JK1 (FAM134B) gene and colorectal cancer : a pilot study on the gene copy number alterations and correlations with clinicopathological parameters

AIMS The aims of the study are to characterize changes in JK-1 (FAM134B) at the DNA level in colorectal adenocarcinoma and adenoma and exploring the possible correlations with clinical and pathological features. METHOD JK-1 gene DNA copy number changes were studied in 211 colorectal carcinomas, 32 colorectal adenoma and 20 colorectal non-cancer colorectal tissue samples by real-time quantitative polymerase chain reaction. The results were correlated with clinical and pathological parameters. RESULTS Colorectal adenomas were more likely to be amplified than deleted with regard to JK-1 (FAM134B) DNA copy number change. The copy number level of JK-1 (FAM134B) DNA in colorectal adenocarcinomas was significantly lower in comparison to colorectal adenomas. Changes in JK-1 (FAM134B) DNA copy number were associated with histological subtypes, and cancer stage. Lower copy numbers were associated with higher tumor stage, lymph node stage and overall pathological stage of cancer. Conversely, higher DNA copy numbers were detected more often in the mucinous adenocarcinoma. CONCLUSIONS This is the first study showing significant correlations of the JK-1 (FAM134B) gene copy number alterations with clinical and pathological features in a large cohort of pre-invasive and invasive colorectal malignancies. The changes in DNA copy number associated with progression of colorectal malignancies reflect that JK-1 (FAM134B) gene could play a role in controlling some steps in development of the invasive phenotypes.

Metachronous carcinomas in colorectum and its clinicopathological significance

PURPOSE The study was designed to examine the significance of colorectal metachronous carcinoma in a large cohort of patients. METHODS Over a mean follow-up period of 10 years, the clinicopathological features, microsatellite instability (MSI) and clinical follow-up of 56 patients with metachronous colorectal carcinoma were analysed. RESULTS The prevalence of metachronous colorectal carcinoma was 2.1 %. The metachronous colorectal carcinomas appeared between 7 and 246 months (mean = 66 months) after surgical resection of the index colorectal carcinomas. Thirty-six per cent (n = 20) of the metachronous carcinoma occurred more than 5 years after the operation of the index carcinoma. Of the 56 patients, 20 % (n = 11) of the metachronous colorectal carcinomas were mucinous adenocarcinoma. Cancers detected in the secondary operations (metachronous colorectal carcinomas), when compared with the primary index cancers, were smaller, showed higher proportions of mucinous adenocarcinoma and more often located in the proximal colon. Patients with metachronous colorectal cancers had higher prevalence of mucinous adenocarcinoma, loss of staining for MSI markers and better survival rates than other patients with colorectal cancers. CONCLUSIONS Patients with metachronous colorectal carcinomas have characteristic features, and attention to these features is important for better management of this group of cancer.

Altered JS-2 expression in colorectal cancers and its clinical pathological relevance

JS-2 is a novel gene located at 5p15.2 and originally detected in primary oesophageal cancer. There is no study on the role of JS-2 in colorectal cancer. The aim of this study is to determine the gene copy number and expression of JS-2 in a large cohort of patients with colorectal tumours and correlate these to the clinicopathological features of the cancer patients. We evaluated the DNA copy number and mRNA expression of JS-2 in 176 colorectal tissues (116 adenocarcinomas, 30 adenomas and 30 non-neoplastic tissues) using real-time polymerase chain reaction. JS-2 expression was also evaluated in two colorectal cancer cell lines and a benign colorectal cell line. JS-2 amplification was noted in 35% of the colorectal adenocarcinomas. Significant differences in relative expression levels for JS-2 mRNA between different colorectal tissues were noted (p = 0.05). Distal colorectal adenocarcinoma had significantly higher copy number than proximal adenocarcinoma (p = 0.005). The relative expression level of JS-2 was different between colonic and rectal adenocarcinoma (p = 0.007). Mucinous adenocarcinoma showed higher JS-2 expression than non-mucinous adenocarcinoma (p = 0.02). Early T-stage cancers appear to have higher JS-2 copy number and lower expression of JS-2 mRNA than later stage cancers (p = 0.001 and 0.03 respectively). Colorectal cancer cell lines showed lower expression of JS-2 than the benign colorectal cell line. JS-2 copy number change and expression were shown for the first time to be altered in the carcinogenesis of colorectal cancer. In addition, genetic alteration of JS-2 was found to be related to location, pathological subtypes and staging of colorectal cancer.

