ZIP4 is a novel diagnostic and prognostic marker in human pancreatic cancer: a systemic comparison between EUS-FNA and surgical specimens.

Aberrant expression of a zinc transporter ZIP4 in pancreatic ductal adenocarcinoma (PDAC) has been shown to contribute to tumor progression and is a potential target for individualized therapy. The overall objective of this study was to determine whether ZIP4 could serve as a novel diagnostic and prognostic marker in human PDAC, and if it can be assessed by minimally invasive sampling using endoscopic ultrasound guided fine needle aspiration (EUS-FNA). Immunohistochemistry was performed to compare ZIP4 expression in the PDAC samples obtained from EUS-FNA and matched surgical tumors (parallel control). Samples were reported by sensitivity, specificity, and predictive values, all with 95% confidence intervals (CI). A total of 23 cases with both FNA and surgical specimens were evaluated. We found that ZIP4 was significantly overexpressed in tumor cells from both sets of samples. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of ZIP4 for the diagnosis of PDAC were 72.9%, 72.5%, 76.1%, and 69.0% in EUS-FNA samples, and were 97.9%, 65.4%, 83.9%, and 94.4% in surgical specimens, respectively. The association between the positive rate of ZIP4 expression in FNA and surgical samples is statistically significant (P=0.0216). Both the intensity and percentage of ZIP4 positive cells from the surgical samples correlated significantly with tumor stage (P=0.0025 and P=0.0002). ZIP4 intensity level in FNA samples was significantly associated with tumor differentiation and patient survival. These results indicate that EUS-FNA is capable of non-operative detection of ZIP4, thus offering the potential to direct pre-operative detection and targeted therapy of PDAC.

Profilin1 sensitizes pancreatic cancer cells to irradiation by inducing apoptosis and reducing autophagy.

Pancreatic cancer has an extremely poor prognosis mainly due to lack of effective treatment options. Radiotherapy is mostly applied to locally advanced cases, although tumor radioresistance limits the effectiveness. Profilin1, a novel tumor suppressor gene, was reported to be down-regulated in various cancers and associated with tumor progression. The objective of this study was to demonstrate how profilin1 affected pancreatic cancer radiosensitivity. We showed profilin1 was down-regulated in pancreatic cancer cells after exposure to radiation, and re-expression of profilin1 suppressed tumor cell viability and increased DNA damage following irradiation. Further studies revealed that up-regulation of profilin1 facilitated apoptosis and repressed autophagy induced by irradiation, which might sensitize pancreatic cancer cells to radiation treatment. Our findings may provide a novel therapeutic strategy for sensitizing pancreatic cancer to radiotherapy.

Interpreting epidemiological research: blinded comparison of methods used to estimate the prevalence of inherited mutations in BRCA1.

While sequence analysis is considered by many to be the most sensitive method of detecting unknown mutations in large genes such as BRCA1, most published estimates of the prevalence of mutations in this gene have been derived from studies that have used other methods of gene analysis. In order to determine the relative sensitivity of techniques that are widely used in research on BRCA1, a set of blinded samples containing 58 distinct mutations were analysed by four separate laboratories. Each used one of the following methods: single strand conformational polymorphism analysis (SSCP), conformation sensitive gel electrophoresis (CSGE), two dimensional gene scanning (TDGS), and denaturing high performance liquid chromatography (DHPLC). Only the laboratory using DHPLC correctly identified each of the mutations. The laboratory using TDGS correctly identified 91% of the mutations but produced three apparent false positive results. The laboratories using SSCP and CSGE detected abnormal migration for 72% and 76% of the mutations, respectively, but subsequently confirmed and reported only 65% and 60% of mutations, respectively. False negatives therefore resulted not only from failure of the techniques to distinguish wild type from mutant, but also from failure to confirm the mutation by sequence analysis as well as from human errors leading to misreporting of results. These findings characterise sources of error in commonly used methods of mutation detection that should be addressed by laboratories using these methods. Based upon sources of error identified in this comparison, it is likely that mutations in BRCA1 and BRCA2 are more prevalent than some studies have previously reported. The findings of this comparison provide a basis for interpreting studies of mutations in susceptibility genes across many inherited cancer syndromes.

Efficacy of bilateral prophylactic mastectomy in BRCA1 and BRCA2 gene mutation carriers.

