Canadian Multicentric Pan-TRK (CANTRK) Immunohistochemistry Harmonization Study.

Tumor-agnostic testing for NTRK1-3 gene rearrangements is required to identify patients who may benefit from TRK inhibitor therapies. The overarching objective of this study was to establish a high-quality pan-TRK immunohistochemistry (IHC) screening assay among 18 large regional pathology laboratories across Canada using pan-TRK monoclonal antibody clone EPR17341 in a ring study design. TRK-fusion positive and negative tumor samples were collected from participating sites, with fusion status confirmed by panel next-generation sequencing assays. Each laboratory received: (1) unstained sections from 30 cases of TRK-fusion-positive or -negative tumors, (2) 2 types of reference standards: TRK calibrator slides and IHC critical assay performance controls (iCAPCs), (3) EPR17341 antibody, and (4) suggestions for developing IHC protocols. Participants were asked to optimize the IHC protocol for their instruments and detection systems by using iCAPCs, to stain the 30 study cases, and to report the percentage scores for membranous, cytoplasmic, and nuclear staining. TRK calibrators were used to assess the analytical sensitivity of IHC protocols developed by using the 2 reference standards. Fifteen of 18 laboratories achieved diagnostic sensitivity of 100% against next-generation sequencing. The diagnostic specificity ranged from 40% to 90%. The results did not differ significantly between positive scores based on the presence of any type of staining vs the presence of overall staining in ≥1% of cells. The median limit of detection measured by TRK calibrators was 76,000 molecules/cell (range 38,000 to >200,000 molecules/cell). Three different patterns of staining were observed in 19 TRK-positive cases, cytoplasmic-only in 7 samples, nuclear and cytoplasmic in 9 samples, and cytoplasmic and membranous in 3 samples. The Canadian multicentric pan-TRK study illustrates a successful strategy to accelerate the multicenter harmonization and implementation of pan-TRK immunohistochemical screening that achieves high diagnostic sensitivity by using laboratory-developed tests where laboratories used centrally developed reference materials. The measurement of analytical sensitivity by using TRK calibrators provided additional insights into IHC protocol performance.

In-situ follicular neoplasia: a clinicopathological spectrum.

AIMS: In-situ follicular neoplasia (ISFN) occurs in approximately 2-3% of reactive lymph nodes, and is currently set apart from 'partial involvement by follicular lymphoma' (PFL). ISFN can progress to overt lymphoma, but precise parameters with which to assess this risk and its association with related diseases remain incompletely understood. The aim of this study was to explore these parameters. METHODS AND RESULTS: We reviewed 11 cases of ISFN and one of PFL between 2003 and 2018. Ten patients had ISFN in the lymph nodes, and one had ISFN in the spleen. Haematoxylin and eosin and immunohistochemical stains were reviewed. Involvement of follicles by ISFN was scored with a three-tier scheme. Of five patients with low ISFN scores, one had chronic myelomonocytic leukaemia, one had mycosis fungoides, and three were free of haematopoietic disease. Among them, four are alive and one was lost to follow-up. Of the six ISFN patients with high scores, two had concurrent marginal zone lymphomas, one had concurrent diffuse large B-cell lymphoma (DLBCL), one had Castleman-like disease, one had progressive transformation of germinal centres with IgG4-related disease, and one had no haematopoietic disease; all are alive except for one who died of concurrent DLBCL. The patient with PFL developed DLBCL 7 years after diagnosis. CONCLUSIONS: On the basis of this limited series, we conclude that only cases with high scores are associated with an overt lymphoma or an abnormal lymphoid process, and that scoring may be a useful parameter with which to assess the risk of associated lymphoma, and deserves further study. We also performed a comprehensive review of the literature.

Recommendations for Specimen and Therapy Selection in Colorectal Cancer.

