Interobserver variation in the classification of tumor deposits in rectal cancer-is the use of histopathological characteristics the way to go?

The focus on lymph node metastases (LNM) as the most important prognostic marker in colorectal cancer (CRC) has been challenged by the finding that other types of locoregional spread, including tumor deposits (TDs), extramural venous invasion (EMVI), and perineural invasion (PNI), also have significant impact. However, there are concerns about interobserver variation when differentiating between these features. Therefore, this study analyzed interobserver agreement between pathologists when assessing routine tumor nodules based on TNM 8. Electronic slides of 50 tumor nodules that were not treated with neoadjuvant therapy were reviewed by 8 gastrointestinal pathologists. They were asked to classify each nodule as TD, LNM, EMVI, or PNI, and to list which histological discriminatory features were present. There was overall agreement of 73.5% (κ 0.38, 95%-CI 0.33-0.43) if a nodal versus non-nodal classification was used, and 52.2% (κ 0.27, 95%-CI 0.23-0.31) if EMVI and PNI were classified separately. The interobserver agreement varied significantly between discriminatory features from κ 0.64 (95%-CI 0.58-0.70) for roundness to κ 0.26 (95%-CI 0.12-0.41) for a lone arteriole sign, and the presence of discriminatory features did not always correlate with the final classification. Since extranodal pathways of spread are prognostically relevant, classification of tumor nodules is important. There is currently no evidence for the prognostic relevance of the origin of TD, and although some histopathological characteristics showed good interobserver agreement, these are often non-specific. To optimize interobserver agreement, we recommend a binary classification of nodal versus extranodal tumor nodules which is based on prognostic evidence and yields good overall agreement.

Outcome reporting in neoadjuvant surgical trials: a systematic review of the literature and proposals for new standards.

BACKGROUND: The use of neoadjuvant therapy before surgery for gastrointestinal cancer is increasing; however, patients may not complete both treatment components. Understanding completion rates of each treatment stage is necessary for treatment evaluation and to inform decision-making. This study evaluates reporting for recent neoadjuvant surgical trials, focusing on treatment progression and other key outcomes. METHODS: Systematic literature searches identified randomized and nonrandomized phase II and III studies evaluating neoadjuvant treatment and surgery for esophageal, stomach, and colorectal cancer, and colorectal liver metastases. Rates of reporting of failure to complete neoadjuvant treatment, nonprogression to surgery after neoadjuvant treatment, and nonresection at planned surgery were assessed. For each measure, reporting was categorized as "full," "partial," and "absent" according to predefined criteria, and reasons for nonprogression at each stage of treatment were examined to inform proposed standards. RESULTS: Of 9854 abstracts, 123 papers were reviewed and 62 articles were included, reporting outcomes for 9126 patients. Details of noncompletion of neoadjuvant treatment and nonprogression to surgery were completely absent in 21 (33.9%) and 19 (30.6%) studies, respectively. Reporting of nonresection at planned surgery was also deficient, with 21 (33.9%) studies providing no information about this outcome. Reasons for noncompletion and nonprogression were similar and included disease progression, treatment toxicity, and patient choice. Common reasons for nonresection were locally advanced disease and the discovery of unsuspected metastases. CONCLUSIONS: Reports of recent neoadjuvant surgical trials often fail to include treatment progression and other key outcomes. These findings support the need for minimum reporting standards.

Clinicopathological and molecular study of penile melanoma.

AIMS: To examine the clinicopathological features of a series of penile melanomas and screen for mutations in the BRAF and KIT genes, which are seen in melanomas from other sites. METHODS AND RESULTS: 12 patients with penile melanoma were identified over a 10-year period in two supra-regional networks in the UK. The 2- and 5-year survival was 61% and 20%, respectively. Half the patients had lymph node involvement at presentation; this was a poor prognostic indicator. KIT exons 11, 13, 17 and 18, and BRAF codons 600 and 601 were analysed for mutations by Sanger sequencing and pyrosequencing, respectively. None of the tumours showed either KIT mutations or the BRAF V600E mutation. CONCLUSION: Penile melanomas are extremely rare and have a similar prognosis to melanomas elsewhere, but they often present late, leading to a poor outcome. The mutations seen in melanomas from other sites appear to be rarely present in these tumours.

