Vijayalakshmi Ananthanarayanan

Evidence for field cancerization of the prostate

Field cancerization, which is not yet well‐characterized in the prostate, occurs when large areas of an organ or tissue surface are affected by a carcinogenic insult, resulting in the development of multi‐focal independent premalignant foci and molecular lesions that precede histological change.

Endoglin suppresses human prostate cancer metastasis

Endoglin is a transmembrane receptor that suppresses human prostate cancer (PCa) cell invasion. Small molecule therapeutics now being tested in humans can activate endoglin signaling. It is not known whether endoglin can regulate metastatic behavior, PCa tumor growth, nor what signaling pathways are linked to these processes. This study sought to investigate the effect of endoglin on these parameters. We used a murine orthotopic model of human PCa metastasis, designed by us to measure effects at early steps in the metastatic cascade, and implanted PCa cells stably engineered to express differing levels of endoglin. We now extend this model to measure cancer cells circulating in the blood. Progressive endoglin loss led to progressive increases in the number of circulating PCa cells as well as to the formation of soft tissue metastases. Endoglin was known to suppress invasion by activating the Smad1 transcription factor. We now show that it selectively activates specific Smad1-responsive genes, including JUNB, STAT1, and SOX4. Increased tumor growth and increased Ki67 expression in tissue was seen only with complete endoglin loss. By showing that endoglin increased TGFβ-mediated suppression of cell growth in vitro and TGFβ-mediated signaling in tumor tissue, loss of this growth-suppressive pathway appears to be implicated at least in part for the increased size of endoglin-deficient tumors. Endoglin is shown for the first time to suppress cell movement out of primary tumor as well as the formation of distant metastasis. It is also shown to co-regulate tumor growth and metastatic behavior in human PCa.

Adjunct therapeutic plasma exchange for anti-N-methyl-D-aspartate receptor antibody encephalitis: a case report and review of literature.

Encephalitis associated with autoantibodies directed against the N‐methyl‐D‐aspartate receptor (NMDAR) is usually a paraneoplastic syndrome that presents in young females with ovarian teratomas. We report a case of a previously healthy 14‐year‐old girl with sudden‐onset paranoia, hallucinations, hyperactivity, increased speech, decreased sleep, seizures, and violent behavior deteriorating to catatonia. Her cerebrospinal fluid tested positive for anti‐NMDAR antibodies. She was treated with five sessions of therapeutic plasma exchange (TPE) after having failed therapy with antibiotics, intravenous steroids, intravenous immunoglobulin (IVIG), one dose of rituximab, and seven sessions of electroconvulsive therapy (ECT). The American Society for Apheresis assigns a Category III (Grade 2C) recommendation for TPE in paraneoplastic neurologic syndromes; however, apheresis specifically for anti‐NMDAR encephalitis has not been well studied. Literature review revealed two case reports describing outstanding improvement in patients with anti‐NMDAR encephalitis following TPE. We report no improvement in our patients symptoms after plasma exchange and discuss possible reasons for why it failed along with review of the literature. J. Clin. Apheresis, 2011.

Morphometric Signature Differences in Nuclei of Gleason Pattern 4 Areas in Gleason 7 Prostate Cancer With Differing Primary Grades on Needle Biopsy

PURPOSE Since clinically significant upgrading of the biopsy Gleason score has an adverse clinical impact, ancillary tools besides the visual determination of primary Gleason pattern are essential to aid in better risk stratification. MATERIALS AND METHODS A total of 61 prostate biopsies were selected in patients with a diagnosis of Gleason score 7 prostatic adenocarcinoma, including 41 with primary Gleason pattern 3 and 20 with primary Gleason pattern 4. Slides from these tissues were stained using Feulgen stain, a nuclear DNA stain. Areas of Gleason pattern in all cases were analyzed for 40 nuclear morphometric descriptors of size, shape and chromatin using a CAS-200 system (BD). The primary outcome analyzed was the ability of morphometric features to identify visually determined primary Gleason pattern 4 on the biopsy. Data were analyzed using logistic regression as well as a C4.5 decision tree with and without preselection. RESULTS Decision tree analysis yielded the best model. Automatic feature selection identified minimum nuclear diameter as the most discriminative feature in a 3-parameter model with 85% classification accuracy. Using a preselected 3-parameter model including minimum diameter, angularity and sum optical density the decision tree yielded a slightly lesser accuracy of around 79%. Bootstrap validation of logistic regression results revealed that there was no unique model that could significantly explain the variance in primary Gleason pattern status, although minimum nuclear diameter was the most frequently selected parameter. CONCLUSIONS In this small cohort of patients with Gleason score 7 disease we report that Gleason pattern 4 nuclei from those with primary Gleason pattern 4 are generally larger with coarser chromatin compared with the Gleason pattern 4 in patients with primary Gleason pattern 3. These findings may aid in better risk stratification of the Gleason score 7 group by supplementing visual estimation of the percent of primary Gleason pattern 3 and 4 in the biopsy.

