Prophylactic irradiation to the contralateral breast for BRCA mutation carriers with early-stage breast cancer.

BACKGROUND: Women who carry germ-line mutations in BRCA1/2 are at very high risk of developing breast and ovarian cancer. Breast conserving therapy is associated with a similar risk of ipsilateral cancer recurrence in BRCA carriers compared with non-carriers. However, the risk of subsequent contralateral breast cancer in carriers is markedly increased. Therefore, mastectomy of the diseased breast along with risk reducing mastectomy of the contralateral breast is often advocated for BRCA carriers who are treated for early breast cancer. Yet, many BRCA carriers forgo this option for fear of harmful effects and choose breast conserving treatment and observation instead. In Israel, BRCA-associated breast cancer is relatively common. Accordingly, a national protocol was devised for this enriched population. PATIENTS AND METHODS: In this Institutional Review Board-approved phase II trial, the option of prophylactic irradiation to the contralateral breast, in addition to standard loco-regional treatment, was offered to BRCA carrier patients treated for early breast cancer who declined contralateral mastectomy. The primary end point was contralateral breast cancer. RESULTS: Between May 2007 and October 2017, 162 patients were enrolled. Eighty-one patients opted for standard loco-regional treatment including surgery and radiation to the involved side (control arm) and 81 patients chose additional contralateral breast irradiation (intervention arm). At a median follow-up of 58 months, 10 patients developed contralateral breast cancer in the control arm at a median of 32 months, as compared with 2 patients in the intervention arm who developed contralateral breast cancer 80 and 105 months after bilateral breast irradiation (log-rank P = 0.011). CONCLUSIONS: Among BRCA carrier patients treated for early breast cancer, the addition of contralateral breast irradiation was associated with a significant reduction of subsequent contralateral breast cancers and a delay in their onset. CLINICAL TRIAL: Phase II, comparative two-arm trial (NCT00496288).

Chemotherapy may eradicate ductal carcinoma in situ (DCIS) but not the associated microcalcifications.

INTRODUCTION: We studied the effect of neoadjuvant chemotherapy (NAC) ± trastuzumab on the ductal carcinoma in situ (DCIS) component in patients with locally advanced breast cancer who achieved pathological complete response (pCR). METHODS: The diagnostic biopsies of 92 consecutive breast cancer patients that were treated with neoadjuvant chemotherapy (NAC) ± trastuzumab were evaluated for the presence of DCIS. Upon completion of NAC, the surgical specimens were evaluated for complete eradication of both the invasive and non-invasive cancer in the breast. The pretreatment mammograms were evaluated for the presence of microcalcifications and compared to the mammograms that were obtained upon completion of therapy prior to surgery. RESULTS: Thirty of 92 patients (33%) had a substantial component of DCIS in the pretreatment biopsy. Thirty nine patients (42%) achieved pCR: 22 (56%) following NAC + trastuzumab, 17 (32%) following chemotherapy only. Ten of 30 patients (33%) with DCIS component achieved pCR: 4 received chemotherapy only, in 6 trastuzumab was added. Multiple microcalcifications on the pretreatment mammograms were observed in 3 of 10 patients with DCIS who achieved pCR. No reduction in the area of calcifications was observed following NAC. CONCLUSIONS: DCIS may be completely eradicated by NAC ± trastuzumab. However, associated microcalcifications probably persist. Patients with locally advanced breast cancer with substantial DCIS may still opt for NAC and breast conservation as the DCIS component may respond and even completely disappear following NAC. Residual widespread microcalcifications after NAC do not necessarily indicate residual cancer. Larger studies are needed to direct the surgical management of these patients.

Normal polymorphism in the incomplete trinucleotide repeat of the arginine-rich protein gene.

