Spatial transcriptomic analysis of virtual prostate biopsy reveals confounding effect of tissue heterogeneity on genomic signatures.

Genetic signatures have added a molecular dimension to prognostics and therapeutic decision-making. However, tumour heterogeneity in prostate cancer and current sampling methods could confound accurate assessment. Based on previously published spatial transcriptomic data from multifocal prostate cancer, we created virtual biopsy models that mimic conventional biopsy placement and core size. We then analysed the gene expression of different prognostic signatures (OncotypeDx®, Decipher®, Prostadiag®) using a step-wise approach with increasing resolution from pseudo-bulk analysis of the whole biopsy, to differentiation by tissue subtype (benign, stroma, tumour), followed by distinct tumour grade and finally clonal resolution. The gene expression profile of virtual tumour biopsies revealed clear differences between grade groups and tumour clones, compared to a benign control, which were not reflected in bulk analyses. This suggests that bulk analyses of whole biopsies or tumour-only areas, as used in clinical practice, may provide an inaccurate assessment of gene profiles. The type of tissue, the grade of the tumour and the clonal composition all influence the gene expression in a biopsy. Clinical decision making based on biopsy genomics should be made with caution while we await more precise targeting and cost-effective spatial analyses.

Calponin and MUC6 complement inhibin as diagnostic immunomarkers of serous cystadenoma in endoscopic ultrasound-guided aspiration/biopsy specimens.

AIMS: Because serous cystadenoma (SCA) does not usually require excision, it is critical to distinguish it from differential diagnoses which do, especially neuroendocrine tumour (NET). The gold standard for diagnosing SCA is assessment of endoscopic ultrasound-guided fine needle aspiration/biopsy (EUS-FNAB) material. Inhibin immunohistochemistry aids this assessment, but such positivity is not absolutely sensitive or specific to SCA. The following is the largest known study of SCA EUS-FNAB specimens and the first to compare four potential SCA immunomarkers between themselves and inhibin, compared against NET. METHODS AND RESULTS: Immunohistochemistry for calponin, mucin 6 (MUC6), glucose transporter 1 (GLUT1) and vascular endothelial growth factor A (VEGFA) was performed on 30 EUS-FNAB and three resection specimens of SCA and 32 EUS-FNAB specimens of NET. GLUT1 and VEGFA were suboptimal as diagnostic immunomarkers of SCA, being expressed by 10 and 44% of NETs, respectively. Further, their expression by cellular constituents of blood which often contaminate EUS-FNAB specimens hampered identification of neoplastic cells, especially in hypocellular samples. While 19% of NETs showed nuclear MUC6 positivity, cytoplasmic expression of the protein showed 100% specificity and sensitivity as an SCA marker. However, assessing MUC6 in EUS-FNAB specimens must also consider the protein's focal expression in physiological pancreatic, gastric or duodenal tissues, which can contaminate these specimens. Calponin was less sensitive (71% versus 100%) but more specific (100% versus 91%) than inhibin, although easier to assess in EUS-FNAB specimens than MUC6. CONCLUSIONS: Of the four potential immunomarkers of SCA suggested by the existing literature, calponin and MUC6 are useful complementary studies to inhibin for application to EUS-FNAB specimens.

Sequencing of prostate cancers identifies new cancer genes, routes of progression and drug targets.

Prostate cancer represents a substantial clinical challenge because it is difficult to predict outcome and advanced disease is often fatal. We sequenced the whole genomes of 112 primary and metastatic prostate cancer samples. From joint analysis of these cancers with those from previous studies (930 cancers in total), we found evidence for 22 previously unidentified putative driver genes harboring coding mutations, as well as evidence for NEAT1 and FOXA1 acting as drivers through noncoding mutations. Through the temporal dissection of aberrations, we identified driver mutations specifically associated with steps in the progression of prostate cancer, establishing, for example, loss of CHD1 and BRCA2 as early events in cancer development of ETS fusion-negative cancers. Computational chemogenomic (canSAR) analysis of prostate cancer mutations identified 11 targets of approved drugs, 7 targets of investigational drugs, and 62 targets of compounds that may be active and should be considered candidates for future clinical trials.