Accessible calculation of multirater kappa statistics for pathologists

Dear Editor: Multirater kappa statistics are relatively underused by pathologists to study interobserver agreement [1, 3]. While several methods are available for measuring agreement when there are only two raters, there is little awareness of the appropriate software programs geared to handle measures of agreement when there are more than two raters or when there are more than three categories to be rated for this kind of analysis. There are two major options for performing this analysis—free online open-source software or routine statistical packages like SPSS, SAS, or STATA to calculate kappa statistics. We personally use SPSS 13 (SPSS Inc., Chicago, IL, USA) for agreement studies; unfortunately, the current version of SPSS does not calculate kappa statistics when there are more than two raters or more than two categories of rating. To circumvent this problem, there is an SPSS macrofile called “MKAPPASC” that is freely available on the Internet for users of SPSS version 6 and above to implement similar studies within the SPSS environment. Still, this method will give only the overall interrater agreement rather than category-specific agreements, which is generally the preferred statistic. Aweighted version of the kappa coefficient is sometimes suggested as a reliability measure for data from ordered scales [1]. The idea is that one should distinguish between different types of disagreement. Specifically, two ratings that differ by a small amount on an ordered scale should be considered a “lesser disagreement” than two scores that are far apart. Weighted kappas can be calculated in STATA software using the “kap” and “kapwgt” commands. A default weights matrix is supplied or one can define one’s own. The SAS procedure PROCFREQ can provide the kappa statistic for two raters and multiple categories, provided that the data are in a 2×2 format. A SAS macro MAGREE, on the other hand, can compute the estimates and tests of agreement between multiple raters/categories. An excellent article is available at http://www2.sas.com/proceedings/ sugi30/155-30.pdf In the interest of other investigators who might wish to perform a similar analysis (unweighted multirater kappa) without using these software, there is a free Excel spreadsheet-based program that calculates category-specific kappa statistics for more than three raters and three categories available at http://www.ccit.bcm.tmc.edu/jking/ homepage/genkappa.doc with confidence interval based on the method of Fleiss [2]. Interested investigators may consider all these options whenever they plan to utilize multirater kappa statistics in the field of pathology.

Morphometric sum optical density as a surrogate marker for ploidy status in prostate cancer: an analysis in 180 biopsies using logistic regression and binary recursive partitioning

We sought to identify morphometric descriptors predictive of nondiploidy in prostatic adenocarcinoma on prostate needle biopsies using logistic regression (LR) and binary recursive partitioning (BRP) and compare the equivalence of both methods. A total of 180 prostate needle biopsies diagnosed as prostatic adenocarcinoma were selected. Deoxyribonucleic acid ploidy and morphometry were performed separately on Feulgen-stained sections from these biopsies using the CAS-200 system and Nuclear Morphometry Suite, respectively. Seven morphometric predictors were tested as predictor variables, including nuclear area, circularity, elongation, sum optical density (OD), configuration run length, coefficient of variation (CV), and angularity. Logistic regression (LR) identified a two-parameter model including sum OD and circularity that had a 93.9% overall correct prediction rate (area under curve = 0.950; 95% CI: 0.913, 0.987). A reduced model including only sum OD was equally good without any significant loss of predictive accuracy (93.3% correct overall classification rate). BRP also selected sum OD as the most predictive parameter; a sum OD cut-off of 7.73 in this model identified 93.3% of the nondiploid cases correctly. Morphometric OD can be used as a surrogate marker of nondiploidy. LR and BRP models are both equivalent in identifying and correctly classifying nondiploid cases of prostate cancer using sum OD as the predictor variable.