Cutaneous metastasis from gastric glomus tumour.

A 58-year-old man presented with a 4-month history of a rapidly enlarging asymptomatic tumour on the scalp vertex. Six years earlier, the patient had undergone subtotal gastrectomy for a presumed gastrointestinal stromal tumour (GIST). The tumour extended to the peritoneal surfaces, and foci of vascular invasion were also seen. No adjuvant treatment was given. Owing to diagnostic difficulties with the scalp tumour, a comparison was made with the previous gastric neoplasm. Both had features consistent with glomus tumour, which suggested a revised diagnosis of primary malignant gastric glomus tumour with subsequent cutaneous metastasis to the scalp some years later. This case highlights the features of glomus tumours and is a reminder that although rare, glomus tumours can be malignant and have the potential to metastasize. To our knowledge, this is the only reported case of a primary gastric glomus tumour with secondary metastasis to the skin.

Subepithelial apoptosis in normal caecal biopsy specimens is primarily an age-related phenomenon.

AIMS: To determine whether prominent subepithelial apoptosis in otherwise normal caecal endoscopic biopsy specimens has any clinical significance. METHODS AND RESULTS: Calculating the average number of subepithelial apoptotic bodies per intercrypt space [i.e. a subepithelial apoptotic index (SAI)] was found to reflect accurately and reproducibly the density of subepithelial apoptosis in a colonic biopsy specimen. The SAIs of 124 histologically normal caecal biopsy specimens from adult patients showed a positively skewed normal distribution without bimodality. The clinical details of patients with specimens in the top quartile of this distribution were compared with those of patients in the bottom quartile. Patients in the top quartile were found to be older and more likely to suffer with ulcerative colitis than those in the bottom quartile. However, controlling for age removed the association between SAI and ulcerative colitis. The SAIs of 144 caecal biopsy specimens (including 20 paediatric cases) showed a positive correlation with patient age. CONCLUSIONS: Subepithelial apoptosis in normal caecal biopsy specimens appears to be an age-related phenomenon with no demonstrable clinical significance.

KIT and PDGFRalpha mutational analyses of mixed cell-type gastrointestinal stromal tumours.

AIMS: To determine whether the epithelioid and spindle components of a mixed cell-type gastrointestinal stromal tumour (GIST) show the same receptor tyrosine kinase mutation and, by inference, the same sensitivity to imatinib. METHODS AND RESULTS: Six mixed cell-type GISTs were identified from 108 gastric GISTs. Clinicopathological and immunohistochemical data of the six neoplasms were collated. For each neoplasm, DNA was extracted separately from the laser-microdissected epithelioid and spindle components and non-neoplastic tissue and sequenced for KIT and platelet-derived growth factor receptor (PDGFR)alpha mutations. The epithelioid component often showed less CD117 and/or CD34 immunoreactivity than the spindle component of the same mixed cell-type GIST. Four mixed cell-type GISTs showed somatic KIT mutations (deletions in exon 11 in three tumours and an insertion in exon 9 in one tumour) and one showed a somatic PDGFRalpha mutation (point mutation in exon 18); in each of the five cases, both epithelioid and spindle components showed identical mutations. CONCLUSIONS: The presence of the same receptor tyrosine kinase mutation in both components of a mixed cell-type GIST suggests that both components should be equally responsive to imatinib treatment, and that such mutation is an early key event in the pathogenesis of these neoplasms.

EpCAM and gpA33 are markers of Barrett's metaplasia.

AIMS: To characterise a specific and sensitive marker of Barrett's metaplasia (BM). METHODS: Cases of normal oesophageal squamous mucosa (11 fresh endoscopic biopsies and 10 formalin fixed, paraffin embedded tissue blocks), BM (11 biopsies and 11 tissue blocks), and normal gastric body mucosa (five biopsies and five tissue blocks) were analysed using reverse transcriptase PCR, Western blotting, and immunohistochemistry for EpCAM, and reverse transcriptase PCR for gpA33. RESULTS: Strong EpCAM mRNA expression was detected in all the BM cases, in contrast to weak expression in all the normal gastric mucosal samples and no expression in any of the normal oesophageal mucosal samples tested. Strong gpA33 mRNA expression was detected in all the BM cases, in contrast to weak expression in a quarter of the normal gastric mucosal samples and no expression in any of the normal oesophageal mucosal samples tested. Western blotting showed EpCAM protein expression in all the BM cases and in none of the normal gastric or oesophageal mucosal samples tested. Immunohistochemistry showed strong EpCAM protein expression in BM and complete absence of expression in normal oesophageal squamous epithelium. Scattered EpCAM expressing cells were found in the gland bases of normal gastric body mucosa. CONCLUSIONS: EpCAM protein and gpA33 mRNA expressions are specific and sensitive markers of BM.