Shared genetics underlying epidemiological association between endometriosis and ovarian cancer

Epidemiological studies have demonstrated associations between endometriosis and certain histotypes of ovarian cancer, including clear cell, low-grade serous and endometrioid carcinomas. We aimed to determine whether the observed associations might be due to shared genetic aetiology. To address this, we used two endometriosis datasets genotyped on common arrays with full-genome coverage (3194 cases and 7060 controls) and a large ovarian cancer dataset genotyped on the customized Illumina Infinium iSelect (iCOGS) arrays (10 065 cases and 21 663 controls). Previous work has suggested that a large number of genetic variants contribute to endometriosis and ovarian cancer (all histotypes combined) susceptibility. Here, using the iCOGS data, we confirmed polygenic architecture for most histotypes of ovarian cancer. This led us to evaluate if the polygenic effects are shared across diseases. We found evidence for shared genetic risks between endometriosis and all histotypes of ovarian cancer, except for the intestinal mucinous type. Clear cell carcinoma showed the strongest genetic correlation with endometriosis (0.51, 95% CI = 0.18–0.84). Endometrioid and low-grade serous carcinomas had similar correlation coefficients (0.48, 95% CI = 0.07–0.89 and 0.40, 95% CI = 0.05–0.75, respectively). High-grade serous carcinoma, which often arises from the fallopian tubes, showed a weaker genetic correlation with endometriosis (0.25, 95% CI = 0.11–0.39), despite the absence of a known epidemiological association. These results suggest that the epidemiological association between endometriosis and ovarian adenocarcinoma may be attributable to shared genetic susceptibility loci.

Defects in tricarboxylic acid cycle enzymes Fumarate hydratase and Succinate dehydrogenase in cancer

Hereditary leiomyomatosis and renal cell cancer (HLRCC) is a recently characterized cancer syndrome which predisposes to cutaneous and uterine leiomyomas as well as renal cell carcinoma (RCC). Uterine leiomyosarcoma (ULMS) has also been observed in certain Finnish HLRCC families. The predisposing gene for this syndrome, fumarate hydratase (FH), was identified in 2002. The well-known function of FH is in the tricarboxylic acid cycle (TCAC) in the energy metabolism of cells. As FH is a novel cancer gene, the role of FH mutations in tumours is in general unknown. Similarly, the mechanisms through which defective FH is associated with tumourigenesis are unclear. The loss of a wild type allele has been observed in virtually all HLRCC patients tumours and the FH enzyme activities are either totally lost or remarkably reduced in the tissues of mutation carrier patients. Therefore, FH is assumed to function as a tumour suppressor. Mutations in genes encoding subunits of other TCAC enzyme SDH have also been reported recently in tumours: mutations in SDHB, SDHC, and SDHD genes predispose to paraganglioma and pheochromocytoma. In the present study, mutations in the SDHB gene were observed to predispose to RCC. This was the first time that mutations in SDHB have been detected in extra-paraganglial tumours. Two different SDHB mutations were observed in two unrelated families. In the first family, the index patient was diagnosed with RCC at the age of 24 years. Additionally, his mother with a paraganglioma (PGL) of the heart and his maternal uncle with lung cancer were both carriers of the mutation. The RCC of the index patient and the PGL of his mother showed LOH. In the other family, an SDHB mutation was detected in two siblings who were both diagnosed with RCC at the ages of 24 and 26 years. Both of the siblings also suffered PGL. All these tumours showed LOH. Therefore, we concluded that mutations in SDHB predispose also for RCC in certain families. Several tumour types were analysed for FH mutations to define the role of FH mutations in these tumour types. In addition, patients with a putative cancer phenotype were analysed to identify new HLRCC families. Three FH variants were detected, of which two were novel. One of the variants was observed in a patient diagnosed with ULMS at the age of 41 years. However, LOH was not detected in the tumour tissue. The FH enzyme activity of the mutated protein was clearly reduced, being 43% of the activity of the normal protein. Together with the results from an earlier study we calculated that the prevalence of FH mutations in Finnish non-syndromic ULMS is around 2.4%. Therefore, FH mutations seem to have a minor role in the pathogenesis on non-syndromic ULMS. Two other germline variants were detected in a novel tumour type, ovarian mucinous cystadenoma. However, tumour tissues of the patients were not available for LOH studies and therefore LOH status remained unclear. Therefore, it is possible that FH mutations predispose also for ovarian tumours but further studies are needed to verify this result. A novel variant form of the FH gene (FHv) was identified and characterized in more detail. FHv contains an alternative first exon (1b), which appeared to function as 5 UTR sequence. The translation of FHv is initiated in vitro from exons two and three. The localization of FHv is both cytosolic and nuclear, in contrast to the localization of FH in mitochondria. FHv is expressed at low levels in all human tissues. Interestingly, the expression was induced after heat shock treatment and in chronic hypoxia. Therefore, FHv might have a role e.g. in the adaptation to unfavourable growth conditions. However, this remains to be elucidated.