Influence of histologic criteria and confounding factors in staging equivocal cases for microscopic perivesical tissue invasion (pT3a): An interobserver study among genitourinary pathologists

Current oncology guidelines and clinical trials consider giving adjuvant chemotherapy to bladder cancer patients with at least microscopic perivesical tissue invasion (MPVTI) (≥pT3a) on cystectomy. The boundary of muscularis propria (MP) and perivesical tissue is commonly ill defined, and hence, when the tumor involves the interface, interpretation of MPVTI is likely to be subjective. In this study, 20 sets of static images that included 1 nontumoral bladder wall for defining MP-perivesical tissue boundary and 19 bladder cancer cases equivocal for MPVTI with confounding factors were sent to 17 expert genitourinary pathologists for review. The confounding factors were “histoanatomic,” as defined by the irregular MP-perivesical tissue boundary, and “tumor related,” such as fibrosis, dense inflammation, tumor cells at the edge of the outermost MP muscle bundle, and lymphovascular invasion. These equivocal cases were divided into 3 categories according to the following factors: (1) histoanatomic only (7/19), (2) histoanatomic+tumor related (7/19), and (3) tumor related only (5/19). Participating genitourinary pathologists used different criteria to assess MPVTI: (A) drawing a straight horizontal line using the outermost MP muscle bundle edge as the MP-perivesical tissue boundary reference (3/17); (B) drawing multiple straight lines interconnecting the outermost MP muscle bundle edges (9/17); (C) following the curves of every outermost MP muscle bundle edge (4/17). In category 1 cases, most pathologists who used the A criterion called for absence (6/7), whereas those who used the C criterion called for presence (5/7) of MPVTI, which resulted in disparity in 4/7 cases. There was no circumstance in which criteria A and C agreed on the presence or absence of MPVTI but was opposed by the B criterion in category 1 cases. Median pairwise agreement among all pathologists (regardless of criteria) for all cases (regardless of category) was only “fair” (&kgr;=0.281). However, when only the B criterion was assessed for category 1 cases, median agreement was “substantial” (&kgr;=0.696), and pairwise rater comparisons included 6/36 (17%) “near perfect,” 13/36 (36%) “substantial,” and 11/36 (31%) “moderate” agreements. When all cases with histoanatomic factors (categories 1 and 2) were combined, median pairwise agreements were: (A) &kgr;=0.588, (B) &kgr;=0.423, and (C) &kgr;=0.512, and the B criterion rater comparisons included 0/36 (0%) “near perfect,” 6/36 (17%) “substantial,” and 16/36 (44%) “moderate” agreements, which showed the confounding effect of tumor-related factors. For category 3 cases, median pairwise agreement for all pathologists was “fair” (&kgr;=0.286), with consensus agreement in only 2/5 of these equivocal cases. Lymphovascular invasion only at the MP-perivesical tissue boundary was not staged as MPVTI by 87.5% of pathologists. In conclusion, this study showed that interpretation of equivocal cases for MPVTI can be made difficult by factors intrinsic to bladder histoanatomy, defined by an irregular MP-perivesical tissue boundary, and factors related to tumor spread. There are at least 3 different approaches to demarcating an irregular outer MP boundary, and agreement is improved on equivocal cases when a common histoanatomic criterion is used. However, inconsistent agreement of anatomic criteria may cause systematic discrepancy in assessing MPVTI. Tumor-related factors such as dense fibrosis or desmoplasia, obscuring inflammation, tumor cells at the edge of the outermost MP muscle bundle, and admixed lymphovascular invasion can also negatively influence the agreement on interpretation of MPVTI. This study highlights the need to adopt common criteria in defining the outer MP boundary. Future studies may identify the most clinically relevant histoanatomic criteria for MPVTI.