Primary peri-anal adenocarcinoma of intestinal type - a new proposed entity.

AIMS: The currently recognised subtypes of anal canal/peri-anal adenocarcinoma are those arising from low rectal mucosa or columnar cuff, fistula-related tumours and anal gland carcinoma. This report presents two examples of a hitherto undescribed subtype of peri-anal adenocarcinoma with an intestinal phenotype. METHODS AND RESULTS: A 74-year-old man had a peri-anal tumour locally excised, whereas a 73-year-old female underwent an abdominoperineal resection for peri-anal Paget's disease with an underlying carcinoma. Neither patient had a history of perineal fistulae, Crohn's disease or previous gastrointestinal neoplasia, and neither showed clinical, radiological or endoscopic evidence of another abdominal or pelvic tumour. Both resection specimens contained adenocarcinoma, which were similar in demonstrating an intestinal morphology and CDX2 immunopositivity. The man has shown a disease-free outcome thus far, but the woman has suffered with nodal and pelvic recurrence within a few months of surgery. CONCLUSIONS: The name 'primary peri-anal adenocarcinoma of intestinal type' is proposed for this previously unrecognised subtype of perineal neoplasia. Awareness of its distinct existence - by recognising its intestinal morphology and immunophenotype while excluding metastasis from the intestinal tract - should help to collate data to determine its specific prognosis and to formulate its best management.

Variation between specialist uropatholgists in reporting extraprostatic extension after radical prostatectomy.

AIMS: Extraprostatic extension of prostate cancer in radical prostatectomy specimens significantly affects patient management. We evaluated the degree of interobserver variation between uropathologists at a tertiary referral teaching hospital in assessing the extraprostatic extension of prostate cancer in radical prostatectomy specimens. METHODS: Histopathological data from a consecutive series of 293 radical prostatectomy specimens (January 2007-December 2012) were reviewed. A subset of 50 consecutive radical prostatectomy cases originally staged as tumours confined to the prostate (pT2) or tumours extending into periprostatic tissue (pT3a) during this period were reviewed by four specialist uropathologists. RESULTS: Five consultant histopathologists reported these specimens with significant differences in the reported stage (p=0.0164) between pathologists. Double-blind review by 4 uropathologists of 50 consecutive radical prostatectomy cases showed a lack of consensus in 16/50 (32%) cases (κ score 0.58, moderate agreement). A consensus meeting was held, but consensus could still not be reached in 9/16 cases. CONCLUSIONS: Our findings highlight variability in the reporting of pT stage in radical prostatectomy specimens even by specialist uropathologists. Assessment of extraprostatic extension has important implications for patient management and there is a need for more precise guidance.

A pathologist's survey on the reporting of sessile serrated adenomas/polyps.

AIM: The purpose of this survey was to ascertain reporting habits of pathologists towards sessile serrated adenomas/polyps (SSA/P). METHODS: A questionnaire designed to highlight diagnostic criteria, approach and clinical implications of SSA/P was circulated electronically to 45 pathologists in the UK and North America. RESULTS: Forty-three of 45 pathologists agreed to participate. The vast majority (88%) had a special interest in gastrointestinal (GI) pathology, had great exposure to GI polyps in general with 40% diagnosing SSA/P at least once a week if not more, abnormal architecture was thought by all participants to be histologically diagnostic, and 11% would make the diagnosis if a single diagnostic histological feature was present in one crypt only, while a further 19% would diagnose SSA/P in one crypt if more than one diagnostic feature was present. The vast majority agreed that deeper sections were useful and 88% did not feel proliferation markers were useful. More than one-third did not know whether, or did not feel that, their clinicians were aware of the implications of SSA/P. CONCLUSIONS: 98% of pathologists surveyed are aware that SSA/P is a precursor lesion to colorectal cancer, the majority agree on diagnostic criteria, and a significant number feel that there needs to be greater communication and awareness among pathologists and gastroenterologists about SSA/P.

Proximal colon cancer and serrated adenomas - hunting the missing 10%.

