Nuclear-specific accumulation of telomerase reverse transcriptase (TERT) mRNA in TERT promoter mutated follicular thyroid tumours visualised by in situ hybridisation: a possible clinical screening tool?

AIMS: Upregulation of the telomerase reverse transcriptase (TERT) gene is a frequent finding in follicular thyroid carcinomas (FTCs) with metastatic features. The augmented expression is usually caused by TERT promoter mutations. As TERT protein immunohistochemistry might not correlate to TERT mRNA levels in follicular thyroid tumours, we therefore sought to determine if visualisation of TERT mRNA through in situ hybridisation could highlight high-risk cases. METHODS: We collected formalin-fixated paraffin-embedded tissues from 26 follicular thyroid tumours; 7 FTCs, 2 follicular thyroid tumours of uncertain malignant potential (FT-UMPs) and a single Hürthle cell carcinoma with established TERT promoter mutations and gene expression, as well as 16 FTCs with no TERT gene aberrancy or gene expression, and assessed them using RNA Scope in situ hybridisation (ISH) and TERT probes targeting the two main TERT transcripts (TERT1 and TERT2). RESULTS: TERT 1 and/or 2 mRNA was found by ISH in 8/10 cases with established promoter mutations and mRNA expression, whereas all 16 cases without TERT gene aberrancies or gene expression were negative (Fisher's exact p<0.001). Strikingly, TERT mRNA was visualised in the nuclear compartment only, thereby corroborating earlier studies suggesting a non-conventional role for TERT in tumour biology. Moreover, TERT mRNA expression was scattered across the tissue sections and only found in a few percentages of tumour nuclei. CONCLUSIONS: TERT mRNA seems to be focally expressed and localised exclusively to the nucleus in TERT promoter mutated follicular thyroid tumours, possibly reflecting a true biological and unorthodox phenomenon worthy of further investigations.

Proteomic Profiling of Diffuse Large B-Cell Lymphomas.

OBJECTIVE: The aim of this study was to identify differences in proteome profiles of diffuse large B-cell lymphoma (DLBCL) of nongerminal center (non-GC) versus GC type in the search for new markers and drug targets. METHODS: Six DLBCL, with 3 repeats for each, were used for the initial study by proteomics: 3 non-GC and 3 GC DLBCL cases. For immunohistochemistry, tissue microarrays were made from 31 DLBCL samples: 16 non-GC de novo lymphomas and 15 GC cases (11 transformed from follicular lymphomas and 4 de novo GC lymphomas). Proteome profiling was performed by two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry. RESULTS: Ninety-one proteins were found differentially expressed in non-GC compared to GC type. The Cytoscape tool was used for systemic analysis of proteomics data, revealing 19 subnetworks representing functions affected in non-GC versus GC types of DLBCL. CONCLUSION: A validation study of 3 selected proteins (BiP/Grp78, Hsp90, and cyclin B2) showed the enhanced expression in non-GC DLBCL, supporting the proteomics data.

Maspin, a Marker of Serrated Colorectal Polyps.

The serine proteinase inhibitor maspin is a tumor-suppressor protein that stimulates apoptosis and inhibits motility, invasion and cancer metastasis. Mutant maspin galvanises partial loss of tumor-suppressor function, reducing susceptibility to apoptosis and facilitating malignant progression. Mutant maspin has been reported in many tumor types. We recently analyzed maspin expression in 128 colorectal lesions: 39 hyperplastic polyps (HPs), 29 sessile serrated adenoma/polyps (SSA/Ps), three traditional serrated adenomas (TSAs), 20 conventional colorectal adenomas (CCRAs), 5 carcinomas evolving from CCRA, 12 active inflammatory bowel disease (IBD), 2 ulcerative colitis (UC) in remission, 4 solitary ulcers (rectum) and 12 normal colorectal mucosa. The topographic distribution of maspin in the cytoplasm was classified into i) extensive, ii) focal, or iii) negative. The intensity of maspin expression in the cytoplasm was classified into i) unquestionable or ii) negative. Cases with faint (questionable) maspin expression were also recorded as negative. Extensive maspin expression was recorded in 95% (39/41) of the HPs, in 100% (29/29) of the SSA/Ps (including one carcinoma arising in a SSA/P), in 66% (2/3) of the TSAs, but only in 10% (2/20) of the CCRAs. None of the specimens with carcinoma arising in CCRA, with UC in remission or with solitary ulcer exhibited extensive maspin expression. Importantly, maspin was not expressed in the normal mucosa (including that adjacent to HP, SSA/P, TSA and CCRA). It is submitted that extensive maspin expression might be a manifestation of mutant maspin in lesions central to the serrated pathway of colorectal carcinogenesis.