CDX1 is an important molecular mediator of Barrett's metaplasia.

The molecular pathogenesis of Barrett's metaplasia (BM) of the esophagus is poorly understood. The change to an intestinal phenotype occurs on a background of esophagitis due to refluxing acid and bile. CDX1, an important regulator of normal intestinal development, was studied as a potential key molecule in the pathogenesis of BM. CDX1 mRNA and protein were universally expressed in all samples of BM tested but not in normal esophageal squamous or gastric body epithelia. This tissue-specific expression was attributable to the methylation status of the CDX1 promoter. Conjugated bile salts and the inflammatory cytokines TNF-alpha and IL-1beta were all found to increase CDX1 mRNA expression in vitro. These effects were primarily mediated by NF-kappaB signaling but only occurred when the CDX1 promoter was unmethylated or partially methylated. The data suggest that CDX1 is a key molecule linking etiological agents of BM to the development of an intestinal phenotype. Although the initial trigger for CDX1 promoter demethylation is not yet identified, it seems likely that demethylation of its promoter may be the key to the induction and maintenance of CDX1 expression and so of the BM phenotype.

Loss of CDX1 expression in colorectal carcinoma: promoter methylation, mutation, and loss of heterozygosity analyses of 37 cell lines.

Expression of the homeobox protein CDX1 is lost or reduced in a significant proportion of colorectal carcinomas (CRCs) but the underlying mechanism for this is unclear. We have demonstrated absence of CDX1 mRNA expression in 7 of 37 CRC cell lines and shown that all 7 cell lines have a methylated CDX1 promoter. Twenty-five cell lines showed both CDX1 mRNA expression and an unmethylated CDX1 promoter. The five remaining cell lines had a partially methylated CDX1 promoter and all expressed CDX1 mRNA; when treated with the demethylating agent, 5-aza-2'-deoxycytidine, these five cell lines all showed increased CDX1 expression. No mutations were found in the promoter and coding regions of CDX1. One polymorphism was demonstrated in each of the promoter, 5' UTR, and coding region of exon 1 of CDX1, but there were no associations between CDX1 mRNA expression and different polymorphic genotypes. Similarly, there was no association between CDX1 mRNA expression and loss of heterozygosity at the CDX1 locus. In conclusion, absence or reduction of CDX1 expression in CRC seems to be primarily regulated by promoter methylation and is probably selected for because of its impact on the differentiation of colonocytes.

SV40 Tag DNA sequences, present in a small proportion of human hepatocellular carcinomas, are associated with reduced survival.

AIMS: To study the association between simian virus 40 (SV40) and human hepatocarcinogenesis. METHODS: Polymerase chain reaction (PCR) to detect SV40 large T antigen (Tag) DNA was performed on: 50 human hepatocellular carcinoma (HCCs) diagnosed between 1978 and 1989 (cohort A); 20 cases of alcoholic liver cirrhosis from the same period; and 20 HCCs diagnosed after 1997 (cohort B). PCR to detect SV40 regulatory sequence and SV40 Tag immunohistochemistry were performed on selected cases from cohorts A and B. Amplified products were directly sequenced. Immunohistochemistry for p53 and pRb and clinicopathological analyses were performed on selected cases from cohorts A and B. Complete survival data were collected for cohort A. RESULT: SV40 Tag DNA was found in five cohort A HCCs but not in alcoholic liver cirrhosis cases or cohort B HCCs. Neither SV40 regulatory sequence nor SV40 Tag protein were demonstrated in Tag DNA positive HCCs. No clinicopathological differences existed between Tag DNA positive and negative HCCs, but the presence of Tag DNA was associated with reduced disease specific survival. Relatively fewer Tag DNA positive than negative HCCs expressed p53, but loss of pRb expression was similar in the two groups. Patients with Tag DNA positive HCCs were unlikely to have received SV40 contaminated poliovirus vaccine. CONCLUSIONS: SV40 Tag DNA is present in a small proportion of historical HCCs and may contribute to their pathogenesis and influence their outcome. The source of the virus is uncertain and more recent HCCs show no evidence of SV40.