INTRODUCTION: Next-generation sequencing has emerged as a clinical tool for the identification of actionable mutations to triage advanced colorectal cancer patients for targeted therapies. The literature is conflicted as to whether primaries or their metastases should be selected for sequencing. Some authors suggest that either site may be sequenced, whereas others recommend sequencing the primary, the metastasis, or even both tumors. Here, we address this issue head on with a meta-analysis and provide for the first time a set of sensible recommendations to make this determination. METHODS: From our own series, we include 43 tumors from 13 patients including 14 primaries, 10 regional lymph node metastases, 17 distant metastases, and two anastomotic recurrences sequenced using the 50 gene Ion AmpliSeq cancer NGS panel v2. RESULTS: Based on our new cohort and a meta-analysis, we found that ~ 77% of patient-matched primary-metastatic pairs have identical alterations in these 50 cancer-associated genes. CONCLUSIONS: Low tumor cellularity, tumor heterogeneity, clonal evolution, treatment status, sample quality, and/or size of the sequencing panel accounted for a proportion of the differential detection of mutations at primary and metastatic sites. The therapeutic implications of the most frequently discordant alterations (TP53, APC, PIK3CA, and SMAD4) are discussed. Our meta-analysis indicates that a subset of patients who fail initial therapy may benefit from sequencing of additional sites to identify new actionable genomic abnormalities not present in the initial analysis. Evidence-based recommendations are proposed.

The morphologic and immunohistochemical spectrum of papillary renal cell carcinoma: study including 132 cases with pure type 1 and type 2 morphology as well as tumors with overlapping features.

Papillary renal cell carcinomas (pRCC) are classically divided into type 1 and 2 tumors. However, many cases do not fulfill all the criteria for either type. We describe the clinical, morphologic, and immunohistochemical (IHC) features of 132 pRCCs to better characterize the frequency and nature of tumors with overlapping features. Cases were reviewed and classified; IHC evaluation of CK7, EMA, TopoIIα, napsin A, and AMACR was performed on 95 cases. The frequencies of type 1, type 2, and "overlapping" pRCC were 25%, 28%, and 47%, respectively. The 2 categories of "overlapping" tumors were: (1) cases with bland cuboidal cells but no basophilic cytoplasm (type A); and (2) cases with predominantly type 1 histology admixed with areas showing prominent nucleoli (type B). The pathologic stage of "overlapping" cases showed concordance with type 1 tumors. Using the 2 discriminatory markers (CK7, EMA), "type A" cases were similar to type 1. Although the high-nuclear grade areas of "type B" tumors showed some staining differences from their low-nuclear grade counterpart, their IHC profile was closer to type 1. Single nucleotide polymorphism array results, although preliminary and restricted to only 9 cases (3 with overlapping features), also seemed to confirm those findings. In conclusion, we demonstrate that variations in cytoplasmic quality and/or presence of high-grade nuclei in tumors otherwise displaying features of type 1 pRCCs are similar in stage and IHC profile those with classic type 1 histology, suggesting that their spectrum might be wider than originally described.

The prognostic role of ERG immunopositivity in prostatic acinar adenocarcinoma: a study including 454 cases and review of the literature.

TMPRSS2/ERG fusion is among the most frequent genetic anomalies in prostate adenocarcinomas. Although positive immunostaining for ERG has been shown to tightly correlate with ERG fusion status, the clinical and prognostic significance of a positive ERG stain remains undetermined. The significance of ERG immunostaining in 454 consecutive prostate adenocarcinomas from radical prostatectomies (RPs) using tissue microarrays, herein, is evaluated. A separate set of 59 cases of incidental prostate adenocarcinoma detected on transurethral resection of prostate with a Gleason score of 6 was also included. ERG translocation was significantly more common in peripheral zone cancer in comparison with cancer of the transitional zone (33% in RP versus 5% in transurethral resection of prostate specimens). In the RP cohort, although ERG positivity was significantly associated with younger age at presentation and lower prostate-specific antigen values, it showed no association with Gleason score or with pathologic stage. In multivariate analysis, biochemical recurrence was only associated with the final RP Gleason score and elevated prostate-specific antigen levels and was unrelated to neither ERG positivity or to its staining intensity. In our hands, ERG positivity was unrelated to either aggressive local tumor characteristics or a worse outcome. Our results, as well as an extensive review of the related literature showing conflicting findings, seem to indicate that ERG immunopositivity cannot be considered as an important prognostic factor in prostate cancer.

HPV testing with cytology triage for cervical cancer screening in routine practice.

