Adela Saco

Validation of whole slide imaging in the primary diagnosis of gynaecological pathology in a University Hospital

Aims Experience in the use of whole slide imaging (WSI) for primary diagnosis in pathology is very limited. We aimed to determine the accuracy of interpretation of WSI compared with conventional light microscopy (CLM) in the diagnosis of routine gynaecological biopsies. Methods All gynaecological specimens (n=452) received over a 2-month period at the Department of Pathology of the Hospital Clinic of Barcelona were analysed blindly by two gynaecological pathologists, one using CLM and the other WSI. All slides were digitised in a Ventana iScan HT (Roche diagnostics) at 200×. All discrepant diagnoses were reviewed, and a final consensus diagnosis was established. The results were evaluated by weighted κ statistics for two observers. Results The level of interobserver agreement between WSI and CLM evaluations was almost perfect (κ value: 0.914; 95% CI 0.879 to 0.949) and increased during the study period: κ value 0.890; 95% CI 0.835 to 0.945 in the first period and 0.941; 95%; CI 0.899 to 0.983 in the second period. Major discrepancies (differences in clinical management or prognosis) were observed in 9 cases (2.0%). All discrepancies consisted of small lesions (8 high grade squamous intraepithelial lesions of the uterine cervix, one lymph node micrometastasis of an ovarian carcinoma) underdiagnosed or missed in the WSI or the CLM evaluation. Discrepancies with no or minor clinical relevance were identified in 3.8% of the biopsies. No discrepancy was related to the poor quality of the WSI image. Conclusions Diagnosis of gynaecological specimens by WSI is accurate and may be introduced into routine diagnosis.

p16 staining has limited value in predicting the outcome of histological low-grade squamous intraepithelial lesions of the cervix

In order to evaluate the usefulness of p16 staining in predicting the outcome of histological low-grade squamous intraepithelial lesion/cervical intraepithelial neoplasia grade 1 (LSIL/CIN1) we prospectively recruited all the patients referred to colposcopy from 2003 to 2011 due to abnormal screening test results and diagnosed with LSIL/CIN1 at biopsy (n=507). All biopsies were stained for p16 and re-evaluated after three years by the same gynecological pathologist using the LAST criteria. Follow-up was conducted every 6 months and included a Pap test (liquid-based cytology), high-risk human papillomavirus testing (Hybrid Capture 2 test), and colposcopy. The mean follow-up was 28 months. An outcome diagnosis of HSIL was defined as a histological diagnosis of high-grade SIL/CIN (HSIL/CIN2-3). The diagnosis of LSIL/CIN1 was confirmed in 416 out of 507 biopsies (82%), whereas 58 (11%) were reclassified as negative and 33 (6%) as HSIL/CIN2-3. During follow-up, 86/507 women initially diagnosed with LSIL/CIN1 (17%) showed an outcome diagnosis of HSIL/CIN2-3, with the rate of HSIL final diagnosis of 3% (2/58) in the women with biopsies reclassified as negative, 17% (70/416) in the group with confirmed LSIL and 42% (14/33) in the women with biopsies reclassified as HSIL (P<0.001). p16 was positive in 245/507 patients (48%) and in 210/416 patients (50%) with confirmed LSIL/CIN1 at re-evaluation. Although positive p16 immunostaining was associated with risk of HSIL/CIN2-3 outcome in the multivariate analysis (Hazard ratio (HR) 1.9; 95% confidence interval (CI): 1.2–3.1; P=0.009) in the overall group of patients with LSIL/CIN1, this association was not verified in the subset of patients with confirmed LSIL/CIN1 after re-evaluation (HR: 1.6; 95% CI: 0.9–2.6; P=0.095). In conclusion, in LSIL/CIN1 lesions p16 should be limited to equivocal cases in which HSIL/CIN2 is included in the differential diagnosis since it has low value in clinical practice as a marker of progression of LSIL/CIN1.

LincRNA-p21 Impacts Prognosis in Resected Non–Small Cell Lung Cancer Patients through Angiogenesis Regulation