There is a 10% shortfall in the number of proximal colorectal cancer cases detected by the UK Bowel Cancer Screening Programme and the actual number of UK-registered proximal colorectal cancers. Sessile serrated adenomas/polyps (SSA/P) are common premalignant lesions in the proximal colon and are notoriously difficult to spot endoscopically. Missed or dismissed SSA/Ps might contribute to this UK proximal colon cancer detection disparity. In Oxfordshire, a service evaluation audit and histological review has shown a linear increase in the detection rate of these lesions over the past 4 years. This is the result of increased endoscopist and pathologist awareness of these lesions and improved interdisciplinary communication. This is the result of increased endoscopist and pathologist awareness of these lesions, together with improved interdisciplinary communication, and we predict that this will lead to a comparable detection increase nationwide. Ongoing surveillance of an increasing number of these premalignant lesions could become a significant endoscopic resource requirement once UK guidelines on serrated lesion follow up are established.

Filiform polyps and filiform polyp-like lesions are common in defunctioned or diverted colorectum resection specimens.

We describe filiform polyps (FPs) in a series of defunctioned rectums with diversion colitis. A 6-year search of all defunctioned rectal resection specimens revealed 8 cases with 17 macroscopically observed FPs. They occurred in 4 females and 4 males aged between 12 and 64 years. Four had defunctioning colostomies for ulcerative colitis, 3 for Crohn disease and 1 for diverticular disease. Multiple lesions were seen in 6 of 8 cases: 1 case having 4 FPs, 1 patient with 3 lesions, and 4 cases with 2 lesions. The FP varied in length from 4 to 11 mm; mean length was 7.8 mm. No evidence of mucosal prolapse was seen in any of the polypoid lesions. In 65 cases without grossly observed polypoid lesions, prominent mucosal polypoid projections in keeping with FP were seen in 47 cases. These were observed in nonulcerated sections and were histologically identical to the 17 macroscopically observed FPs ranging from 3 to 8 mm, and 3 to 5 such polypoid lesions were seen in 20 cases. We suggest that FP and FP-like lesions are commonly encountered in defunctioned resection specimens.

Reporting trends of right-sided hyperplastic and sessile serrated polyps in a large teaching hospital over a 4-year period (2009-2012).

AIM: An audit of serrated polyps diagnosed over a 4-year period: 2009 to 2012 was undertaken to ascertain the reporting trends of sessile serrated polyps (SSP). METHODS: All right sided hyperplastic polyps (HP) proximal to the splenic flexure and all polyps designated SSP were retrieved from the study period. Three pathologists blinded to the original diagnosis re-examined the slides. Recent American College of Gastroenterology guidelines for the diagnosis of SSP was utilised. RESULTS: No cases of SSP were diagnosed in 2009. In 2010, 32 right-sided cases were encountered, 83 confirmed in 2011 and 134 confirmed in 2012. The vast majority of these were right-sided. With regards to right-sided HP that were re-classified as SSP the data is as follows: 20 of 66 in 2009 (30%); 58 of 91 in 2010 (64%); 42 of 106 (40%) in 2011 and 69 of 206 in 2012 (33%). CONCLUSIONS: This study has demonstrated an almost exponential increase in the diagnosis of SSP over a 4-year period. In addition, 30 to 64% of right-sided HP were re-classified as SSP over the 4-year period suggesting that greater awareness of the diagnostic criteria for SSP is required. SSP is an important precursor lesion in the serrated pathway of colorectal cancer. Its recognition is important for surveillance and therapeutic strategies.

Gastric neuroendocrine cell hyperplasia and type 1 tumours occurring within gastric hyperplastic polyps.