The arginine-rich protein (ARP) gene was recently cloned and localized to human chromosome band 3p21. Recent reports have suggested that ARP is mutated in a high percentage of different human tumors. We amplified and sequenced the multiple arginine coding area of the ARP gene in primary head and neck, non-small cell lung, and renal cell cancers. We found a high frequency of genetic changes in this region, including a single base pair substitution and deletions of arginine repeats in primary tumors. However, these changes were always present in matched normal controls. Thus, the variations in the ARP trinucleotide repeat region represent normal polymorphisms rather than tumor-specific mutations.

CpG methylation as a basis for breast tumor-specific loss of NES1/kallikrein 10 expression.

The normal epithelial cell-specific-1 (NES1)/kallikrein 10 gene is expressed in normal mammary epithelial cells, but its expression is dramatically decreased in breast cancer cell lines. Now, we have cloned and characterized the active promoter region of NES1. Using a luciferase reporter system, we demonstrate that most tumor cell lines are able to support full or partial transcription from the NES1 promoter, suggesting a role for promoter-independent cis-acting mechanisms of loss of NES1 expression. We show that hypermethylation of the NES1 gene represents one such mechanism. Using methylation-specific PCR and sequence analysis of sodium bisulfite-treated genomic DNA, we demonstrate a strong correlation between exon 3 hypermethylation and loss of NES1 mRNA expression in a panel of breast cancer cell lines and in primary tumors. Treatment of NES1-nonexpressing cells with a demethylating agent led to reexpression of NES1, suggesting an important role of hypermethylation in the loss of NES1 expression. We suggest that hypermethylation is responsible for tumor-specific loss of NES1 gene expression. Our results also suggest that hypermethylation of the NES1 gene may serve as a potential marker for breast cancer.

Loss of cyclin D2 expression in the majority of breast cancers is associated with promoter hypermethylation.

Cyclin D2 is a member of the D-type cyclins, implicated in cell cycle regulation, differentiation, and malignant transformation. It was noted previously that cyclin D2 is not expressed in the majority of breast cancer cell lines, whereas abundant expression was detected in finite life span human mammary epithelial cells. By reverse transcription-PCR and Western blot analysis, we extended this finding to primary breast carcinomas and show that the majority of these tumors lack expression of cyclin D2 mRNA (18 of 24) and protein (10 of 13). In contrast, both luminal and myoepithelial subpopulations of normal breast tissues expressed cyclin D2. Hypermethylation of the CpG island in the promoter was detected by methylation-specific PCR in nearly half of the breast cancers (49 of 106) and was associated with silencing of cyclin D2 gene expression. Promoter hypermethylation was also detected in ductal carcinoma in situ, suggesting that loss of cyclin D2 expression is an early event in tumorigenesis. Our results suggest that loss of cyclin D2 expression is associated with the evolution of breast cancer.

Wilms' tumor suppressor gene (WT1) is expressed in primary breast tumors despite tumor-specific promoter methylation.

We analyzed Wilms' tumor suppressor 1 (WT1) expression and its regulation by promoter methylation in a panel of normal breast epithelial samples and primary carcinomas. Contrary to previous reports, WT1 protein was strongly expressed in primary carcinomas (27 of 31 tumors) but not in normal breast epithelium (1 of 20 samples). Additionally, the WT1 promoter was methylated in 6 of 19 (32%) primary tumors, which nevertheless expressed WT1. The promoter is not methylated in normal epithelium. Thus, although tumor-specific methylation of WT1 is established in primary breast cancer at a low frequency, other transcriptional regulatory mechanisms appear to supercede its effects in these tumors. Our results demonstrate expression of WT1 in mammary neoplasia, and that WT1 may not have a tumor suppressor role in breast cancer.

Analysis of PTEN/MMAC1 alterations in aerodigestive tract tumors.