Introduction: Long intergenic noncoding RNA‐p21 (lincRNA‐p21) is a long noncoding RNA transcriptionally activated by tumor protein p53 (TP53) and hypoxia inducible factor 1 alpha subunit (HIF1A). It is involved in the regulation of TP53‐dependent apoptosis and the Warburg effect. We have investigated the role of lincRNA‐p21 in NSCLC. Methods: LincRNA‐p21 expression was assessed in tumor and normal tissue from 128 patients with NSCLC and correlated with time to relapse and cancer‐specific survival (CSS). H23, H1299, and HCC‐44 cell lines were cultured in hypoxic conditions after silencing of lincRNA‐p21. The TaqMan human angiogenesis array was used to explore angiogenesis‐related gene expression. Levels of the protein vascular endothelial growth factor A were measured by enzyme‐linked immunosorbent assay in the cell supernatants. Angiogenic capability was measured by human umbilical vein endothelial cell tube formation assay. Microvascular density in tumor samples was analyzed by immunohistochemistry. Results: LincRNA‐p21 was down‐regulated in tumor tissue, but no association was observed with TP53 mutational status. High lincRNA‐p21 levels were associated with poor CSS in all patients (p = 0.032). When patients were classified according to histological subtypes, the impact of lincRNA‐p21 was confined to patients with adenocarcinoma in both time to relapse (p = 0.006) and CSS (p < 0.001). To explain the poor outcome of patients with high lincRNA‐p21 expression, we studied the role of lincRNA‐p21 in angiogenesis in vitro and observed a global downregulation in the expression of angiogenesis‐related genes when lincRNA‐p21 was inhibited. Moreover, supernatants from lincRNA‐p21–inhibited cells were significantly less angiogenic and had lower levels of secreted vascular endothelial growth factor A than controls did. Finally, tumor samples with high lincRNA‐p21 levels had higher microvascular density. Conclusions: Our findings suggest that lincRNA‐p21 affects outcome in patients with NSCLC adenocarcinoma through the regulation of angiogenesis.

Validation of Whole-Slide Imaging for Histolopathogical Diagnosis: Current State

Rapid advances in informatics and technological improvements have led to the development of high-throughput whole-slide imaging (WSI) scanners able to produce high-quality digital images, which allow achieving a correct diagnosis of the biopsies using virtual viewers. This technology is currently prepared to be introduced in the departments of pathology for routine diagnosis. The aim of this review is to analyze the current evidence regarding the use of WSI in primary or routine diagnosis in the different subspecialties of pathology. An increasing number of studies have shown almost perfect inter- and intraobserver agreement between the diagnoses obtained with WSI and the classical diagnoses based on conventional light microscopy. The only exception seems to be cytology, which still requires some technological development. Although validation studies are needed in some areas of pathology, growing evidence indicates that WSI is a reliable tool for routine diagnosis. Pathologists have a positive perception of the ergonomics of the workstations, the low magnification of WSI and the possibility of making annotations and measurements. WSI can be used from any device and anywhere, thereby providing great opportunities for teleconsultation. New technologies such as the recognition of histopathology patterns using image analysis may facilitate diagnosis and improve the reproducibility among pathologists in the future.

Virtual microscopy in the undergraduate teaching of pathology.

Background: Little evidence is available concerning the impact of virtual microscopy (VM) in the undergraduate teaching of pathology. We aimed: (1) to determine the impact in student scores when moving from conventional microscopy (CM) to VM; (2) to assess the students′ impressions and changes in study habits regarding the impact of this tool. Methods: We evaluated two groups taking the discipline of pathology in the same course, one using CM and the other VM. The same set of slides used in the CM classes was digitized in a VENTANA iScan HT (Roche Diagnostics, Sant Cugat, Spain) at ×20 and observed by the students using the Virtuoso viewer (Roche Diagnostics). We evaluated the skill level reached by the students with an online test. A voluntary survey was undertaken by the VM group to assess the students′ impressions regarding the resource. The day and time of any accession to the viewer were registered. Results: There were no differences between the two groups in their marks in the online test (mean marks for the CM and the VM groups: 9.87 ± 0.34 and 9.86 ± 0.53, respectively; P = 0.880). 86.6% of the students found the software friendly, easy-to-use and effective. 71.6% of the students considered navigation easier with VM than with CM. The most appreciated feature of VM was the possibility to access the images anywhere and at any time (93.3%). 57.5% of the accesses were made on holidays and 41.9% later than 6:00 pm. Conclusions: Virtual microscopy can effectively replace the traditional methods of learning pathology, providing mobility and convenience to medical students.

Current Status of Whole-Slide Imaging in Education

Conventional light microscopy (CLM) has classically been the basic tool to teach histology and pathology. In recent years, whole-slide imaging (WSI), which consists of generating a high-magnification digital image of an entire histological glass slide, has emerged as a useful alternative to CLM offering a myriad of opportunities for education. Navigation through the digitized slides closely simulates viewing glass slides with a microscope and is also referred to as virtual microscopy. WSI has many advantages for education. Students feel more comfortable with its use, and it can be used in any classroom as it only requires a computer with Internet access and it allows remote access from anywhere and from any device. WSI can be used simultaneously by a large number of people, stimulating cooperation between students and improving the interaction with the teachers. It allows making marks and annotations on specific fields, which enable specific directed questions to the teacher. Finally, WSI supports are cost-effective compared with CLM. Consequently, WSI has begun to replace CLM in many institutions. WSI has shown to be an extremely useful tool for undergraduate education (medical, dental and veterinary schools), for the training of residents of pathology, tele-education and in tumor boards.

