DNA methylation in breast cancers: Differences based on estrogen receptor status and recurrence.

DNA methylation plays a role in the etiology of primary breast cancers. We analyzed paired primary and second breast tumors to elucidate the role of methylation in recurrence. Methylation profiles from paired primary and second breast tumors of 23 women were assessed using the HumanMethylation450 BeadChip. Twelve women had estrogen receptor positive (ERpos) primary and second tumors, five had estrogen receptor negative (ERneg) primary and second tumors, and six had an ERpos primary tumor but an ERneg second tumor. Stratifying tumors by occurrence revealed that the greater methylation previously associated with ERpos tumors, is more pronounced in primary tumors than in second tumors. Further, ERneg second tumors are more methylated than ERpos second tumors among women who had ERpos primary tumors. Pathway analyses using gene lists generated from comparisons of methylation in ERpos primary tumors from the paired sets with ERpos tumors from six women without recurrences, identified differences between groups based on the ER status of the second tumor. Hypermethylated genes of significantly enriched pathways were differentially associated with survival. DNA methylation profiles of ERpos primary breast tumors support the development and use of tumor methylation profiles for stratifying women with breast cancer both for prognosis and therapy.

TROP2 methylation and expression in tamoxifen-resistant breast cancer.

BACKGROUND: The DNA methyltransferase 1 inhibitor, 5-Aza-2'-deoxycytidine (5-Aza-dC) is a potential treatment for breast cancer. However, not all breast tumors will respond similarly to treatment with 5-Aza-dC, and little is known regarding the response of hormone-resistant breast cancers to 5-Aza-dC. METHODS: We demonstrate that 5-Aza-dC-treatment has a stronger effect on an estrogen receptor-negative, Tamoxifen-selected cell line, TMX2-28, than on the estrogen receptor-positive, MCF7, parental cell line. Using data obtained from the HM450 Methylation Bead Chip, pyrosequencing, and RT-qPCR, we identified a panel of genes that are silenced by promoter methylation in TMX2-28 and re-expressed after treatment with 5-Aza-dC. RESULTS: One of the genes identified, tumor associated calcium signal transducer 2 (TACSTD2), is altered by DNA methylation, and there is evidence that in some cancers decreased expression may result in greater proliferation. Analysis of DNA methylation of TACSTD2 and protein expression of its product, trophoblast antigen protein 2 (TROP2), was extended to a panel of primary (n = 34) and recurrent (n = 34) breast tumors. Stratifying tumors by both recurrence and ER status showed no significant relationship between TROP2 levels and TACSTD2 methylation. Knocking down TACSTD2 expression in MCF7 increased proliferation however; re-expressing TACSTD2 in TMX2-28 did not inhibit proliferation, indicating that TACSTD2 re-expression alone was insufficient to explain the decreased proliferation observed after treatment with 5-Aza-dC. CONCLUSIONS: These results illustrate the complexity of the TROP2 signaling network. However, TROP2 may be a valid therapeutic target for some cancers. Further studies are needed to identify biomarkers that indicate how TROP2 signaling affects tumor growth and whether targeting TROP2 would be beneficial to the patient.

ELF5 and DOK7 regulation in anti-estrogen treated cells and tumors.