Human trypsinogens in the pancreas and in cancer

Human pancreatic juice contains two major trypsinogen isoenzymes called trypsinogen-1 and -2, or cationic and anionic trypsinogen, respectively. Trypsinogen isoenzymes are also expressed in various normal and malignant tissues. We aimed at developing monoclonal antibodies (MAbs) and time-resolved immunofluorometric methods recognizing human trypsinogen-1 and -2, respectively. Using these MAbs and methods we purified, characterized and quantitated trypsinogen isoenzymes in serum samples, ovarian cyst fluids and conditioned cell culture media. In sera from healthy subjects and patients with extrapancreatic disease the concentration of trypsinogen-1 is higher than that of trypsinogen-2. However, in acute pancreatitis we found that the concentration of serum trypsinogen-2 is 50-fold higher than in controls, whereas the difference in trypsinogen-1 concentration is only 15-fold. This suggested that trypsinogen-2 could be used as a diagnostic marker for acute pancreatitis. In human ovarian cyst fluids tumor-associated trypsinogen-2 (TAT-2) is the predominant isoenzyme. Most notably, in mucinous cyst fluids the levels of TAT-2 were higher in borderline and malignant than in benign cases. The increased levels in association with malignancy suggested that TAT could be involved in ovarian tumor dissemination and breakage of tissue barriers. Serum samples from patients who had undergone pancreatoduodenectomy contained trypsinogen-2. Trypsinogen-1 was detected in only one of nine samples. These results suggested that the expression of trypsinogen is not restricted to the pancreas. Determination of the isoenzyme pattern by ion exchange chromatography revealed isoelectric variants of trypsinogen isoenzymes in serum samples. Intact trypsinogen isoenzymes and tryptic and chymotryptic trypsinogen peptides were purified and characterized by mass spectrometry, Western blot analysis and N-terminal sequencing. The results showed that pancreatic trypsinogen-1 and -2 are sulfated at tyrosine 154 (Tyr154), whereas TAT-2 from a colon carcinoma cell line is not. Tyr154 is located within the primary substrate binding pocket of trypsin, thus Tyr154 sulfation is likely to influence substrate binding. The previously known differences in charge, substrate specificity and inhibitor binding between pancreatic and tumor-associated trypsinogens are suggested to be caused by sulfation of Tyr154 in pancreatic trypsinogens.

Dual-colour HER2/chromosome 17 chromogenic in situ hybridisation enables accurate assessment of HER2 genomic status in ovarian tumours

Ovarian cancer is a leading cause of gynaecological cancer-related morbidity and mortality. There has been increasing interest in the potential utility of anti-human epidermal growth factor receptor 2 (anti-HER2) agents in the treatment of this disease, with the attendant need to identify suitable predictive biomarkers of response to treatment.