Nuclear grade and necrosis predict prognosis in malignant epithelioid pleural mesothelioma: a multi-institutional study

A recently described nuclear grading system predicted survival in patients with epithelioid malignant pleural mesothelioma. The current study was undertaken to validate the grading system and to identify additional prognostic factors. We analyzed cases of epithelioid malignant pleural mesothelioma from 17 institutions across the globe from 1998 to 2014. Nuclear grade was computed combining nuclear atypia and mitotic count into a grade of I–III using the published system. Nuclear grade was assessed by one pathologist for three institutions, the remaining were scored independently. The presence or absence of necrosis and predominant growth pattern were also evaluated. Two additional scoring systems were evaluated, one combining nuclear grade and necrosis and the other mitotic count and necrosis. Median overall survival was the primary endpoint. A total of 776 cases were identified including 301 (39%) nuclear grade I tumors, 354 (45%) grade II tumors and 121 (16%) grade III tumors. The overall survival was 16 months, and correlated independently with age (P=0.006), sex (0.015), necrosis (0.030), mitotic count (0.001), nuclear atypia (0.009), nuclear grade (<0.0001), and mitosis and necrosis score (<0.0001). The addition of necrosis to nuclear grade further stratified overall survival, allowing classification of epithelioid malignant pleural mesothelioma into four distinct prognostic groups: nuclear grade I tumors without necrosis (29 months), nuclear grade I tumors with necrosis and grade II tumors without necrosis (16 months), nuclear grade II tumors with necrosis (10 months) and nuclear grade III tumors (8 months). The mitosis–necrosis score stratified patients by survival, but not as well as the combination of necrosis and nuclear grade. This study confirms that nuclear grade predicts survival in epithelioid malignant pleural mesothelioma, identifies necrosis as factor that further stratifies overall survival, and validates the grading system across multiple institutions and among both biopsy and resection specimens. An alternative scoring system, the mitosis–necrosis score is also proposed.

Deubiquitinase OTUD6B Isoforms Are Important Regulators of Growth and Proliferation

Deubiquitinases (DUB) are increasingly linked to the regulation of fundamental processes in normal and cancer cells, including DNA replication and repair, programmed cell death, and oncogenes and tumor suppressor signaling. Here, evidence is presented that the deubiquitinase OTUD6B regulates protein synthesis in non–small cell lung cancer (NSCLC) cells, operating downstream from mTORC1. OTUD6B associates with the protein synthesis initiation complex and modifies components of the 48S preinitiation complex. The two main OTUD6B splicing isoforms seem to regulate protein synthesis in opposing fashions: the long OTUD6B-1 isoform is inhibitory, while the short OTUD6B-2 isoform stimulates protein synthesis. These properties affect NSCLC cell proliferation, because OTUD6B-1 represses DNA synthesis while OTUD6B-2 promotes it. Mutational analysis and downstream mediators suggest that the two OTUD6B isoforms modify different cellular targets. OTUD6B-2 influences the expression of cyclin D1 by promoting its translation while regulating (directly or indirectly) c-Myc protein stability. This phenomenon appears to have clinical relevance as NSCLC cells and human tumor specimens have a reduced OTUD6B-1/OTUD6B-2 mRNA ratio compared with normal samples. The global OTUD6B expression level does not change significantly between nonneoplastic and malignant tissues, suggesting that modifications of splicing factors during the process of transformation are responsible for this isoform switch. Implications: Because protein synthesis inhibition is a viable treatment strategy for NSCLC, these data indicate that OTUD6B isoform 2, being specifically linked to NSCLC growth, represents an attractive, novel therapeutic target and potential biomarker for early diagnosis of malignant NSCLC. Mol Cancer Res; 15(2); 117–27. ©2016 AACR.

Recurrent NAB2-STAT6 gene fusions and oestrogen receptor-α expression in pulmonary adenofibromas

Pulmonary adenofibromas are rare benign fibroepithelial tumours of the lung with unknown histogenesis and an indolent clinical behaviour. Their stroma resembles that of solitary fibrous tumours, whereas the glands are composed of respiratory epithelium organized in a phyllodes‐like architecture. Differentiation of pulmonary adenofibromas from other more aggressive intrathoracic tumours is clinically relevant. However, their biology is unknown. Here, we sought to characterize pulmonary adenofibromas at a clinicopathological level and to define whether they could be underpinned by a highly recurrent somatic genetic alteration akin to tumours with similar morphology.