Ex vivo confocal microscopy detects basic patterns of acute and chronic lesions using fresh kidney samples.

Background: Ex vivo confocal microscopy is a real-time technique that provides high-resolution images of fresh, non-fixed tissues, with an optical resolution comparable to conventional pathology. The objective of this study was to investigate the feasibility of using ex vivo confocal microscopy in fusion mode (FuCM) and the haematoxylin and eosin (H&E)-like digital staining that results for the analysis of basic patterns of lesion in nephropathology. Methods: Forty-eight renal samples were scanned in a fourth-generation ex vivo confocal microscopy device. Samples were subjected to confocal microscopy imaging and were then processed using conventional pathology techniques. Concordance between the techniques was evaluated by means of the percentage of agreement and the κ index. Results: Agreement between conventional microscopy and H&E-like digital staining was strong (κ = 0.88) in the evaluation of acute tubular damage and was substantial (κ = 0.79) in the evaluation of interstitial fibrosis, interstitial inflammation, arterial and arteriolar lesions. H&E-like digital staining also allows rapid identification of extracapillary proliferation (κ = 0.88), necrosis and segmental sclerosis (κ = .88) in the glomerular compartment, but the results reported here are limited because of the small number of cases with these glomerular findings. Conclusions: FuCM proved to be as effective as conventional techniques in evaluating the presence of acute tubular necrosis and interstitial fibrosis changes, but in fresh tissue. The ease of acquisition of ex vivo confocal microscopy images suggests that FuCM may be useful for rapid evaluation of kidney biopsies and to restructure the clinical workflow in renal histopathology.

The Density of Renal Lymphatics Correlates With Clinical Outcomes in IgA Nephropathy.

Introduction: IgA nephropathy (IgAN) is the most common primary glomerulonephritis (GN) worldwide. The disease course fluctuates, and the most important challenge is the considerable variation in the time lag between diagnosis and the development of a hard clinical end point, such as end-stage kidney disease (ESKD). The reaction of renal tissue to damage resembles the common wound-healing response. One part of this repair in IgAN is the expansion of lymphatic vessels known as lymphangiogenesis. The aim of this work was to establish the prognostic value of the density of lymphatic vessels in the renal biopsy at the time of diagnosis, for predicting the risk of ESKD in a Spanish cohort of patients with IgAN. Methods: We performed a retrospective multicenter study of 76 patients with IgAN. The end point of the study was progression to ESKD. The morphometric analysis of lymphatic vessels was performed on tissue sections stained with antipodoplanin antibody. Results: Density of lymphatic vessels was significantly higher in patients with IgAN with mesangial hypercellularity >50%, segmental sclerosis, higher degrees of interstitial fibrosis, and tubular atrophy. Patients with more lymphatic vessels had significantly higher values of proteinuria and lower estimated glomerular filtration rate (eGFR). A density of lymphatic vessels ≥8 per mm2 was associated with a significantly higher rate of progression to ESKD at 3 years from biopsy. After adjustment for the International IgAN prediction score, at the multivariate logistic regression, high density of lymphatic vessels (≥8 per mm2) remained significantly associated with a higher rate of early progression to ESKD. Conclusion: This study contributes to the understanding of the natural history of the progression to ESKD in patients with IgAN revealing the density of lymphatics vessels may optimize the prognostic value of the International IgA predicting tool to calculate the risk of ESKD, favoring the evaluation of new targeted therapies.

Gene expression profiles of breast cancer metastasis according to organ site.

In advanced breast cancer, biomarker identification and patient selection using a metastatic tumor biopsy is becoming more necessary. However, the biology of metastasis according to the organ site is largely unknown. Here, we evaluated the expression of 771 genes in 184 metastatic samples across 11 organs, including liver, lung, brain, and bone, and made the following observations. First, all PAM50 molecular intrinsic subtypes were represented across organs and within immunohistochemistry-based groups. Second, HER2-low disease was identified across all organ sites, including bone, and HER2 expression significantly correlated with ERBB2 expression. Third, the majority of expression variation was explained by intrinsic subtype and not organ of metastasis. Fourth, subtypes and individual subtype-related genes/signatures were significantly associated with overall survival. Fifth, we identified 74 genes whose expression was organ-specific and subtype-independent. Finally, immune profiles were found more expressed in lung compared to brain or liver metastasis. Our results suggest that relevant tumor biology can be captured in metastatic tissues across a variety of organ sites; however, unique biological features according to organ site were also identified and future studies should explore their implications in diagnostic and therapeutic interventions.