Le cancer épithélial des ovaires à cellules claires et de type mucineux exprime un niveau élevé de HYAL-1 : corrélation inverse avec l’expression des récepteurs d’estrogène et de progestérone

Le cancer épithélial des ovaires (CEO) est classifié en sous types histopathologiques identifiés tel que séreux, endométrioide, à cellules claires et mucineux. Une analyse génétique réalisée au niveau moléculaire a suggéré un rôle pour des gènes suppresseurs de tumeur localisés sur le bras court du chromosome 3p21.3 dans la pathogénèse du CEO de type séreux. Notre objectif était d’évaluer le profil d’expression de HYAL-1, localisé dans cette même région, dans les différents sous types du CEO, et de vérifier une éventuelle corrélation avec l’expression des récepteurs d’hormones stéroïdiennes. Pour se faire, nous avons analysé par RT-PCR quantitative l’expression de l’ARNm de HYAL-1, des récepteurs d’estrogène (ER-α et ER-β) et du récepteur de progestérone (PR) dans des échantillons de tissus extraits de tumeurs du CEO provenant de deux cohortes indépendantes et dans des lignées cellulaires. Nous avons également réalisé des analyses bioinformatiques à partir de l’expression de ces gènes en ayant recours à une base de données de microarray disponible en ligne et ouverte au public. Par la suite, nous avons mesuré l’activité enzymatique de HYAL-1 dans des lignées cellulaires du CEO et dans des échantillons de plasma. Nos résultats ont montré que l’expression de l’ARNm de HYAL-1 était élevée dans le type à cellules claires et mucineux mais non dans les types séreux et endométrioides, autant dans les échantillons sains que de ceux provenant de tumeurs bénignes. De façon cohérente, le niveau d’ARNm et l’activité enzymatique de HYAL-1 étaient élevés dans les lignées cellulaires à cellules claires et mucineuses. Nous avons aussi démontré qu’il y avait une corrélation inverse entre les niveaux de l’ARNm de HYAL-1 et ceux d’ER-α et PR dans les échantillons de tissus de CEO du type mucineux et à cellules claires. De façon similaire, nous avons noté que l’activité de HYAL-1 était élevée dans le plasma de ces mêmes patients. En conséquence nos travaux proposent HYAL-1 en tant que biomarqueur potentiel dans le cas des CEO de type à cellules claires et mucineux présentant un faible niveau d’expression d’ER-α et PR.

Régulation de l’expression de HYAL-1 par le récepteur de l’oestrogène alpha

HYAL-1 (hyaluronidase-1) appartient à la famille des hyaluronidases connues pour leur rôle dans la dégradation de l’acide hyaluronique. L’expression de HYAL-1 est élevée dans de nombreux type de cancers, notamment dans le cancer de la prostate, de la vessie, des reins et du sein où il est impliqué dans la croissance tumorale et les métastases. Récemment notre laboratoire a aussi démontré une expression élevée de HYAL-1 dans le cancer épithélial de l’ovaire (CEO) de type mucineux et à cellules claires, expression qui est inversement corrélée à celle du récepteur de l’oestrogène alpha (REα). Cependant, malgré le fait que le rôle de HYAL-1 dans le cancer soit bien établit, le mécanisme de sa régulation reste encore inconnu. Le REα est un facteur de transcription qui suite à sa liaison avec son ligand va réguler l’expression de plusieurs gènes. Le REα ainsi stimulé par l’hormone va activer la transcription de ces gènes cibles mais il est connu maintenant qu’une grande partie des gènes régulés par le REα sont en réalité réprimés par ce récepteur. Dans ce travail nous proposons d’étudier le mécanisme de la régulation du gène HYAL-1 par le REα dans le CEO à cellules claires et dans le cancer du sein. L’expression ectopique du REα dans la lignée TOV21G (RE-) de même que le traitement de la lignée MCF-7 (RE+) avec de l’oestrogène a induit une diminution du niveau d’expression de l’ARN m de HYAL-1. Ces résultats nous ont permis de confirmer que HYAL-1 est un gène cible du REα. Il est aussi connu que le REα peut exercer son action par différents mécanismes d’action, entre autres en interagissant avec une séquence d’ADN appelée élément de réponse à l’oestrogène (ERE), retrouvé sur le promoteur des gènes cibles ou bien indirectement par des interactions protéine-protéine en se liant à d’autres facteur de transcription tels que Sp1. Après avoir identifiés de telles séquences sur le promoteur proximal de HYAL-1, (1 ERE proximal à -900 pb, 3 distaux à -32350 pb, 48430, -50130 pb du site d’initiation de la transcription) en plus des 2 Sp1 connus (-60 et – 1020pb), nous avons démontrés par immunoprécipitation de la chromatine que le REα est recruté sur le promoteur de HYAL-1 au niveau de l’ERE proximal -900 pb et du distal -32350 pb de même que sur le site Sp1 -1020 pb. De plus, l’activité biologique de l’ERE -900 pb et du ii Sp1-1020pb à été confirmée par des essais de gènes rapporteurs à la luciférase. Avec son rôle connu dans la tumorigenèse, l’identification de HYAL-1 comme gène cible du REα pourrait être une avenue intéressante pour le traitement des cancers hormono-indépendants.