BACKGROUND: In women with a family history of breast cancer, bilateral prophylactic mastectomy is associated with a decreased risk of subsequent breast cancer of approximately 90%. We examined the association between bilateral prophylactic mastectomy and breast cancer risk in women at high risk for breast cancer who also had mutations in BRCA1 and BRCA2 genes. METHODS: We obtained blood samples from 176 of the 214 high-risk women who participated in our previous retrospective cohort study of bilateral prophylactic mastectomy. We used conformation-sensitive gel electrophoresis and direct sequence analysis of the blood specimens to identify women with mutations in BRCA1 and BRCA2. The carriers' probabilities of developing breast cancer were estimated from two different penetrance models. RESULTS: We identified 26 women with an alteration in BRCA1 or BRCA2. Eighteen of the mutations were considered to be deleterious and eight to be of uncertain clinical significance. None of the 26 women has developed breast cancer after a median of 13.4 years of follow-up (range, 5.8-28.5 years). Three of the 214 women are known to have developed a breast cancer after prophylactic mastectomy. For two of these women, BRCA1 and BRCA2 screening was negative, and no blood specimen was available for the third. Estimations of the effectiveness of prophylactic mastectomy were performed, considering this woman as both a mutation carrier and a noncarrier. These calculations predicted that six to nine breast cancers should have developed among the mutation carriers, which translates into a risk reduction, after bilateral prophylactic mastectomy, of 89.5%-100% (95% confidence interval = 41.4% to 100%). CONCLUSIONS: Prophylactic mastectomy is associated with a substantial reduction in the incidence of subsequent breast cancer not only in women identified as being at high risk on the basis of a family history of breast cancer but also in known BRCA1 or BRCA2 mutation carriers.

Identifying and managing hereditary risk of breast and ovarian cancer.

In the past, all women with a family history of breast or ovarian cancer were considered to be at increased risk of cancer themselves. The discovery of BRCA1 and BRCA2 demonstrated that susceptibility to breast and ovarian cancer can be inherited by women as a single-gene autosomal dominant disorder. For such women, evaluation of family history is an important screening tool to identify the possibility of hereditary cancer risk but only genetic testing can provide definitive, individualized risk assessment. Women who have inherited mutations in BRCA1 or BRCA2 now have several medical management options to address their increased risk of cancer. A well-educated community of health care providers and patients can use hereditary risk assessment, including genetic testing, to improve health care.

Hereditary cancer syndromes.

OBJECTIVE: To summarize the biological basis, clinical implications, identification, and medical management of syndromes associated with increased risk of common adult cancers. DATA SOURCES: Recent studies and data available from molecular and clinical analysis of genes responsible for autosomal-dominant inheritance of cancer risk. DATA SYNTHESIS: Several hereditary cancer syndromes have been identified for which there are increasingly effective diagnostic and management options. Specific hereditary susceptibility syndromes have been characterized that increase the risk of malignancies of the breast, ovary, colon, endometrium, and endocrine organs. Following a summary of the biological basis of hereditary cancer risk in adults, the identification of such syndromes by clinical and laboratory means is reviewed. Finally, management options for individuals with these syndromes are summarized. CONCLUSIONS: Advances in gene discovery have allowed the diagnosis of recently characterized hereditary cancer syndromes to enhance medical management for individuals with inherited susceptibility to common cancers.

Incidence of BRCA1/2 germ line alterations in a high risk cohort participating in a phase II chemoprevention trial.

It is unknown what proportion of women at high risk for breast cancer, entering phase II chemoprevention trials, have BRCA1/2 alterations, and whether their initial biomarker patterns or response to preventive interventions will differ between carriers and non-carriers. As part of a 6-month phase II chemoprevention trial of diflouromethlyornithine (DFMO), high-risk subjects (family history, prior precancerous breast disease or prior breast cancer), who had random peri-areolar fine needle evidence of epithelial hyperplasia with or without atypia, were offered genetic counselling and testing at the completion of their study participation. 97% of the 119 women eligible for testing underwent BRCA1/2 gene sequencing, 3 declined. 26 (22%) of the 116 women had an alteration in BRCA1/2. Known deleterious mutations were present in 3 (3%), uncertain significance mutations in 19 (16%), and probable polymorphisms in 6 (5%). There does not appear to be a difference in initial biomarker distribution between participants with and without germ line alterations.

The predictive value of BRCA1 and BRCA2 mutation testing.

Genetic testing for mutations in BRCA1 and BRCA2, two genes predisposing to breast and ovarian cancers, is available to women with a relevant family history. The aim of this study was to estimate the positive and negative predictive value of clinical sequence analysis of these genes. A reference graph showing positive and negative predictive values over a range of pre-test risk was derived, taking into account the sensitivity and specificity of a full-sequence analysis test. High positive and negative predictive values were found for women with pre-test risk between 4% and 40%, a range of risk commonly seen in clinical testing. The predictive value of full sequence and single-site analysis of BRCA1 and BRCA2, therefore, compares favorably with other diagnostic medical tests. Our results provide a numerical estimate of the predictive value of BRCA testing, and as such, provide a valuable tool to healthcare providers and families as they interpret BRCA1 and BRCA2 test results.

Hereditary susceptibility to breast cancer: significance of age of onset in family history and contribution of BRCA1 and BRCA2.

