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Epithelial to mesenchymal transition (EMT) is typically defined by the acquisition of a spindle cell morphology in combination with loss of E-cadherin and upregulation of mesenchymal markers. However, by studying E-cadherin inactivation in 38 human breast cancer cell lines, we noted that not all cell lines that had undergone EMT had concomitantly lost E-cadherin expression. We further investigated this discrepancy functionally and in clinical breast cancer specimens. Interestingly, reconstitution of wild-type E-cadherin cDNA in a E-cadherin negative cell line that had undergone EMT (MDA-MB-231) did not revert the spindle morphology back to an epithelial morphology. Neither were changes observed in the expression of several markers known to be involved in the EMT process. Similarly, upregulation of E-cadherin via global DNA demethylation in eleven cell lines that had undergone EMT did not induce a change in cell morphology, nor did it alter the expression of EMT markers in these cells. Next, we extracted genes differentially expressed between cell lines that had undergone EMT versus cell lines that had not undergone EMT. Caveolin-1 was identified to be an excellent marker for EMT, irrespective of E-cadherin status (specificity and sensitivity of 100 %). Consistent with our observations in the breast cancer cell lines, expression of Caveolin-1 identified a subset of basal breast cancers, particularly of metaplastic pathology, and only 50 % of these lacked E-cadherin expression. The discrepancy between E-cadherin loss and EMT was thus reproduced in clinical samples. Together, these results indicate that in human breast cancer loss of E-cadherin is not causal for EMT and even not a necessity.

A family history of breast cancer is one of the major risk factors for breast cancer. It is estimated that hereditary factors play a causative role in more than 25% of patients. Currently, many genetic alterations associated with highly (4 to 10 ), moderately (2 to 4 ), and slightly (1 to 2 ) increased risk for developing breast cancer have been identified. However, in daily clinical practice, mutations in a few high-risk genes are mainly being determined (ie, BRCA1, BRCA2, TP53, and PTEN). Mutations in these genes are roughly associated with a cumulative life-time risk (CLTR) of 50% to 85% and are characterized by early onset of disease, starting from the age of 25. However, such highly dominant genetic alterations occur in only a small minority (approximately 5%) of patients with breast cancer. In women with a family history of breast cancer without a proven gene mutation, the CLTR of breast cancer varies between 15% and 50% depending on the pedigree and other risk factors. Women with a moderately to highly increased genetic risk of breast cancer have the option to reduce their risk by prophylactic surgery or chemoprevention. Prophylactic bilateral mastectomy reduces the risk of breast cancer by nearly 100% and prophylactic bilateral salpingo-oophorectomy by approximately 50%. Chemoprevention for 5 years reduces the risk by 40% to 50%, but is used by only a very small minority ( 10%) of high-risk women, mainly in North-America. A promising strategy to reduce the risk of breast cancer death in women with a genetic predisposition is early diagnosis by intensive surveillance, usually from the age of 25. In the 1990s, several retrospective and prospective studies were performed assessing the efficacy of yearly mammography screening, sometimes in combination with (semi-annual) clinical breast examination (CBE) and/or ultrasound. Indeed, it was possible to detect breast cancer in a significant number of asymptomatic women, but the sensitivity (40% to 60%) appeared to be low in comparison with the results of population-based mammographic screening in women between 50 and 75 years of age. Especially in carriers of BRCA1/2 gene mutations, interval cancers frequently occurred (ie, in approximately 40% to 60% of breast cancers). Since the late 1990s, a new approach has involved the addition of magnetic resonance imaging (MRI) to yearly mammographic screening. In the past 10 years, results of different pilot studies and large prospective clinical trials investigating this approach have been published—all nonrandomized because of practical and ethical reasons. The first results of five large prospective studies (with at least 20 breast cancers detected) have shown that MRI appears to be about twice as sensitive as mammography in detecting tumors in women with a genetic susceptibility to breast cancer (Table 1 ); in the pooled analysis of Sardanelli involving 3,571 women the sensitivity of MRI was 81% versus 40% similar to the review of Warner of 11 studies (77% v 39%). Subsequently, the application of MRI has been incorporated in national guidelines in various Western countries, but no consensus on the optimal screening protocol exists for all risk groups. Meanwhile, inconsistent results of the sensitivity of MRI in detecting ductal carcinoma in situ (DCIS) have been reported. Currently, in daily clinical practice, the combination of yearly MRI and mammography is being used in women with a genetic risk of breast cancer because mammography adds significantly to the diagnostic accuracy of MRI, especially in DCIS. In Journal of Clinical Oncology, Kuhl et al previously reported on the results of a large single center study performed at the University of Bonn, Germany. In the current issue of JCO, the results of a multicenter trial (the EVA EVAluation of imaging methods for secondary prevention of familial breast cancer trial) coordinated by Kuhl in four German centers are reported. The main objective of the EVA trial was to compare the respective efficacy in terms of cancer yield and stage at diagnosis of CBE, mammography, ultrasound, and quality-assured breast MRI, used alone and in different combinations, for screening of women at elevated risk for breast cancer quantified using the BRCAPRO model. A secondary objective was to investigate the cancer yield of additional half-yearly screening with ultrasound and CBE in a subgroup of 371 women. Six hundred eighty-seven asymptomatic women with a family history of breast cancer (CLTR 20%) were included. Median age was 44 years (range, 25 to 71) and median follow-up was 2.4 years (range, 1.1 to 3.3). In 65 women (9.5%), a BRCA1/2 mutation was documented, while 212 women (including 26 BRCA1/2 mutation carriers) had a personal history of breast cancer (31%). Twenty-seven women were diagnosed with breast cancer (11 of 27 had DCIS) resulting in an overall detection rate of 15.5 per 1,000 woman-years. Nine breast cancers occurred in women with a personal history of breast cancer and five breast cancers occurred in BRCA1/2 mutation carriers. No interval cancers occurred and most tumors were in an early stage. All women were diagnosed with the combination of MRI and mammography. JOURNAL OF CLINICAL ONCOLOGY E D I T O R I A L VOLUME 28 NUMBER 9 MARCH 2