Clinical, pathological, and PAM50 gene expression features of HER2-low breast cancer.

Novel antibody-drug conjugates against HER2 are showing high activity in HER2-negative breast cancer (BC) with low HER2 expression (i.e., 1+ or 2+ and lack of ERBB2 amplification). However, the clinical and molecular features of HER2-low BC are yet to be elucidated. Here, we collected retrospective clinicopathological and PAM50 data from 3,689 patients with HER2-negative disease and made the following observations. First, the proportion of HER2-low was higher in HR-positive disease (65.4%) than triple-negative BC (TNBC, 36.6%). Second, within HR-positive disease, ERBB2 and luminal-related genes were more expressed in HER2-low than HER2 0. In contrast, no gene was found differentially expressed in TNBC according to HER2 expression. Third, within HER2-low, ERBB2 levels were higher in HR-positive disease than TNBC. Fourth, HER2-low was not associated with overall survival in HR-positive disease and TNBC. Finally, the reproducibility of HER2-low among pathologists was suboptimal. This study emphasizes the large biological heterogeneity of HER2-low BC, and the need to implement reproducible and sensitive assays to measure low HER2 expression.

Prognostic implications of genotyping and p16 immunostaining in HPV-positive tumors of the uterine cervix.

Human papillomaviruses (HPVs) are the causative agents of carcinoma of the uterine cervix. A number of HPV genotypes have been associated with cervical cancer and almost all tumors associated with HPV show strong p16 expression. However, there is little information on the possible impact of the HPV genotype and p16 immunostaining on the clinicopathological features or their prognostic value in cervical carcinoma. We evaluated a series of 194 patients with HPV-positive cervical cancers treated at our institution, focusing on the clinicopathological features and the relationship of the HPV genotypes and p16 immunostaining with the prognosis. A single HPV type was identified in 149 (77%) tumors, multiple HPV infection was detected in 30 cases (15%), and undetermined HPV type/s were identified in 15 (8%) carcinomas. HPV 16 and/or 18 were detected in 156 (80%) tumors. p16 was positive in 186 (96%) carcinomas, but eight tumors (4%) were negative for p16 (seven squamous cell carcinomas, one adenocarcinoma); 5/8 caused by HPV 16 and/or 18. Patients with HPV 16 and/or 18 were younger (49 ± 15 vs. 57 ± 17 years, p < 0.01) and more frequently had nonsquamous tumors than patients with other HPV types (24% [37/156] vs. 0% [0/38]; p = 0.01). Neither the HPV type nor multiple infection showed any prognostic impact. Patients with p16-negative tumors showed a significantly worse overall survival than women with p16-positive carcinomas (45 vs. 156 months, p = 0.03), although no significant differences in disease-free survival were observed. In the multivariate analysis, negative p16 immunostaining was associated with a worse overall survival together with advanced FIGO stage and lymph node metastases. In conclusion, the HPV genotype has limited clinical utility and does not seem to have prognostic value in cervical cancer. In contrast, a negative p16 result in patients with HPV-positive tumors is a prognostic marker associated with a poor overall survival.

mRNA Detection in Anal Cytology: A Feasible Approach for Anal Cancer Screening in Men Who Have Sex with Men Living With HIV.