Epstein-Barr virus infection in colorectal neoplasms associated with inflammatory bowel disease: detection of the virus in lymphomas but not in adenocarcinomas.

Epstein-Barr virus (EBV) is associated with several lymphoid and epithelial human malignancies. The latter include gastric adenocarcinomas, while sporadic colorectal adenocarcinomas (CRCs) have been reported to be EBV-negative. Recently, increased numbers of EBV-infected B lymphocytes have been detected in intestinal mucosal samples affected by ulcerative colitis (UC) and, to a lesser extent, Crohn's disease (CD). Both CRC and colorectal non-Hodgkin's lymphoma (NHL) are recognized complications of inflammatory bowel disease (IBD), but it is unclear to what extent EBV contributes to the development of these neoplasms. Seventeen cases of IBD-associated CRC and nine cases of IBD-associated colorectal NHL were therefore studied for the presence of EBV by in situ hybridization. EBV-positive cases were further studied for the expression of the EBV-encoded nuclear antigen (EBNA) 2 and the latent membrane protein (LMP) 1 of EBV by immunohistochemistry. Four out of seven cases of colorectal NHL associated with UC were shown to be EBV-positive. In addition, two of two colorectal NHLs developing in patients with CD were EBV-positive. Of the EBV-positive lymphomas, three displayed a pattern of EBV latent gene expression consistent with type I latency (EBNA2(-)/LMP1(-)), two a type II pattern (EBNA2(-)/LMP1(+)), and one a type III pattern (EBNA2(+)/LMP1(+)). These findings suggest that EBV infection is involved in the pathogenesis of a proportion of colorectal NHLs developing in IBD. Iatrogenic immunosuppression may contribute to the development of these lymphomas. By contrast, all 17 IBD-associated CRCs were EBV-negative, including a case of CRC occurring synchronously with an EBV-positive NHL. In conjunction with previous reports on sporadic CRCs, this suggests that EBV is not involved in the pathogenesis of CRC.

Prognostic indicators for gastrointestinal stromal tumours: a clinicopathological and immunohistochemical study of 108 resected cases of the stomach.

AIMS: Whether immunohistochemical markers increase accuracy in predicting prognosis for gastrointestinal stromal tumours (GISTs) remains uncertain. However, past studies have used only small, heterogeneous patient groups. Our aim was to test previously studied and more novel morphological features as well as four immunohistochemical markers as prognostic indicators amongst a large cohort of surgically resected, gastric GISTs. METHODS AND RESULTS: Tissues from 127 gastric mesenchymal tumours were collected retrospectively and subjected to repeat histological assessment and immunophenotyping. Further immunohistochemistry was performed for Ki67, p53, Bcl-2 and cyclin D1. Complete follow-up data were collected for 108 patients with immunophenotyped diagnoses of GIST (i.e. c-kit+ tumours). At the census point, 52 patients were alive, 24 had died from their GISTs and the remainder of other causes. Univariate analysis showed the following predicted for shorter disease-specific survival: size > or =50 mm; necrosis, no intratumoral lymphocytes; mitotic count > or =5/50 high power fields; Ki67 labelling index > or =5%; p53 immunopositivity. Of these variables, multivariate analyses showed only mitotic count and, to a lesser extent, Ki67 labelling to be independent prognostic indicators. CONCLUSIONS: Mitotic count remains the best predictor of outcome following surgical resection of gastric GISTs. Ki67 immunohistochemistry does not provide better prognostication and p53, Bcl-2 and cyclin D1 immunohistochemistry provide no additional prognostication.

An immunohistochemical study and review of potential markers of human intestinal M cells.