Purpose The Dutch MRI Screening Study on early detection of hereditary breast cancer started in 1999. We evaluated the long-term results including separate analyses of BRCA1 and BRCA2 mutation carriers and first results on survival. Patients and Methods Women with higher than 15% cumulative lifetime risk (CLTR) of breast cancer were screened with biannual clinical breast examination and annual mammography and magnetic resonance imaging (MRI). Participants were divided into subgroups: carriers of a gene mutation (50% to 85% CLTR) and two familial groups with high (30% to 50% CLTR) or moderate risk (15% to 30% CLTR). Results Our update contains 2,157 eligible women including 599 mutation carriers (median follow-up of 4.9 years from entry) with 97 primary breast cancers detected (median follow-up of 5.0 years from diagnosis). MRI sensitivity was superior to that of mammography for invasive cancer (77.4% v 35.5%; P < .00005), but not for ductal carcinoma in situ. Results in the BRCA1 group were worse compared to the BRCA2, the high-, and the moderate-risk groups, respectively, for mammography sensitivity (25.0% v 61.5%, 45.5%, 46.7%), tumor size at diagnosis ≤ 1 cm (21.4% v 61.5%, 40.9%, 63.6%), proportion of DCIS (6.5% v 18.8%, 14.8%, 31.3%) and interval cancers (32.3% v 6.3%, 3.7%, 6.3%), and age at diagnosis younger than 30 years (9.7% v 0%). Cumulative distant metastasis-free and overall survival at 6 years in all 42 BRCA1/2 mutation carriers with invasive breast cancer were 83.9% (95% CI, 64.1% to 93.3%) and 92.7% (95% CI, 79.0% to 97.6%), respectively, and 100% in the familial groups (n = 43). Conclusion Screening results were somewhat worse in BRCA1 mutation carriers, but 6-year survival was high in all risk groups.

Mutations of E-cadherin have been identified in half of lobular breast cancers and diffuse-type gastric cancers, two tumor subtypes with remarkably similar pathological appearances including small rounded cells with scant cytoplasm and a diffuse growth pattern. A causal role for E-cadherin gene mutations in the lobular breast cancer phenotype was recently demonstrated in E-cadherin knock-out mice. These observations suggested that another gene in the E-cadherin tumor suppressor pathway might be mutated in lobular breast cancers with wild-type E-cadherin genes. Here, we identified E-cadherin gene mutations exclusively in human breast cancer cell lines that grow with a rounded cell morphology. Using expression analyses and gene mutation analyses, we have identified four biallelic inactivating alpha-catenin mutations among 55 human breast cancer cell lines. All four alpha-catenin mutations predicted premature termination of the encoded proteins, and concordantly, none of the four mutant cell lines expressed alpha-catenin proteins. Importantly, three of the alpha-catenin mutant cell lines had the rounded cell morphology and all 14 cell lines with the rounded cell morphology had mutations of either E-cadherin or alpha-catenin. As anticipated, loss of alpha-catenin protein expression was associated with the lobular subtype in primary breast cancers. Together, our observations suggest that alpha-catenin may be a new tumor suppressor gene that operates in the E-cadherin tumor suppressor pathway.

The use of gene-expression microarray analysis to assess the expression levels of all the genes in the genome has tremendous potential. Important information has been obtained about many disease processes, particularly in classifying tumors in different subtypes and risk groups. Combining gene-expression data with other genomic information and the use of sophisticated bioinformatic tools enables the discovery of potential new targets for treatment, and is helpful for high-throughput drug screening and for designing new classes of drugs for targeted therapy. Here, we provide a short overview of the recent, promising developments in the field with emphasis on breast cancer.