Histological characteristics of HPV-associated and -independent squamous cell carcinomas of the vulva: A study of 1594 cases

There are at least two different etio‐pathogenic pathways for the development of vulvar squamous cell carcinoma (VSCC): one associated with infection by human papillomavirus (HPV) and another independent of HPV. We aimed to describe the histological characteristics of HPV‐associated and ‐independent tumors and to determine the best strategy to identify HPV in VSCC. A single paraffin block was available for review from a series of 1,594 VSCCs. In all cases HPV DNA detection was analyzed using the SPF10PCR/DEIA/LiPA25 system and p16 immunohistochemistry (IHC). A tumor was considered as unquestionably HPV‐associated if both HPV DNA and p16 IHC were positive. A tumor was considered indisputably HPV‐independent if both HPV DNA and p16 IHC were negative. Two groups of tumors were classified as non‐conclusive: (1) HPV DNA+/p16− and (2) HPV DNA−/p16+. WHO typing and a thorough histological evaluation were conducted in all cases. Four hundred and forty‐one tumors were HPV DNA+ with 367 cases (23.0%) being HPV DNA+/p16+. The latter tumors were more frequently basaloid or warty (49.8%), but 36.5% were of the keratinizing type; 1,153 tumors were HPV DNA−, with 1,060 cases (66.5%) being HPV DNA−/p16−. These HPV DNA−/p16− tumors were mostly keratinizing (81.2%) but were occasionally basaloid or warty (5.2%). The features of HPV DNA−/p16+ cases (n = 93) were similar to those of the HPV‐associated VSCC, and HPV DNA+/p16− (n = 74) cases had a more diverse profile, although they were more similar to HPV‐independent tumors. Several histological characteristics were more frequently associated with HPV‐related VSCC (koilocytotic‐like change, necrosis, moderate to marked pleomorphism, invasive front in nests; p < 0.001), however, none of these characteristics allowed differentiation between HPV‐associated and ‐independent VSCC. In conclusion, histological criteria do not allow differentiation between HPV‐associated and ‐independent VSCC. p16 Alone is a clinically easy strategy to determine HPV status in VSCC.

Validation of whole-slide imaging in the primary diagnosis of liver biopsies in a University Hospital

BACKGROUND Experience in the use of whole slide imaging (WSI) for primary diagnosis is limited and there are no comprehensive reports evaluating this technology in liver biopsy specimens. AIMS To determine the accuracy of interpretation of WSI compared with conventional light microscopy (CLM) in the diagnosis of needle liver biopsies. METHODS Two experienced liver pathologists blindly analyzed 176 consecutive biopsies from the Pathology Department at the Hospital Clinic of Barcelona. One of the observers performed the initial evaluation with CLM, and the second evaluation with WSI, whereas the second observer performed the first evaluation with WSI and the second with CLM. All slides were digitized in a Ventana iScan HT at 400× and evaluated with the Virtuoso viewer (Roche diagnostics). We used kappa statistics (κ) for two observations. RESULTS Intra-observer agreement between WSI and CLM evaluations was almost perfect (96.6%, κ=0.9; 95% confidence interval [95% CI]: 0.9-1 for observer 1, and 90.3%, κ=0.9; 95%CI: 0.8-0.9 for observer 2). Both native and transplantation biopsies showed an almost perfect concordance in the diagnosis. CONCLUSION Diagnosis of needle liver biopsy specimens using WSI is accurate. This technology can reliably be introduced in routine diagnosis.

Role of Human Papillomavirus in Vulvar Cancer

Human papillomavirus (HPV) is involved in one of the at least 2 pathways leading to vulvar squamous cell carcinoma (VSCC). Inactivation of p53 and retinoblastoma by the viral products E6 and E7 is involved in malignant transformation. The percentage of HPV-positive VSCCs ranges from 18% to 75%, depending on the geographical area. HPV-associated tumors affect relatively young women and arise from high-grade intraepithelial lesions, identical to other HPV-associated premalignant lesions of the anogenital tract. HPV-independent tumors tend to affect older women and usually arise in a background of inflammatory skin disorders and a subtle variant of in situ lesion called differentiated vulvar intraepithelial neoplasia. HPV-positive tumors tend to be of basaloid or warty types, whereas HPV-independent tumors tend to be of keratinizing type, but there is frequent overlap between histologic types. There is no conclusive evidence yet on the best strategy in terms of determining HPV attribution. HPV DNA detection is generally considered the gold standard although there is some concern about misclassification when using this technique alone. p16 immunostaining has shown to be an excellent surrogate marker of HPV infection. Positive results for both techniques are considered the best evidence for HPV-association. The prognostic role of HPV in VSCC is still contradictory, but increasing evidence suggests that HPV-associated tumors are less aggressive. Currently, there are no differences in treatment between HPV-associated and HPV-independent VSCC, but novel immunological strategies based on anti-HPV antigens are being evaluated in clinical trials.

Spontaneous Abortion Associated with Zika Virus Infection and Persistent Viremia

We report a case of spontaneous abortion associated with Zika virus infection in a pregnant woman who traveled from Spain to the Dominican Republic and developed a rash. Maternal Zika viremia persisted at least 31 days after onset of symptoms and 21 days after uterine evacuation.