Maspin highlights colorectal serrated polyps: preliminary findings.

AIMS: Maspin, a 42-kDa serine proteinase inhibitor, is a tumor suppressor protein that stimulates apoptosis and inhibits motility, invasion and cancer metastasis. Mutant maspin leads to partial loss of tumor suppressor function, decreased susceptibility to apoptosis and to malignant progression. We recently found maspin expression (ME) in the cytoplasm of serrated colonic lesions, such as hyperplastic polyps (HP) and sessile serrated adenoma/polyps (SSA/P). MATERIALS AND METHODS: ME was investigated in 10 colorectal lesions: three HP, three SSA/P and four conventional colorectal adenomas (CCRA). RESULTS: Widespread cytoplasmic ME (comprising the entire height and width of the lesion) was present in the three HP and in the three SSA/P. In contrast, ME was only focally expressed in the four CCRA. ME was not present in the normal mucosa adjacent to those lesions. CONCLUSION: These preliminary findings suggest that maspin might be of help in discriminating between serrated colonic lesions (HP and SSA/P) and CCRA.

ß-catenin Helices in the cytoplasm of sporadic and FAP duodenal adenomas.

BACKGROUND: Initiation and progression in conventional adenomas is triggered by deregulation of Wnt/ß-catenin signaling. In the absence of Wnt signal (off-state), ß-catenin prevents phosphorylation of glycogen synthase kinase (GSK)-3ß leading to aberrant nuclear accumulation in human tumors. While investigating the nuclear expression of ß-catenin in biopsies from duodenal adenomas, we observed a non-previously reported phenomenon, namely the presence of ß-catenin cytoplasmic helices (coils). MATERIALS AND METHODS: Sections from 39 biopsies were immunostained with ß-catenin: 25 from duodenal adenomas and the remaining 14 had normal duodenal mucosa (n=11) or polypoid gastric duodenal metaplasia (n=3). RESULTS: Eighteen out of the 25 duodenal adenomas (72%) showed ß-catenin helices; in contrast, none of the 33 control biopsies (including those with normal duodenal mucosa, gastric duodenal metaplasia and normal mucosa adjacent to 19 adenomas) showed ß-catenin helices (p<0.05). The review of diagnostic H&E-stained sections and of ß-catenin-stained nuclei revealed that the dysplastic nuclei were arranged in a picket fence-like fashion along the basement membrane of the glands and not as loops within the dysplastic glands; the nuclei of the dysplastic glands were not forming part of the ß-catenin helices. DISCUSSION: If these ß-catenin coils are unrelated to an abnormal nuclear distribution at the base of the dysplastic glands, the rational explanation might be that the helices highlight changes taking place in the cytoplasm of affected glandular cells. CONCLUSION: According to some authors, mutations in the ß-catenin genes are always associated with a morphologically neoplastic course. It is herein proposed that ß-catenin helices in duodenal adenomas might uncover a novel cytoplasmic phenomenon ensuing during the adenoma-carcinoma pathway.

ß-catenin helices in the cytoplasm of sessile serrated adenoma/polyps and conventional colorectal adenomas.