PTEN/MMAC1 is a candidate tumor suppressor gene recently identified at chromosomal band 10q23. It is mutated in sporadic brain, breast, and prostate cancer and in the germ line of patients with hereditary Cowden disease. We searched for genetic alterations of the PTEN/MMAC1 gene in 39 primary head and neck cancers (HNSCCs), 42 primary non-small cell lung cancers (NSCLCs), 80 pancreatic cancer xenografts, and 37 cell lines and xenografts from colon, lung, and gastric cancers. Microsatellite analysis revealed loss of heterozygosity at markers near the gene in 41% of primary HNSCCs, 50% of NSCLCs, and 39% of the pancreatic cancers. Three cases of HNSCCs displayed homozygous deletion involving the gene. We sequenced the entire coding region of the PTEN/MMAC1 gene in the remaining tumors displaying loss of heterozygosity and found one terminating mutation in a HNSCC sample. Thus, a second inactivation event was observed in 4 of 39 primary HNSCC cases. By use of a protein truncation assay, one terminating mutation was also identified in one of eight NSCLC cell lines. Our results suggest that PTEN/MMAC1 gene inactivation plays a role in the genesis of some tumor types.

Hypermethylation of 14-3-3 sigma (stratifin) is an early event in breast cancer.

We have identified 14-3-3 sigma (sigma) as a gene whose expression is lost in breast carcinomas, primarily by methylation-mediated silencing. In this report, we investigated the timing of loss of sigma gene expression during breast tumorigenesis in vivo. We analysed the methylation status of sigma in breast cancer precursor lesions using microdissection for selective tissue sampling. We found hypermethylation of sigma in 24 of 25 carcinomas (96%), 15 of 18 (83%) of ductal carcinoma in situ, and three of eight (38%) of atypical hyperplasias. None of the five hyperplasias without atypia showed sigma-hypermethylation. Unexpectedly, patients with breast cancer showed sigma hypermethylation in adjacent histologically normal breast epithelium, while this was never observed in individuals without evidence of breast cancer. Also, samples of periductal stromal breast tissue were consistently hypermethylated, underscoring the importance of selective tissue sampling for accurate assessment of 14-3-3-sigma methylation in breast epithelium. These results suggest that hypermethylation of 14-3-3-sigma occurs at an early stage in the progression to invasive breast cancer, and may occur in apparently normal epithelium adjacent to breast cancer. These results provide evidence that loss of expression of sigma is an early event in neoplastic transformation.

Point mutation and homozygous deletion of PTEN/MMAC1 in primary bladder cancers.

A new tumor suppressor gene PTEN/MMAC1 was recently isolated at chromosome 10q23 and found to be inactivated by point mutation or homozygous deletion in glioma, prostate and breast cancer. PTEN/MMAC1 was also identified as the gene predisposing to Cowden disease, an autosomal dominant cancer predisposition syndrome associated with an increased risk of breast, skin and thyroid tumors and occasional cases of other cancers including bladder and renal cell carcinoma. We screened 345 urinary tract cancers by microsatellite analysis and found chromosome 10q to be deleted in 65 of 285 (23%) bladder and 15 of 60 (25%) renal cell cancers. We then screened the entire PTEN/MMAC1 coding region for mutation in 25 bladder and 15 renal cell primary tumors with deletion of chromosome 10q. Two somatic point mutations, a frameshift and a splicing variant, were found in the panel of bladder tumors while no mutation was observed in the renal cell carcinomas. To screen for homozygous deletion, we isolated two polymorphic microsatellite repeats from genomic BAC clones containing the PTEN/MMAC1 gene. Using these new informative markers, we identified apparent retention at the gene locus indicative of homozygous deletion of PTEN/MMAC1 in four of 65 bladder and 0 of 15 renal cell tumors with LOH through chromosome 10q. Identification of the second inactivation event in six bladder tumors with LOH of 10q implies that the PTEN/MMAC1 gene is occasionally involved in bladder tumorigenesis. However, the low frequency of biallelic inactivation suggests that either PTEN/MMAC1 is inactivated by other mechanisms or it is not the only target of chromosome 10q deletion in primary bladder and renal cell cancer.

Idiopathic calcitriol-induced hypercalcemia. A new disease entity?