There is growing interest in anal cancer screening strategies. However, cytological/molecular evaluation of anal samples is challenging. We aimed to determine the feasibility of detecting, in anal liquid-based cytologies, the expression of biomarkers involved in the cell cycle disturbance elicited by human papillomavirus (HPV). The accuracy of this approach in the identification of high-grade squamous intraepithelial lesions/anal intraepithelial neoplasia grade2-3 (HSIL/AIN2-3) was also evaluated. 215 anal cytologies from men having sex with men living with human immunodeficiency virus were evaluated. Patients showing concordant cytological and anoscopy-directed biopsy diagnosis were selected: 70 with negative cytology and HPV test, 70 with low-grade SIL (LSIL/AIN1) cytology and biopsy, and 75 with cytology and biopsy of HSIL/AIN2-3. CDKN2A/p16, MKI67 and TOP2A mRNA expression was analyzed. HPV detection was performed with Xpert HPV Assay (Cepheid, Sunnyvale, CA, USA). HSIL/AIN2-3 showed higher expression for the biomarkers than LSIL/AIN1 or negative samples. The specificity for HSIL/AIN2-3 detection for a sensitivity established at 70% was 44.7% (95%confidence interval [CI] 36.5-53.2) for TOP2A and MKI67 and 54.5% (95%CI 46.0-62.8%) for CDKN2A/p16. mRNA detection of cell biomarkers in anal liquid-based cytology is feasible. Further studies are warranted to confirm if strategies based on mRNA detection have any role in anal cancer screening.

CADM1, MAL, and miR124 Promoter Methylation as Biomarkers of Transforming Cervical Intrapithelial Lesions.

BACKGROUND: Squamous intraepithelial lesions/cervical intraepithelial neoplasias (SIL/CIN) are high-risk human papilloma virus (hrHPV)-related lesions which are considered as high grade (HSIL/CIN2-3) or low grade (LSIL/CIN1) lesions according to their risk of progression to cervical cancer (CC). Most HSIL/CIN2-3 are considered as transforming hrHPV infections, so truly CC precursors, although some clear spontaneously. hrHPV testing has a high sensitivity for the detection of HSIL/CIN2-3 but a relatively low specificity for identifying transforming lesions. We aimed to determine whether the combination of CADM1, MAL and miR124 promoter methylation status assessed in histological samples can be used as a biomarker in the identification of transforming HSIL/CIN lesions. DESIGN: 131 cervical biopsies, including 8 cases with no lesion and a negative hrHPV test result (control group), 19 low-grade (L)SIL/CIN1, 30 HSIL/CIN2, 60 HSIL/CIN3, and 14 CC were prospectively collected. hrHPV was detected and genotyped using the polymerase chain reaction (PCR)-based technique SPF10 HPV LIPA. A multiplex quantitative methylation-specific PCR (qMSP) was used to identify the methylation status of the CADM1, MAL, and miR124 promoter genes. RESULTS: Significantly higher methylation levels of CADM1, MAL and miR-124 were found in HSIL/CIN2-3 and CC compared with normal and LSIL lesions. DNA methylation of at least one gene was detected in 12.5% (1/8) of normal samples, 31.5% (6/19) of LSIL/CIN1, 83.3% (25/30) of HSIL/CIN2, 81.6% (49/60) of HSIL/CIN3 and 100% (14/14) of CC (p < 0.001). The sensitivity and specificity for HSIL/CIN2-3 and CC of having at least one methylated gene were 84.6% and 74.0%, respectively. The sensitivity and specificity of the combination of at least one methylated gene and a positive hrHPV test were 80.7% and 85.1% for HSIL/CIN2-3 and CC, respectively. CONCLUSIONS: The methylation rate of CADM1, MAL and miR124 increases with the severity of the lesion. Further research is warranted to evaluate the usefulness of these biomarkers for the identification of transforming HSIL/CIN.

HPV-negative tumors of the uterine cervix.

Human papillomaviruses (HPV) are the causative agents of virtually all cervical carcinomas. Nevertheless, a small proportion of cervical cancer are negative for HPV, although the significance of this finding remains unclear. We aimed to provide insight into the differential clinico-pathological characteristics of this unusual subset of HPV-negative cervical cancer. We performed HPV-DNA detection using a highly sensitive PCR test (SPF10) and p16 immunostaining in 214 cervical carcinomas specimens from women treated at the Gynecological Oncology Unit of the Hospital Clinic (Barcelona, Spain) from 2012 to 2015. The clinical and pathological characteristics, including disease-free survival and overall survival, of HPV-negative and -positive cervical carcinomas were compared. Twenty-one out of 214 tumors (10%) were negative for HPV DNA. HPV-negative tumors were more frequently of the non-squamous type (9/21, 43% vs. 37/193, 19%; p < 0.01) and showed negative p16 staining (9/21; 43% vs. 7/193; 4%; p < 0.01). HPV-negative tumors were more frequently diagnosed at advanced FIGO stage (19/21, 91% vs. 110/193, 57%; p < 0.01) and more frequently had lymph node metastases (14/21, 67% vs. 69/193, 36%; p < 0.01). Patients with HPV-negative cervical cancer had a significantly worse disease-free survival (59.8 months, 95% confidence interval 32.0-87.6 vs. 132.2 months, 95% confidence interval 118.6-145.8; p < 0.01) and overall survival (77.0 months, 95% confidence interval 47.2-106.8 vs. 153.8 months, 95% confidence interval 142.0-165.6; p = 0.01) than women with HPV-positive tumors. However, only advanced FIGO stage and lymph node metastases remained associated with a poor disease-free survival and overall survival on multivariate analysis. In conclusion, our results suggest that a low percentage of cervical cancer arise via an HPV-independent pathway. These HPV-negative tumors are diagnosed at advanced stages, show higher prevalence of lymph nodes metastases and have an impaired prognosis.