M cells are found in intestinal follicle associated epithelium. Studies into the physiological and pathological roles of human M cells have been hampered by the lack of well-substantiated, specific markers for these cells. A critical literature review suggests the following molecules may potentially serve as such markers: CK7, FcaR (CD89), S100, CD1a, CD21, CD23, sialyl Lewis A, and cathepsin E. Normal ileum, appendix and colorectum were studied using paraffin-embedded, formalin-fixed tissue and immunohistochemistry for these 8 markers. Cathepsin E immunohistochemistry was also performed on cases of colorectal adenocarcinoma, colorectal adenoma, colorectal hyperplastic/metaplastic polyp, lymphocytic colitis, collagenous colitis, pseudomembranous colitis and active ulcerative colitis. Of the 8 markers tested, only cathepsin E appeared to be specific to follicle associated epithelium (expressed by cells with and without M cell morphology) and follicular crypt epithelium; this specificity was limited to the colorectum. Focal epithelial expression of cathepsin E was seen in adenocarcinoma, adenoma, hyperplastic/metaplastic polyp, ulcerative colitis and pseudomembranous colitis. In conclusion, cathepsin E is a specific marker of normal colorectal follicle associated epithelium and follicular crypt epithelium though is not specific to M cells within these compartments. None of the other 7 markers studied is exclusively expressed by human M cells.

Cyclin D1 overexpression in colorectal carcinoma in vivo is dependent on beta-catenin protein dysregulation, but not k-ras mutation.

Cyclin D1 protein overexpression is commonly found in colorectal carcinomas (CRCs) and is associated with a poorer prognosis, but the mechanism underlying overexpression remains uncertain. Both dysregulation of beta-catenin protein expression and k-ras mutation have recently been shown to promote cyclin D1 expression in human in vitro and rodent in vivo studies. In this study, 53 sporadic CRCs were examined by immunohistochemistry for cyclin D1 and beta-catenin protein expression, and with PCR and direct DNA sequencing for k-ras gene status. The study also addressed whether cyclin Dl overexpression might associate with poorer prognosis because of a relationship with poorer response to 5-fluorouracil (5FU) chemotherapy. Cyclin D1 overexpression was demonstrated in 34/53 (64%) CRCs, was significantly associated with higher Dukes' stage, and was particularly prominent at the invasive edges of carcinomas. Furthermore, cyclin D1 overexpression was always and only seen in association with nuclear expression of beta-catenin. There were no significant associations between cyclin D1 overexpression and k-ras mutation or response to 5FU. Amongst 17 microsatellite unstable CRCs, a smaller proportion of tumours showed cyclin D1 overexpression (18%), but again cyclin D1 overexpression was only seen in cases showing nuclear beta-catenin expression. In conclusion, beta-catenin protein dysregulation, but not k-ras mutation, appears to be required for cyclin D1 overexpression in colorectal carcinoma in vivo.

Nuclear thymidylate synthase expression, p53 expression and 5FU response in colorectal carcinoma.

Thymidylate synthase (TS) is a key enzyme in DNA synthesis and is inhibited by metabolites of the chemotherapeutic agent 5-fluorouracil (5FU). Nuclear expression of TS in human tissue in vivo has not been characterised and its clinicopathological correlates in malignancy are unknown. 52 cases of primary colorectal carcinoma (CRC) and 24 cases of matched metastatic carcinoma were studied immunohistochemically using the monoclonal antibody TS106. The degree of nuclear TS immunostaining correlated closely with levels of TS mRNA expression amongst 10 CRCs studied. Strong nuclear immunostaining was seen in normal basal crypt colonocytes and germinal centre cells, and in a varying proportion of adenocarcinoma cells. Amongst the primary carcinomas, higher TS nuclear expression was associated with prominent extracellular mucin production and right-sided location. Higher TS nuclear expression also showed a significant association with poorer response to protracted venous infusional 5FU therapy. There was no clear association between TS nuclear expression and Ki67 or p53 expression assessed immunohistochemically. There was a strong positive correlation between TS nuclear expression in primary and metastatic CRC but the latter generally showed higher expression than matched primary tumour tissue. These findings confirm the nuclear expression of TS protein in human cells in vivo and provide new insight into how such expression may relate to the behaviour of CRCs.