We describe 3 patients with a prolonged history of hypercalcemia. The patients did not take any vitamin supplements. All patients had low parathyroid hormone (PTH), high calcitriol (1,25(OH)2D3), and high angiotensin-converting enzyme (ACE) blood levels. There was no evidence of sarcoidosis of any other underlying disease in the results of an extensive workup. Treatment with prednisone resulted in normalization of calcium levels in all patients. They remained dependent on low-dose prednisone on a subsequent prolonged follow-up.

Primary multifocal lymphoma of bone presenting as hypercalcemic crisis: report of a rare manifestation of extranodal lymphoma.

Primary non-Hodgkin's lymphoma of bone is uncommon and usually manifests clinically as localized bone pain. Here we report a woman who presented with hypercalcemic crisis and extensive investigation revealed a primary multifocal lymphoma of bone. The course of the disease was very aggressive and despite intensive supportive care and urgent chemotherapy the patient died within 1 month. Since her blood PTH and calcitriol levels were suppressed and her parathyroid-hormone-related peptide (PTHrp) was mildly elevated, we believe that release of cytokines combined with PTHrp, as well as extensive osteolytic lesions, were the causes of the hypercalcemia. This is an unusual presenting symptom of lymphomas and to the best of our knowledge severe symptomatic hypercalcemia and crisis has never been reported in primary lymphoma of bone before.

[Purulent pericarditis].

Purulent pericarditis is diagnosed when pus is drained from the pericardial space or when bacteria are cultured from the pericardial fluid. This rare disease is often diagnosed late, when severe hemodynamic compromise develops due to pericardial tamponade. It is usually a complication of pneumonia, especially if there is empyema as well, and often follows chest surgery or chest wall infections. It sometimes appears in patients with septicemia, especially when they are debilitated or immuno-compromised. Diagnosis is aided by echocardiography. Pericardiocentesis and drainage of the pus, as well as prolonged antibiotic treatment, are mandatory. Delay in diagnosis and treatment often results in death. Some surviving patients may develop constrictive pericarditis and require pericardiectomy. We report a 73-year-old man with pulmonary lymphoma who suffered from purulent pericarditis secondary to sepsis with methicillin-resistant Staphylococcus aureus. Pericardial drainage and appropriate antibiotic treatment eventually resulted in complete recovery.

[Budd-Chiari syndrome].

Budd Chiari syndrome is a rare disorder resulting from occlusion of hepatic venous drainage by hepatic vein thrombosis or by a membranous web in the inferior vena cava. In western countries the commonest causes are myeloproliferative disorders and hypercoagulable states. Presentation may be acute with rapid accumulation of ascites and hepatic failure, or subacute with symptoms developing over a few months. A chronic progressive form has also been described. On presentation there is usually abdominal pain, ascites, and hepatosplenomegaly; hepatic encephalopathy is found in about a third. Noninvasive, ultrasound-Doppler is recommended in diagnosis, and has a high correlation with hepatic venography. Liver biopsy is required for therapeutic decisions. Those with advanced hepatic failure or severe fibrosis on liver biopsy are referred for hepatic transplantation. When biopsy shows only hepatic congestion and inflammatory infiltrates, portosystemic shunting is recommended. We present a 61-year-old woman with ascites and hepatosplenomegaly that had developed over the courses of a few months. Budd-Chiari syndrome with chronic myelofibrosis and congenital protein C deficiency were diagnosed. Portosystemic shunt was performed but death from sepsis followed shortly.

Acute renal failure complicating organophosphate intoxication.

Organophosphates are an extremely uncommon cause of acute renal failure. The mechanism is unknown. We present a case and refer to earlier reports.

HCV hepatitis associated with anticardiolipin antibody and a cerebrovascular accident. Response to interferon therapy.