We wish to highlight the unusual occurrence of gastric neuroendocrine cell hyperplasia and type I neuroendocrine tumours within three hyperplastic polyps. In all cases, the neuroendocrine component was present within and between the hyperplastic foveolar glands of the polyps and overall formed the minor part of the polyps. Two of the patients presented with epigastric pain and the other with fatigue from anaemia, and on endoscopy, all three were found to have superficially ulcerated gastric polyps in the body (cases 1 and 2) and fundus (case 3). Two of the cases had serologically proven autoimmune atrophic gastritis, while the third case had histological evidence of an atrophic gastritis, most likely also autoimmune in aetiology. Cases 1 and 3 had single hyperplastic polyps, while case 2 had three polyps. All polyps showed linear neuroendocrine cell hyperplasia within hyperplastic foveolar epithelium both at the surface and within deeper-situated glands. Neuroendocrine immunohistochemistry highlighted the neuroendocrine cell hyperplasia. The bulk of the neuroendocrine component was restricted to hyperplastic mucosa forming the polyps. Non-hyperplastic adjacent mucosa showed less prominent neuroendocrine cell hyperplasia. It is unclear whether the two pathologies occurred simultaneously or independently. The common feature and causal link is atrophic gastritis, which predisposed the gastric mucosa to the development of both neuroendocrine cell hyperplasia and tumours, and hyperplastic polyps.

The handling and sampling of radical prostatectomy specimens for reporting and research: the Oxford approach.

This article reviews previously described methods of fresh tissue sampling from radical prostatectomy specimens for research and describes a method used in Oxford which is simple, logical and cost effective. The method utilises a systematic zonal approach to tissue procurement in order to meet the increasing requirement for research samples with detailed morphological information such as zone of origin, tumour stage and Gleason grade. The described method involves punch biopsy sampling from a 4mm thick transverse slice cut 8mm superior to the apex. 9 biopsies are taken from each specimen in the following zonal distribution: Mid anterior, right lateral, right peripheral zone lateral, right peripheral zone mid, left peripheral zone mid, left peripheral zone lateral, left lateral, left transition zone and right transition zone. The method was validated by successfully sampling tumour in 7 out of 8 cases (88%). In 6 of the positive cases, tumour was present in more than 1 punch biopsy. The mean time from receipt of the specimen to completion of the biopsy freezing was 23.5 minutes. Tumour stage, zone and Gleason grade were determined for all positive biopsies. All cases were reported to RCPath guidelines with no compromising of margins. A logical systematic method of fresh tissue sampling from radical prostatectomy specimens is presented, which balances the need for accurate routine histopathological reporting with the requirement for increasingly complex research samples to be taken with attention to morphological details such as zone and stage.

International study group on rectal cancer regression grading: interobserver variability with commonly used regression grading systems.

The aim of this study was to ascertain the level of concordance among gastrointestinal pathologists for regression grading in rectal cancers treated with neoadjuvant chemoradiation. Seventeen gastrointestinal pathologists participated using the Mandard, Dworak, and modified rectal cancer regression grading systems to grade 10 representative slides that were selected from 10 cases of rectal cancer treated with long-course neoadjuvant chemoradiation. The slides were scanned with a whole-slide scanner generating dynamic digitized images. The results showed very little concordance across the 3 grading systems, with κ values of 0.28, 0.35, and 0.38 for the Mandard, Dworak, and modified rectal cancer regression grading systems, respectively. In only 1 of 10 study cases was there unanimous grading concordance using the modified rectal cancer regression grading system. It was felt that these systems lacked precision and clarity for reproducible, accurate regression grading. The study concluded that there was a need for a simple, reproducible regression grading system with clear criteria, a cumulative or composite score taking into account all sections of the tumor bed that is sampled rather than the worst section (highest grade), and there should be a uniform method of sampling of these specimens.

A multi-centre pathologist survey on pathological processing and regression grading of colorectal cancer resection specimens treated by neoadjuvant chemoradiation.

To ascertain the approach and degree of consensus of pathologists in the handling and regression grading of colorectal cancer resection specimens treated with neoadjuvant chemoradiation, a ten-part questionnaire was circulated to 18 gastrointestinal pathologists in eight countries. The questions were specific and addressed pertinent issues related to colorectal cancer with neoadjuvant chemoradiation. There is a lack of consensus on how to handle the specimen, number of sections taken, correlation with pre- and post-operative radiological imaging, and especially, regression grading schema employed. Consensus in the form of guidelines is required so that the pathological assessment of these specimens will provide clinically relevant information for patient management, irrespective of location.