Initiation and progression in conventional adenomas is triggered by deregulation of WNT/ß-catenin signaling. In the absence of WNT signal (off-state), ß-catenin prevents phosphorylation of GSK3ß, leading to aberrant nuclear accumulation in human tumors. It has been postulated that mutations in the ß-catenin gene are always associated with a morphologically-neoplastic course. While investigating the nuclear expression of ß-catenin in 170 colorectal biopsies, we observed a non-previously reported phenomenon, namely the presence of ß-catenin cytoplasmic helices in 29% (n=7) of 24 sessile serrated adenoma/polyps (SSA/P), in 24% (n=13) of 54 adenomas, in 8% (n=3) of 38 specimens with IBD, but in none (0/54) with normal mucosa. The earliest ß-catenin helices were found at the bottom of SSA/P glands (the domain of stem cells in the colorectal mucosa). It is submitted that ß-catenin helices might highlight a non-previously described cytoplasmic phenomenon evolving during the serrated-carcinoma pathway in SSA/P, and during the adenoma-carcinoma pathway in conventional adenomas.

Paucity of synaptophysin-expressing cells in Barrett's mucosa.

AIMS: To assess synaptophysin expression in columnar-lined oesophageal mucosa showing either goblet cells, known as intestinal metaplasia, or with accompanying oxyntic glands or pyloric glands. METHODS AND RESULTS: Of 159 biopsies, 53 were oesophageal (19 had intestinal metaplasia, 13 oxyntic glands, and 21 pyloric glands), 77 gastric (12 had goblet cells and 27 no goblet cells) and 29 duodenal. Synaptophysin-positive goblet cells were found in all biopsies from the normal duodenum, in 53% of the oesophageal biopsies showing intestinal metaplasia, but only in 8% of gastric biopsies showing intestinal metaplasia. Synaptophysin-positive Paneth cells occurred in all duodenal biopsies, and in nine of the gastric biopsies showing intestinal metaplasia, but in only one of the oesophageal biopsies showing intestinal metaplasia. A continuous synaptophysin-positive neck cell zone was found in all biopsies from the normal antrum, but in none of the oesophageal biopsies with pyloric glands or with chronic antritis. CONCLUSIONS: The paucity or absence of synaptophysin-positive cells in all three phenotypes of Barrett's mucosa might mirror a sequela of chronic inflammation caused by the particular pathogenic bacteria present in the immediate oesophageal microenvironment.

A method to assess the distribution and frequency of plasma cells and plasma cell precursors in autoimmune hepatitis.

Chronic inflammation exhibiting interface hepatitis and plasma cells in hematoxylin-eosin (H&E)-stained sections is typical of autoimmune hepatitis (AIH), a non-resolving inflammatory liver disease of unidentified cause. Some biopsies may only reveal lymphocytes and occasional granulocytes but no plasma cells. Recent studies on liver biopsies showed that the antibody against multiple myeloma oncogene-1 (MUM1) stained plasma cells (PC), and plasma cell precursors (PCP). Here, liver biopsies from 86 patients were stained with H&E, as well as for MUM1. The portal triad with the highest degree of chronic inflammation (hot-spot PTCI) was chosen for assessing both the topographic distribution and the frequency of MUM1-positive cells. In the 12 untreated AIH cases, MUM1-positive cells were found organized in an irregular ring-like fashion at the peripheral domain of the PTCI, but in none of the three medically-treated AIH cases. Only one out of the remaining 71 liver biopsies exhibited a similar ring-like arrangement, but the PTCI outline was sharp and the number of MUM1-positive cells was low. The highest mean number of MUM1-positive cells at the peripheral domain of the PTCI (59.2 cells) was found in AIH cases (AIH vs. other liver ailments, p<0.05). The highest mean number of MUM1-labelled cells in the core of the PTCI (83.3 cells) was found in PBC cases (PBC vs. other liver ailments p<0.05). Anti-MUM1 permits assessment of qualitative and quantitative PC/PCP changes evolving in autoimmune liver diseases. It is suggested that MUM1 may be of help in the histological differential diagnosis between autoimmune liver diseases and other liver ailments.