CDX2, a highly sensitive and specific marker of adenocarcinomas of intestinal origin - An immunohistochemical survey of 476 primary and metastatic carcinomas

CDX2 is a recently cloned homeobox gene that encodes an intestine-specific transcription factor, expressed in the nuclei of epithelial cells throughout the intestine, from duodenum to rectum. While expression of CDX2 protein in primary and metastatic colorectal carcinomas has been previously documented, neither the sensitivity nor the specificity of CDX2 expression, as determined by immunohistochemistry, for colorectal adenocarcinoma has been determined. We performed an immunohistochemical survey of 476 tumors with a monoclonal antibody, CDX2-88, including 89 tumors from the colon and duodenum and 95 tumors from other gastrointestinal sites, including the esophagus, stomach, pancreatobiliary system, gastrointestinal carcinoids, and liver. CDX2 was expressed uniformly (that is, in 76-100% of tumor cells) in all but one of the evaluated colorectal and duodenal tumors. High-level expression of CDX2 was also found, however, in mucinous ovarian carcinomas and adenocarcinomas primary to the urinary bladder of which 64% and 100% were positive, respectively. Gastric, gastroesophageal, and pancreatic adenocarcinomas and cholangiocarcinomas all showed similar, heterogeneous patterns of CDX2 expression. Most tumors in each group showed CDX2 expression by a minority of cells, whereas a substantial minority of cases in each group was completely negative and a smaller minority was uniformly positive. Gastrointestinal carcinoids gave similarly varied results, but the majority (58%) was negative. Hepatocellular carcinomas showed no expression of CDX2. Only very rare examples of carcinomas of the genitourinary and gynecologic tracts, breast, lung, and head and neck showed significant levels of CDX2 expression. In this study of primary and metastatic epithelial tumors, uniform CDX2 expression is demonstrated to be an exquisitely sensitive and highly, but incompletely, specific marker of intestinal adenocarcinomas. Compared with villin, a previously described marker of GI adenocarcinomas, CDX2 demonstrated superior sensitivity and comparable specificity. CDX2 expression can be seen, however, in selected non-GI adenocarcinomas such as mucinous ovarian carcinomas and adenocarcinomas of the urinary bladder.

Cistoadenoma mucinoso do apêndice: relato de caso

O cistoadenoma mucinoso do apêndice ceca l, um tipo de mucocele do apêndice, é uma neoplasia benigna caracterizada pela metaplasia focal ou difusa do epitélio superficial da mucosa, associada à dilatação do apêndice e produção de muco para a luz apendicular. No presente estudo os autores relatam um caso de cistoadenoma mucinoso do apêndice observado em uma mulher de 65 anos de idade, com dor e massa palpável no quadrante inferior direito do abdome, cujo quadro teve início há 1 ano. O diagnóstico de cistoadenoma do apêndice foi suspeitado através da ultrassonografia e tomografia computadorizada do abdome, e confirmado pelo exame histopatológico. A paciente também apresentava cálculos na vesícula biliar e um cisto renal à direita. A laparotomia foi indicada sendo realizada a apendicectomia, a colecistectomia e a exérese do cisto renal. Na presente discussão esperamos contribuir para o diagnóstico precoce dessa doença e a melhor compreensão de sua classificação e patogenia.