OBJECTIVE: To correlate mutations in BRCA1 and BRCA2 with family history of breast cancer in a first-degree relative for women diagnosed with breast cancer before age 45 who do not have a personal or family history of ovarian cancer. METHODS: Family history for women with breast cancer diagnosed before age 45 was provided by ordering physicians via a test requisition form designed for this purpose. Gene analysis was performed by dye primer sequencing for the entire coding regions of BRCA1 and BRCA2. Because a personal and family history of ovarian cancer are known to be significantly associated with mutations, women with either were excluded from analysis. RESULTS: Overall, deleterious mutations in BRCA1 or BRCA2 were identified in 85 of 440 women (19%) with breast cancer under 45. Mutations were identified in 73 of 276 women (26%) with a first degree family history of breast cancer compared to 12 of 164 without (7%) (P < .0001). When results were analyzed by the age of diagnosis in first degree relatives, mutations were identified in 56 of 185 women (30%) with at least one first degree relative with breast cancer diagnosed before age 50 compared with 17 of 91 women (19%), where the first degree family history of breast cancer was at or over age 50 (P = .042). CONCLUSION: Among women with breast cancer diagnosed before age 45, a first-degree relative diagnosed with the disease under age 50 is an indicator of a mutation in BRCA1 or BRCA2 even in the absence of a family history of ovarian cancer. Therefore, women diagnosed with early-onset breast cancer should be asked about the age of onset in any first-degree relative diagnosed with the disease, as well as about any family history of ovarian cancer. Mutations in BRCA2 account for a substantial proportion of hereditary breast cancer. Therefore, studies that are limited to BRCA1 or that do not analyze by age of onset of breast cancer in relatives may underestimate the contribution of mutations in BRCA1 and BRCA2 to women with early onset breast cancer.

Laboratory determination of hereditary susceptibility to breast and ovarian cancer.

Inherited mutations in the genes BRCA1 and BRCA2 are associated with a significantly increased risk of breast cancer, particularly before the age of 50 years, as well as an increased risk of ovarian cancer. Patients with early-onset breast cancer or ovarian cancer at any age with a family history of either disease are at higher risk of carrying a mutation in BRCA1 or BRCA2. Laboratory analysis of these genes can determine whether a patient has inherited an increased risk of breast and ovarian cancer. In the absence of a mutation that has been previously identified in a family member, most tests for hereditary breast-ovarian cancer risk analyze the entire coding sequences of BRCA1 and BRCA2. The gene sequencing process itself can be automated, but the data must be interpreted by an individual with training in molecular diagnostics. Management options generally available to individuals with hereditary susceptibility to breast and ovarian cancer include heightened surveillance, prophylactic surgery, and chemoprevention. The use of genetic techniques to identify women with increased risk of cancer demonstrates the application of recent advances in the understanding of the genetic basis of malignancy to laboratory medicine and clinical care.

The pros and cons of genetic testing for breast and ovarian cancer risk.

Nearly 10% of breast and ovarian cancers develop as a direct consequence of an inherited flaw in the genes BRCA1 and BRCA2. The protein products of these genes suppress the development of cancer, in part by repairing damage in other genes. Women who inherit a nonfunctioning copy of either BRCA1 or BRCA2 therefore have a significantly elevated lifetime risk of breast cancer, especially at an early age. Identification of hereditary breast and ovarian cancer susceptibility allows optimized medical management of an individual's increased risk of breast and ovarian cancer. Significantly, testing may also identify women in "high-risk" families who did not themselves inherit cancer susceptibility, allowing them to avoid unnecessary medical intervention. Choosing to be tested for breast and ovarian cancer risk is a complicated task, however. It takes into account concerns about insurance liability, family dynamics, and an individual's psychological needs. From the limited research, evidence suggests that for individuals in high-risk families it is more beneficial to know than not to know one's genetic status. Education and counseling may improve public perception about genetic testing for breast cancer.

Comparison of alterations of chromosome 17 in carcinoma of the ovary and of the breast.

Breast and ovarian carcinomas share a region of allelic loss on chromosome 17q25, suggesting that these tumours may arise by similar molecular pathways. We analysed paraffin-embedded tissues from 84 sporadic ovarian carcinomas and 42 sporadic infiltrating ductal carcinomas of the breast for abnormalities on chromosome 17. Loss of heterozygosity (LOH) of at least one informative marker on 17q was identified in 49 of 82 (60%) ovarian carcinomas, as against only 6 of 40 (15%) informative breast carcinomas (P<0.0001). In ovarian carcinoma, LOH was most commonly observed for GH on 17q23 (56%), and was also frequently observed at 17q21 (46%). In contrast, LOH of D17S 1330/CTT16 on 17q25 was observed in only 19% of ovarian tumours. LOH in breast carcinomas was most frequently observed at 17q21 (16%), less frequently at 17q23 (7%) and not identified at all at 17q25 in any breast cancers. Immunohistochemical analysis demonstrated overexpression of the p53 gene product in 38 of 84 (45%) ovarian carcinomas, as against 10 of 42 (24%) breast carcinomas (P = 0.0195). p53 immunoreactivity was significantly associated with LOH in ovarian and breast cancers. Immunohistochemical expression of HER2/neu was observed in 6 of 84 (7%) ovarian and 3 of 42 (7%) breast carcinomas. There was no relationship between HER2/neu immunoreactivity and LOH. Although sporadic carcinomas of breast and ovary share some regions of allelic loss on chromosome 17q, differences in other alterations on this chromosome suggest divergent pathways of tumour development.