BACKGROUND: Most women with primary breast cancers that express estrogen receptor alpha (ER or ESR1) are treated with endocrine therapies including the anti-estrogen tamoxifen, but resistance to these anti-endocrine therapies often develops. This study characterizes the expression of hormone receptors, and the mRNA and DNA methylation levels of docking protein 7 (DOK7), and E74-like factor 5 (ELF5), in 21 novel tamoxifen-resistant cell lines and extends the findings to primary and recurrent human breast tumors. METHODS: Twenty-one tamoxifen-selected cell lines were developed through cloning by limiting dilution of an MCF-7 cell culture treated with 1 µM tamoxifen for 6 months. The parent (MCF-7) and tamoxifen-selected cell lines were characterized for protein expression of ER, progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) using immunohistochemistry (IHC). The mRNA levels of ER, DOK7, and ELF5 were assessed using quantitative RT-PCR. Promoter methylation levels of DOK7 and ELF5 were determined by pyrosequencing of bisulfite-modified DNA. The relationship between hormone receptor status and promoter methylation of DOK7 and ELF5 was further examined using available methylation array data (Illumina HM450) from a set of paired primary and second breast tumors from 24 women. RESULTS: All 21 of the novel tamoxifen-selected cell lines are ER-positive, and HER2-negative, and 18 of the cell lines are PR-negative while the MCF-7 cells were scored as ER-positive, modestly PR-positive and HER2 negative. Expression of DOK7 and ELF5 is significantly up-regulated in half of the tamoxifen-selected cell lines as compared to the parental MCF-7. In contrast, the previously established ER-negative TMX2-28 cell line has decreased expression of both DOK7 and ELF5 and increased DNA methylation in the transcriptional start site region of these genes. ELF5 methylation was lower in second versus primary tumors in women who received anti-estrogen treatment, in PR-negative versus PR-positive tumors, and in the subset of PR-positive first tumors from the group of women who had second PR-negative tumors as compared to those who had second PR-positive tumors. CONCLUSIONS: The distinct ELF5 methylation of PR-positive primary tumors from women who had a PR-negative recurrence indicates the possibility of stratification of women for tailored treatment in the early stages of disease.

Association Between Loss of SATB2 Expression in Inflammatory Bowel Disease Indefinite for Dysplasia and a Diagnosis of Definitive Dysplasia on Follow-up.

Special AT-rich sequence-binding protein 2 (SATB2) is a sensitive and specific biomarker for sporadic colonic adenocarcinomas. Previous studies have found that SATB2 is lost in some adenocarcinomas and dysplasias associated with inflammatory bowel disease (IBD). In establishing these findings, the prior studies did not examine cases of IBD interpreted as indefinite for dysplasia. We examined SATB2 expression in this diagnostic category to determine if any potential loss is associated with a diagnosis of definitive dysplasia on follow-up. To investigate this possibility, we collected 87 biopsies of IBD indefinite for dysplasia from 62 patients and stained them with SATB2. Among patients' indefinite for dysplasia, we found SATB2 loss in 6/62 (9.7%). Among those with follow-up (n=51), we observed 5/6 (83%) with a future dysplasia in those with SATB2 loss compared with 10/45 (22%) in those with SATB2 retention, absolute difference 61.1% (95% confidence interval=28.9%-93.3%). We conclude that loss of SATB2 on biopsies otherwise interpreted as IBD indefinite for dysplasia may mark a population at high risk for showing definitive dysplasia on future biopsies.

Enrichment of CpG island shore region hypermethylation in epigenetic breast field cancerization.

While changes in DNA methylation are known to occur early in breast carcinogenesis and the landscape of breast tumour DNA methylation is profoundly altered compared with normal tissue, there have been limited efforts to identify DNA methylation field cancerization effects in histologically normal breast tissue adjacent to tumour. Matched tumour, histologically normal tissue of the ipsilateral breast (ipsilateral-normal), and histologically normal tissue of the contralateral breast (contralateral-normal) were obtained from nine women undergoing bilateral mastectomy. Laser capture microdissection was used to select epithelial cells from normal tissue, and neoplastic cells from tumour for genome-scale measures of DNA methylation with the Illumina HumanMethylationEPIC array. We identified substantially more CpG loci that were differentially methylated between contralateral-normal and tumour (63,271 CpG loci q < 0.01), than between ipsilateral-normal and tumour (38,346 CpG loci q < 0.01). We identified differential methylation in ipsilateral-normal relative to contralateral-normal tissue (9,562 CpG loci p < 0.01). In this comparison, hypomethylated loci were significantly enriched for breast cancer-relevant transcription factor binding sites including those for ESR1, FoxA1, and GATA3 and hypermethylated loci were significantly enriched for CpG island shore regions. In addition, progression of shore hypermethylation was observed in tumours compared to matched ipsilateral normal tissue, and these alterations tracked to several well-established tumour suppressor genes. Our results indicate an epigenetic field effect in surrounding histologically normal tissue. This work offers an opportunity to focus investigations of early DNA methylation alterations in breast carcinogenesis and potentially develop epigenetic biomarkers of disease risk. ABBREVIATIONS: DCIS: ductal carcinoma in situ; GO: gene ontology; OR: odds ratio; CI: confidence interval; TFBS: transcription factor binding site; LOLA: Locus Overlap Analysis.

