24 m OS of 44.5%. Molecular biomarkers showed no correlation with efficacy. CONCLUSIONS: The addition of crizotinib to standard RT and TMZ for newly diagnosed GBM was safe and the efficacy was encouraging, warranting prospective validation in an adequately powered, randomized controlled study.

In silico validation of RNA-Seq results can identify gene fusions with oncogenic potential in glioblastoma.

RNA-Sequencing (RNA-Seq) can identify gene fusions in tumors, but not all these fusions have functional consequences. Using multiple data bases, we have performed an in silico analysis of fusions detected by RNA-Seq in tumor samples from 139 newly diagnosed glioblastoma patients to identify in-frame fusions with predictable oncogenic potential. Among 61 samples with fusions, there were 103 different fusions, involving 167 different genes, including 20 known oncogenes or tumor suppressor genes (TSGs), 16 associated with cancer but not oncogenes or TSGs, and 32 not associated with cancer but previously shown to be involved in fusions in gliomas. After selecting in-frame fusions able to produce a protein product and running Oncofuse, we identified 30 fusions with predictable oncogenic potential and classified them into four non-overlapping categories: six previously described in cancer; six involving an oncogene or TSG; four predicted by Oncofuse to have oncogenic potential; and 14 other in-frame fusions. Only 24 patients harbored one or more of these 30 fusions, and only two fusions were present in more than one patient: FGFR3::TACC3 and EGFR::SEPTIN14. This in silico study provides a good starting point for the identification of gene fusions with functional consequences in the pathogenesis or treatment of glioblastoma.

Detection of HPV by in situ hybridization in thin-layer (ThinPrep) cervicovaginal samples.

We have studied an automated in situ hybridization (ISH) method as a possible alternative approach for detecting high-risk human papillomavirus (HPV) in monolayer (ThinPrep) cervico-vaginal samples, comparing the results with those obtained by polymerase chain reaction (PCR) using consensus primers and studying the relationship between the ISH staining pattern and the viral integration in HPV 16-positive cases. Eighty atypical squamous cells of undetermined significance (ASCUS) and low-grade squamous intraepithelial lesion (LSIL) cases were used for our purposes. The patients were monitored through periodic cytologies. ISH with was performed with an automated Ventana System, analysis by PCR was performed with consensus primers and integration of HPV16 was performed by realtime PCR analyzing E2 and E6 genes. Additionally, 27 HSIL cases were also studied to observe the ISH staining patterns. HPV infection was detected by ISH in 21.7% of the ASCUS cases and 55.8% of the LSIL cases. Two distinct staining patterns were observed: multipunctated (MP) and diffuse (DI). In some cases, a mixed pattern (MP + DI) was observed and these cases were considered as MP. The MP pattern increased with the degree of lesion and seemed to have a prognostic value in ASCUS/LSIL cases. The lesion in MP pattern cases persisted throughout the entire study in 77% of cases, whereas in cases with a DI staining pattern, only 41% of them showed persistence of the lesion (p <0.001). No correlation was found between HPV integration and the ISH staining pattern. Given the lower sensitivity and negative predictive value of ISH and its incapacity to demonstrate the integration of high-risk HPV in ASCUS and LSIL cases using liquid-based cytology, we do not recommend this technique for the triage of ASCUS and LSIL cases.

Human papillomavirus genotype distribution and human papillomavirus 16 and human papillomavirus 18 genomic integration in invasive and in situ cervical carcinoma in human immunodeficiency virus-infected women.

BACKGROUND: Women infected with human immunodeficiency virus (HIV) are at increased risk of developing precancerous and cancerous lesions in cervix because of persistence of oncogenic human papillomavirus (HPV) infection. Scarce information about the HPV genotypes attributed to cervical cancer in the HIV-infected population is available, especially in countries with a low prevalence of this pathology. OBJECTIVE: The objective of the study was to assess the prevalence and distribution of HPV types, and the viral integration of HPV-16 and HPV-18 in cervical squamous cell carcinoma of HIV-infected and HIV-negative women. METHODS: A total of 140 formaldehyde-fixed paraffin-embedded specimens from 31 HIV-infected and 109 matched HIV-negative women, with a diagnosis of in situ or invasive cervical carcinoma, were identified between 1987 and 2010 from different hospitals of the Barcelona area, Spain. Human papillomavirus genotyping and integration were analyzed by standardized polymerase chain reaction. RESULTS: Similar prevalence and distribution of HPV genotypes were detected in cervical cancers (in situ and invasive) regardless of HIV condition. The most common types were as follows: HPV-16 (58% in HIV-positive vs 72% in HIV-negative) and HPV-33 (16% vs 8%). In invasive cervical carcinoma, HPV-18 was significantly more prevalent in HIV-positive women (14% vs 1%; P = 0.014). The proportion of samples with integrated forms of HPV-16 (39% vs 45%) and HPV-18 (50% vs 50%) was similar in both groups. CONCLUSIONS: The prevalence and distribution of principal HPV types involved in the carcinogenesis process of the cervix were similar in HIV-infected and noninfected women, although a tendency toward a lower HPV-16 and a higher HPV-18 prevalence in invasive cervical carcinoma was detected in HIV-positive women. Similar percentage of HPV-16 and HPV-18 viral integration was found in formaldehyde-fixed paraffin-embedded specimens of cervical cancer regardless of the HIV infection status.