Late administration of a specific COX-2 inhibitor does not treat and/or prevent progression of gastric tumors in rats submitted to duodenogastric reflux procedure

PURPOSE:To assess whether late introduction of a specific COX-2 inhibitor (Meloxicam) can treat and/or prevent the progression of tumors in the stomach of rats submitted to duodenogastric reflux. METHODS: Seventy five male Wistar rats, weighing 150 grams, were submitted to the induction of duodenogastric reflux through the pylorus. At 36 weeks of follow-up were established three experimental groups: DGR36 sacrificed immediately, DGR54 and DGR54MLX both sacrificed at 54th week of follow-up . The animals of the latter group were fed with a rat chow premixed with Meloxicam (2.0 mg/ kg feed; 0.3 mg / kg bw / day) and the other two with standard rat chow. The lesions found in the pyloric mucosa and gastrojejunal anastomosis were analyzed macroscopically and histologically. For statistical analysis was adjusted a generalized linear model assuming a binomial distribution with LOGIT link function. RESULTS: No significant differences were found when comparing the incidences of benign tumor lesions (Adenomatous Hyperplasia), p=0.4915, or malignant (Mucinous Adenocarcinoma), p=0.2731, among groups. CONCLUSION: Late introduction of specific COX-2 inhibitor (Meloxicam) did not treat and was not able to prevent the progression of tumoral lesions induced by duodenogastric reflux in the rat stomachs.

REVERSIBILITY OF ADENOMATOUS HYPERPLASIA IN THE GASTRIC STUMP AFTER DIVERSION OF BILE REFLUX IN RATS

Morphological and phenotypical patterns of proliferative epithelial lesions induced in the gastric stump mucosa by duodenal content reflux after Billroth II partial gastrectomy (BII) were evaluated in rats. Control animals were either sham-operated or submitted at different times after BII to Roux-en-Y (RY) surgical procedure which prevents duodenal reflux. The lesions were analysed using routine haematoxylin and eosin staining, immunohistochemical staining for pepsinogen isoenzyme 1 and histochemical procedures for mucins (paradoxical Concanavalin A, galactose oxidase Schiff and sialidase galactose oxidase Schiff reactions). Mucosal hyperplasia (H) was observed in the group submitted to BII procedure 6 weeks after surgery. Adenomatous hyperplasia (AH) also appeared 6 weeks after induction of the reflux and its incidence and size increased until the 54th week of the experiment. RY procedure performed in the normal animals at the beginning of the experiment or at the 24th week after BII gastrectomy led to a significantly lower incidence of AH which was related to the moment of surgery. Most of H was due to pyloric mucosal hyperplasia. AH consisted mainly of gastric type glands but in some animals glands of the intestinal type Were present probably originating from the intestinal mucosa. Six mucinous adenocarcinomas were observed, all of them of intestinal type. This study demonstrates that AH induced by BII procedure is a reversible lesion and that the anomalous epithelial proliferation in the stoma may lead to adenocarcinomas.

COORDINATE EXPRESSION OF CYTOKERATIN-7 AND CYTOKERATIN-20 DEFINES UNIQUE SUBSETS OF CARCINOMAS

We tested the hypothesis that the coordinate expression of cytokeratin 7 (CK 7) and cytokeratin 20 (CK 20) could distinguish among carcinomas arising from different primary sites. A total of 384 cases of carcinomas primary to various organs, as well as 16 cases of malignant mesothelioma, were evaluated using commercially available monoclonal antibodies and an avidin-biotin immunoperoxidase technique. The subset of tumors strongly expressing both CK 7 and CK 20 included virtually all bladder transitional cell carcinomas and the majority of pancreatic adenocarcinomas; the tumors negative for both CK 7 and CK 20 were largely restricted to hepatocellular, prostate, and renal cell carcinomas in addition to squamous cell and neuroendocrine carcinomas of lung. The CK 7-/CK 20+ immunophenotype, however, was highly characteristic of adenocarcinomas of colorectal origin, whereas CK 7+/CK 20- immunophenotype was typically seen in the vast majority of carcinomas arising from other sites, including ovary, endometrium, breast, and lung, as well as malignant mesothelioma. Gastric carcinomas were the most heterogeneous subgroup with respect to CK 7/CK 20 immunophenotype. In the subset of mucinous tumors, striking immunophenotypic differences were noted among those primary to the breast (CK 7+/CK 20-), gastrointestinal tract (CK 7-/CK 20+), and ovary (CK 7+/CK 20+). In all cases investigated, this CK immunophenotype was invariant in metastatic vs. primary tumors. It is concluded that, in the appropriate clinical setting, the CK 7/CK 20 immunophenotype of carcinomas is a valuable diagnostic marker in the determination of primary site of origin.

Reappraisal of immunohistochemical profiling of special histological types of breast carcinomas: a study of 121 cases of eight different subtypes

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)