A 54-year-old man with chronic hepatitis C virus (HCV) developed quadrihemianopsia caused by lacunar brain infarction. Extensive evaluation revealed high titers of anticardiolipin antibodies (ACA). Following interferon treatment (6 x 10(6), three times a week for 2 months and 3 x 10(6) for another 7 months), liver transaminase levels decreased to normal, HCV RNA in blood was no longer detectable, concomitantly with the disappearance of the ACA. The patient remained clinically stable without evidence for either HCV activity (RNA) or ACA or further thromboembolic events.

Polymyositis, arthritis, and proteinuria in a patient with adult celiac disease.

A 37-year-old woman developed polymyositis and arthritis concomitantly with proteinuria and watery diarrhea. Repeated duodenal biopsies and serological evaluation established the diagnosis of adult celiac disease. Treatment with gluten-free diet resolved all clinical and laboratory abnormalities. We believe that this is the first report of adult celiac disease presenting as a multisystem disease involving kidneys, joint, and muscles.

Compromised HOXA5 function can limit p53 expression in human breast tumours.

Expression of the p53 gene protects cells against malignant transformation. Whereas control of p53 degradation has been a subject of intense scrutiny, little is known about the factors that regulate p53 synthesis. Here we show that p53 messenger RNA levels are low in a large proportion of breast tumours. Seeking potential regulators of p53 transcription, we found consensus HOX binding sites in the p53 promoterS. Transient transfection of Hox/HOXA5 activated the p53 promoter. Expression of HOXA5 in epithelial cancer cells expressing wild-type p53, but not in isogenic variants lacking the p53 gene, led to apoptotic cell death. Moreover, breast cancer cell lines and patient tumours display a coordinate loss of p53 and HOXA5 mRNA and protein expression. The HOXA5 promoter region was methylated in 16 out of 20 p53-negative breast tumour specimens. We conclude that loss of expression of p53 in human breast cancer may be primarily due to lack of expression of HOXA5.

Correlation between gold-induced enterocolitis and the presence of the HLA-DRB1*0404 allele.

OBJECTIVE: In recent years we have treated 4 rheumatoid arthritis (RA) patients who developed gold-induced enterocolitis, a well-recognized, although rare, complication of chrysotherapy. The aim of the present study was to seek any genetic predisposition for this complication. METHODS: HLA DNA typing was done on fresh white blood cells from the 4 patients. RESULTS: Three of the 4 patients (75%) exhibited the DRB1*0404 allele, whereas the prevalence of this allele among the Ashkenazi Jewish population of RA patients without colitis was 9.2% and 10.2% in 2 different studies. CONCLUSION: The results indicate that the DRB1*0404 may be associated with risk for the development of gold-induced enterocolitis in this population and suggest that HLA DNA typing should be considered in Jews who may be undergoing chrysotherapy.

Somatic mutations of the PTEN tumor suppressor gene in sporadic follicular thyroid tumors.

The PTEN (MMAC1/TEP1) tumor suppressor gene was recently isolated and mapped to human chromosome band 10q23. Homozygous deletions and mutations of PTEN were observed in cell lines and sporadic cancers of the breast, kidney, and central nervous system. Germline mutations in PTEN were recently found in Cowden disease, an autosomal dominant inherited syndrome, previously mapped to chromosome bands 10q22-23. This disease is associated with a wide variety of malignancies and hamartomas of ectodermal, mesodermal, and endodermal origin. The most common neoplasms in Cowden disease patients arise in the breast, skin, and thyroid (follicular subtype). To determine the involvement of PTEN in sporadic follicular thyroid tumors, we first analyzed sporadic follicular adenomas and carcinomas for deletions of the PTEN gene. Loss of heterozygosity was found in 7/26 (27%) follicular carcinomas and 2/27 (7%) follicular adenomas, one of which was a small hemizygous deletion (approximately 3 cm). Sequence analysis of the entire PTEN coding region revealed two mutations in carcinomas with 10q loss. Our findings suggest that the PTEN tumor suppressor gene is occasionally inactivated in sporadic follicular thyroid tumors.