Hepatic Sarcoidosis Complicated with Pancreatic Adenocarcinoma.

Sarcoidosis is a systemic noncaseous granulomatous disease. The liver is a common location but usually asymptomatic. Current literature suggests an association between sarcoidosis and cancers. However, there is a lack of definite evidence. We present a case of a 59-year-old man with jaundice and acutely elevated alkaline phosphatase. The diagnosis was confirmed by obtaining a liver biopsy and was treated with 6 months of steroids. A year later, he had a recurrence of jaundice. MRCP showed biliary dilatation and a mass in the pancreatic head, confirmed by biopsy to be adenocarcinoma. This is the first case to be reported of hepatic sarcoidosis associated with pancreatic cancer.

Sexually transmitted infections of the lower gastrointestinal tract.

The World Health Organization estimates that there is greater than one million new cases of sexually transmitted infections (STIs) every day. In many countries, STIs are at an unprecedented high, including the USA, where nearly 20 million new cases were reported in 2016. Although morbidity associated with STIs is usually seen in the context of genitourinary disease, these pathogens may also affect the gastrointestinal tract and cause anal pain, abdominal pain, or diarrhea. It is important to recognize patterns of injury associated with these pathogens, especially those that may mimic other gastrointestinal diseases, such as idiopathic inflammatory bowel disease (IBD). This review focuses upon STIs of the lower gastrointestinal tract, organized by the most common site of involvement: the anus, rectum, and colon.

Isolated infective endocarditis of the pulmonary valve: an autopsy analysis of nine cases.

INTRODUCTION: Infective endocarditis (IE) of the pulmonary valve is uncommon and usually occurs in conjunction with tricuspid and/or left-sided valvular endocarditis. There have been only sporadic reports of isolated pulmonary valvular infective endocarditis (PVIE). This report documents the pathological features of nine such cases at autopsy. METHODS: Among 155 cases of IE encountered in a 14-year period, we selected nine cases that had isolated PVIE for analysis. The clinical records were reviewed for the patient demographics, presence or absence of underlying cardiac disease or other predisposing factors, and modes of presentation; these were correlated with the relevant investigations. A detailed study of the heart was done in all with special attention to the pulmonary valve morphology. RESULTS: The nine cases of isolated PVIE formed 5.8% of the IE cases, seen in six males and three females, largely adults. Seven patients (77.8%) had admissions for about 24 h. Hospital admission was sought for mainly progressive shortness of breath (66.7%) and fever (44.4%). Congenital heart disease was seen in seven (77.8%); two (an infant and an adult) had normal hearts. A single blood sample for blood culture in two patients was negative. Two-dimensional echocardiography, performed in eight, revealed vegetations on the pulmonary valve in four. The pulmonary valve was tricuspid in six cases, bicuspid in two, and unicuspid in one. The vegetations (active in three, active and healing in two, healed in four) were accompanied by concomitant thickening, shortening, perforations, or complete destruction of the cusps. Involvement of the right ventricular outflow tract or the main pulmonary artery was identified in five hearts. Five patients (55.6%) developed pulmonary complications, related to the endocarditis. CONCLUSIONS: Detection of PVIE, especially the isolated type, may be underdiagnosed. This condition should be kept in mind during evaluation of patients especially with cardiac anomalies, who present with fever, prominent respiratory symptoms, and negative blood cultures.

Isolated pericardial and intracardiac hydatidosis: presentation as congestive cardiac failure and fatal pulmonary embolism.

Localization of hydatid cysts in the heart is a rare phenomenon, with an incidence of 0.5-2%. In almost half these cases, the heart is the sole organ to be involved. We report a case of massive pericardial hydatidosis in a female patient who presented with features of congestive cardiac failure. Cysts in the endocardium of right side of the heart resulted in fatal pulmonary embolism.