CKS1B amplification is a frequent event in cutaneous squamous cell carcinoma with aggressive clinical behaviour.

Genetic mechanisms giving rise to the development of cutaneous squamous cell carcinoma (cSCC) are poorly understood and development of genomic high resolution techniques has led to a better knowledge of the genetic basis of several human cancers. In this study, 16 cSCC were analyzed using array comparative genomic hybridization (arrayCGH). The most common aberrations found were gains of 3q11q13, 1q21.3q25, 13q34, and 19p13, and losses of 1p36p31, 3p24p21, 10p15q22, and 13q11q21. We detected gains (3/16) and amplification (1/16) of the 1q21.1q21.3 region. A potential candidate gene in this region, CKS1B (1q21.2), was selected for validation in an independent cohort and correlations with clinicopathological features were carried out. CKS1B gene and protein status were analyzed using fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) in a series of 53 cSCC, 22 actinic keratoses (AK), and 10 normal skin samples. cSCC presented a higher frequency of chromosome 1 polysomy than AK (70% vs. 46%, P = 0.047). Association between CKS1B protein overexpression and both polysomy and amplification was demonstrated in cSCC (P < 0.001). Regarding amplifications, 11 cSCC patients (21%) presented CKS1B gene amplification. Interestingly, 8/11 (73%) patients who showed a CKS1B amplification had presented metastatic spread (mcSCC). Differences between the presence of CKS1B amplification and the presence or absence of mcSCC were observed (mcSCC [8/14] vs. cSCC [3/39]) (P < 0.001). Several drugs targeting CKS1B have been reported and may be useful for treating patients with cSCC and CKS1B amplifications.

Influence of age on treatment and prognosis of invasive cervical cancer.

OBJECTIVE: Invasive cervical cancer is considered a young women's disease, however up to 20 % of cases develop cervical cancer at advanced ages. The aim was to characterize invasive cervical cancer in women aged 65 and older assessing age-specific survival differences. STUDY DESIGN: A retrospective study including cervical cancer patients was conducted at Hospital del Mar Barcelona from July-2007 to December-2016. Women were stratified: <65 or ≥65years. Clinical and pathological data were collected. Multivariate analysis was used to compare outcomes. Adjusted hazard ratios with 95 % confidence intervals for disease-free survival, and overall survival were estimated using Cox proportional hazards models. RESULTS: 124 patients with invasive cervical cancer (n = 87 < 65years and n = 37 ≥ 65years) were included. At diagnosis, 48.3 % of <65years patients were diagnosed at advanced stages, while 64.9 % in ≥65years (p = 0.018). Standard treatment was given to 83.9 % of patients in <65years group compared to 62.2 % in ≥65years (p = 0.015). Disease-free survival and overall survival showed no significant differences between groups. Age ≥65 did not predict worse disease-free survival (HR: 0.3 95 %CI, 0.04-3.1, p = 0.347) or overall survival (HR: 0.82 95 %CI, 0.3-2.3, p = 0.729). CONCLUSION: Invasive cervical cancer was diagnosed at advanced stages and was treated less frequently with radical intention in patients ≥65years; overall survival and disease-free survival were similar to those cervical cancer diagnosed at younger ages.

Developing indicators for quality assurance in cytopathology. Catalan Society of Cytopathology.

BACKGROUND: Quality control in cytology must be established through reliable and easily measurable indicators. METHODS: From the Catalan Society of Cytopathology a group of experts has been established to write a document with 13 indicators that cover the entire cytological process, based on its Cytopathology Quality Guide. It has been elaborated through guides and documents with scientific evidence and DELPHI methodology in order to reach a structured consensus on the opinions of a group of experts. RESULTS: Thirteen indicators, covering all the cytologic process are expressed in worksheets specifying all their characteristics. CONCLUSION: This document allows the control of all stages of the cytological process.