OBJECTIVE: The purpose of this study was to evaluate the feasibility and effectiveness of Viral Testing Alone with Pap (Papanicolaou) Triage for Screening Cervical Cancer in Routine Practice (VASCAR) in a publicly funded university-affiliated hospital in Montreal, Canada. STUDY DESIGN: Women who are 30-65 years old are screened with the Hybrid Capture-2 assay. Women with negative results are retested at 3-year intervals; women with positive results are triaged with conventional cytologic methods. Women with Papanicolaou positive test results (≥atypical squamous cells of undetermined significance) are referred to colposcopy; women with Papanicolaou negative test results are retested with Hybrid Capture-2 assay and a Papanicolaou test in 1 year. Results were compared with a historic era (annual cytology with ≥atypical squamous cells of undetermined significance threshold for colposcopy referral) in the 3 years before VASCAR. RESULTS: VASCAR included 23,739 eligible women, among whom 1646 women (6.9%) tested positive for the human papillomavirus (HPV). Because of the need for subsequent sampling for cytologic testing, follow-up evaluation for cytologic triage was relatively poor; only 46% and 24% of HPV-positive women were Papanicolaou-triaged and underwent biopsy, respectively. Protocol violations occurred mainly in the early phases of implementation (12%). Detection of high-grade cervical intraepithelial neoplasia increased nearly 3-fold (rate ratio, 2.78; 95% confidence interval [CI], 2.1-3.7) during VASCAR, mostly because of a doubling in the rate of high-grade cervical intraepithelial neoplasia (34.0%; 95% CI, 21.2-48.8) compared with the historic cytology-only era (16.3%; 95% CI, 13.2-19.8). VASCAR reduced the median time to colposcopy from a positive screen from 11 months (95% CI, 10.48-11.50) to 3 months (95% CI, 2.64-3.80). CONCLUSION: VASCAR is feasible; however, it requires cosampling for HPV and cytology and for continuous education of healthcare providers of the HPV-Papanicolaou triage protocol. Efficacy in disease detection and reduction in time to colposcopy referrals compared with the historic cytology era is encouraging but should be considered preliminary because of the small number of patients who were tested.

Human papillomavirus and cancer prevention: gaps in knowledge and prospects for research, policy, and advocacy.

The recognition that human papillomavirus (HPV) infection is the central, necessary cause of cervical cancer paved the way to new fronts of prevention via improved screening methods and HPV vaccination. Much has been learned in all fronts, from the molecular basis of our understanding of how HPV causes disease to the health economics of preventive strategies at the individual and population levels. Progress in other areas of cancer control has yet to show the same multi- and trans-disciplinary gains seen in research on HPV-associated malignancies, which is one of the unequivocal success stories in disease prevention. Yet, as an embarrassment of riches, much more research is needed to fill the gaps in knowledge that remain before we are able to reap the benefits from the knowledge translation from all fronts. Public health research on setting-specific implementation of HPV-based preventive strategies and more concerted advocacy to counter barriers facing the adoption of these strategies are likely to yield major dividends in reducing the burden of HPV-associated diseases. This article forms part of a special supplement entitled "Comprehensive Control of HPV Infections and Related Diseases" Vaccine Volume 30, Supplement 5, 2012.

Epidemiology and burden of HPV infection and related diseases: implications for prevention strategies.

Human papillomavirus (HPV) infection is a necessary, although not sufficient cause of cervical cancer. Globally, HPV infection accounts for an estimated 530,000 cervical cancer cases (~270,000 deaths) annually, with the majority (86% of cases, 88% of deaths) occurring in developing countries. Approximately 90% of anal cancers and a smaller subset (<50%) of other cancers (oropharyngeal, penile, vaginal, vulvar) are also attributed to HPV. In total, HPV accounts for 5.2% of the worldwide cancer burden. HPVs 16 and 18 are responsible for 70% of cervical cancer cases and, especially HPV 16, for a large proportion of other cancers. Prophylactic vaccination targeting these genotypes is therefore expected to have a major impact on the burden of cervical cancer as well as that of other HPV-related cancers. Over the past 50 years, organized or opportunistic screening with Papanicolaou (Pap) cytology has led to major reductions in cervical cancer in most developed countries. However, due to lack of resources or inadequate infrastructure, many countries have failed to reduce cervical cancer mortality through screening. HPV DNA testing recently emerged as a likely candidate to replace Pap cytology for primary screening. It is less prone to human error and more sensitive than Pap in detecting high-grade cervical lesions. For countries with national vaccination programs, HPV testing may also serve as a low cost strategy to monitor long term vaccine efficacy. Introduction of well organized vaccination and screening programs should be a priority for all countries. Increased support from donors is needed to support this cause.