Value of HPV 16/18 Genotyping and p16/Ki-67 Dual Staining to Predict Progression to HSIL/CIN2+ in Negative Cytologies From a Colposcopy Referral Population.

OBJECTIVES: To assess the prognostic value of human papillomavirus (HPV) 16/18 genotyping and p16/Ki-67 dual staining cytology in high-risk HPV (hrHPV)-positive women with no lesion or minor abnormalities. METHODS: We evaluated progression to high-grade squamous intraepithelial lesion/cervical intraepithelial neoplasia grades 2 to 3 or cervical cancer (HSIL/CIN2+), persistence/regression of hrHPV infection in women referred to colposcopy showing hrHPV infection, histology diagnosis different from HSIL/CIN2+, and negative cytology. HPV 16/18 genotyping and dual staining were performed in liquid-based cytologic specimens obtained on the first visit. RESULTS: Progression was observed in 16 (8.0%) of 200 women. Those with HPV 16/18 infection had an increased risk of progression compared with women infected by other hrHPV types, and they also showed more persistence. However, no association was observed between progression or persistence and the result of the dual staining. CONCLUSIONS: HPV 16/18-positive women with no lesions or minor abnormalities are at high risk of progression to HSIL/CIN2+ and hrHPV persistence.

Colposcopy Evaluation at the Time of Loop Electrosurgical Excision Procedure May Avoid Unnecessary Treatment.

OBJECTIVE: The aim of the study was to assess the accuracy of colposcopy evaluation at the time of the loop electrosurgical excision procedure (LEEP) to identify women with a previous confirmatory diagnosis of squamous intraepithelial lesion/cervical intraepithelial neoplasia (SIL/CIN) with low probability of dysplasia in the LEEP specimen. MATERIALS AND METHODS: We prospectively recruited a cohort of 162 women undergoing LEEP for histological high-grade SIL/CIN 2-3 or low-grade SIL/CIN 1 with high-grade SIL cytology showing a fully visible squamocolumnar junction in the colposcopy evaluation at the time of LEEP. At the referral visit cervical cytology, human papillomavirus and genotype detection, digital colposcopy, colposcopical lesion measurement, and 1 or more biopsies of the transformation zone were obtained. The uterine cervix was colposcopically evaluated intraoperatively. RESULTS: Thirty-four women (21.0%) had a normal colposcopy evaluation at the time of the LEEP, whereas the remaining 128 women showed abnormal findings. Absence of SIL/CIN in the LEEP specimen was confirmed in 28 (82.3%) of the 34 women with a normal colposcopy at the time of LEEP group and 8 (3.1%) of the 128 women showing abnormal colposcopy at the time of LEEP group (p < .001). A normal colposcopic evaluation at the time of LEEP was associated with an increase in the risk of absence of lesion in the cone specimen compared with cases presenting an abnormal colposcopy (95% CI = 33.8-1,555.1, p < .001). The colposcopy evaluation at the time of LEEP had a positive predictive value of 82.3% (95% CI = 66.5-91.5) and a negative predictive value of 96.9% (95% CI = 92.2-98.8) to predict low probability of SIL/CIN in the specimen. CONCLUSIONS: Colposcopic evaluation at the time of LEEP seems to be accurate to identify SIL/CIN postbiopsy regression; thus, its performance would be considered at the time of the treatment.