Comparison of mutations of Ki-RAS and p53 immunoreactivity in borderline and malignant epithelial ovarian tumors.

Ovarian tumors of low malignant potential ("borderline tumors") have been proposed variably to represent a distinctive type of malignancy, precursors of frank ovarian malignancy, or a nonmalignant process. We analyzed 81 malignant and 39 borderline ovarian tumors for p53 immunoreactivity and alterations in codon 12 of Ki-RAS in order to correlate these alterations with tumor and cell type. Diffuse p53 immunoreactivity was significantly more prevalent among malignant (36 of 81, 44%) than among borderline (3 of 39, 8%) tumors and was particularly prevalent among serous invasive carcinomas (16 of 26, 62%). Conversely, mutations in codon 12 of Ki-RAS were significantly more prevalent in borderline (16 of 39, 41%) than in malignant (9 of 81, 11%) ovarian tumors and were most prevalent among mucinous tumors. This preliminary molecular analysis suggests that serous borderline tumors have some molecular features usually associated with malignancy but are unlikely to represent a precursor of invasive serous carcinoma. In contrast, mucinous borderline tumors may represent a precursor or variant of mucinous carcinoma of the ovary.

Frequency of germline and somatic BRCA1 mutations in ovarian cancer.

Germline mutations in the BRCA1 tumor suppressor gene are thought to be the most common cause of hereditary ovarian cancer. The aim of this study was to explore further the role of BRCA1 alterations in the development of ovarian cancers. We sought to determine whether somatic BRCA1 mutations are ever present in ovarian cancers and whether mutation is always accompanied by loss of the wild-type allele. The entire coding region and intronic splice sites of BRCA1 were sequenced using genomic DNA samples from 103 unselected ovarian cancers. Thirteen clearly deleterious BRCA1 mutations and two variants of uncertain significance were found. Blood DNA was available in all but two cases and demonstrated that 4 of 13 mutations and both variants of uncertain significance were germline alterations, whereas in seven cases the mutation was a somatic change present only in the cancer. Using four microsatellite markers, loss of heterozygosity at the BRCA1 locus was found in all 15 ovarian cancers with BRCA1 sequence alterations, compared with only 58% of ovarian cancers that did not have BRCA1 mutations. BRCA1-associated ovarian cancers were characterized by serous histology and moderate histological grade. These data confirm prior reports suggesting that germline mutations in BRCA1 are present in about 5% of women with ovarian cancer. In addition, somatic mutations in BRCA1 occur in the development of some sporadic cases. The finding that both germline and somatic BRCA1 mutations are accompanied by loss of heterozygosity, suggests that loss of this tumor suppressor gene is a critical event in the development of these cancers.

Sequence analysis of BRCA1 and BRCA2: correlation of mutations with family history and ovarian cancer risk.

PURPOSE: Previous studies of mutations in BRCA1 or BRCA2 have used detection methods that may underestimate the actual frequency of mutations and have analyzed women using heterogeneous criteria for risk of hereditary cancer. PATIENTS AND METHODS: A total of 238 women with breast cancer before age 50 or ovarian cancer at any age and at least one first- or second-degree relative with either diagnosis underwent sequence analysis of BRCA1 followed by analysis of BRCA2 (except for 27 women who declined analysis of BRCA2 after a deleterious mutation was discovered in BRCA1). Results were correlated with personal and family history of malignancy. RESULTS: Deleterious mutations were identified in 94 (39%) women, including 59 of 117 (50%) from families with ovarian cancer and 35 of 121 (29%) from families without ovarian cancer. Mutations were identified in 14 of 70 (20%) women with just one other relative who developed breast cancer before age 50. In women with breast cancer, mutations in BRCA1 and BRCA2 were associated with a 10-fold increased risk of subsequent ovarian carcinoma (P = .005). CONCLUSION: Because mutations in BRCA1 and BRCA2 in women with breast cancer are associated with an increased risk of ovarian cancer, analysis of these genes should be considered for women diagnosed with breast cancer who have a high probability of carrying a mutation according to the statistical model developed with these data.