Background Patient prognosis and response to endocrine therapy in breast cancer correlate with protein expression of both estrogen receptor (ER) and progesterone receptor (PR), with poorer outcome in patients with ER+/PR− compared to ER+/PR+ tumors. Methods To better understand the underlying biology of ER+/PR− tumors, we examined RNA expression (n > 1000 tumors) and DNA copy number profiles from five previously published studies of human breast cancers with clinically assigned hormone receptor status (ER+/PR+, ER+/PR−, and ER−/PR−). Results We identified an expression “signature” of genes with either elevated or diminished RNA levels specifically in ER+/PR+ compared to ER−/PR− and ER+/PR− tumors. We similarly identified a gene signature specific to ER−/PR− tumors. ER+/PR− tumors, on the other hand, were a mixture of three different subtypes: tumors manifesting the ER+/PR+ signature, tumors manifesting the ER−/PR− signature, and tumors not associating with ER+/PR+ or ER−/PR− tumors (which we considered “true” ER+/PR−). In analyses of both tamoxifen-treated and untreated patients, ER+/PR− breast cancers defined by RNA profiling were associated with poor patient outcome, worse than those with pure ER+/PR+ patterns; these differences were not observed when using clinical assays to assign ER and PR status. ER+/PR− tumors also showed twice as many DNA copy number gains or losses compared to ER+/PR+ and ER−PR− tumors. Targets of transcriptional up-regulation by specific oncogenic pathways, including PI3 K/Akt/mTOR, were enriched in both ER+/PR− and ER−/PR− compared to ER+/PR+ tumors. Conclusion ER+/PR− tumors as defined by RNA profiling represent a distinct subset of breast cancer with aggressive features and poor outcome, despite being clinically ER+. Multigene assays derived from our gene signatures could conceivably provide an improved clinical assay for inferring PR status for prognostic and therapeutic purposes.

Purpose: We previously discovered an extracellular matrix (ECM) gene cluster associated with resistance to first-line tamoxifen therapy of patients with metastatic breast cancer. In this study, we determined whether the six individual ECM genes [collagen 1A1 (COL1A1), fibronectin 1 (FN1), lysyl oxidase (LOX), secreted protein acidic cysteine-rich (SPARC), tissue inhibitor of metalloproteinase 3 (TIMP3), and tenascin C (TNC)] were associated with treatment response, prognosis, or both. Experimental Design: In 1,286 primary breast tumors, mRNA expression (quantitative real-time PCR) was related to clinicopathologic factors and disease outcome in univariate and multivariate analysis including traditional factors. Results: TIMP3, FN1, LOX, and SPARC expression levels (continuous variables) were significantly associated with distant metastasis-free survival (MFS) in 680 lymph node–negative untreated patients (P < 0.03). Using a calculated linear prognostic score, these patients were evenly divided into five prognostic groups with a significant difference in 10-year MFS of ∼40% between the two extreme prognostic groups. Furthermore, high TNC expression as continuous variable was associated with (a) shorter MFS in 139 estrogen receptor–positive and lymph node–positive patients who received adjuvant tamoxifen therapy (hazard ratio, 1.53; P = 0.001), and (b) no clinical benefit (odds ratio, 0.81; P = 0.035) and shorter progression-free survival (hazard ratio, 1.19; P = 0.002) in 240 patients in whom recurrence was treated with tamoxifen as first-line monotherapy. These results were also significant in multivariate analyses. Conclusion: FN1, LOX, SPARC, and TIMP3 expression levels are associated with the prognosis of patients with breast cancers, whereas TNC is associated with resistance to tamoxifen therapy. Further validation and functional studies are necessary to determine the use of these ECM genes in decisions regarding treatment and whether they can serve as targets for therapy.

Purpose: The pathogenic CHEK2 1100delC variant is firmly established as a breast cancer susceptibility allele. Dutch CHEK2 1100delC breast cancer families frequently also include colorectal cancer cases, and the variant is particularly prevalent among breast cancer families with hereditary breast and colorectal cancer. Yet, it is still unclear whether CHEK2 1100delC also confers a colorectal cancer risk independent of its breast cancer risk. Experimental Design:CHEK2 1100delC was genotyped in the index cases of 369 Dutch colorectal cancer families that had been excluded for familial breast cancer. The cohort included 132 cases with familial adenomatous polyposis (FAP) and FAP-related disease, and 237 cases with hereditary nonpolyposis colorectal cancer (HNPCC) and HNPCC-related disease. Results: None of the FAP/FAP-related cases carried the CHEK2 1100delC variant. In contrast, CHEK2 1100delC was present in 10 of 237 (4.2%) HNPCC/HNPCC-related cases that was significantly more prevalent than the 1.0% Dutch population frequency (odds ratio, 4.3; 95% confidence interval, 1.7-10.7; P = 0.002). Nine of the 10 CHEK2 1100delC colorectal cancer cases met the revised Amsterdam and/or Bethesda criteria. The 10 CHEK2 1100delC colorectal cancer families had a high-risk cancer inheritance pattern, including 35 colorectal cancer cases, 9 cases with polyps, and 21 cases with other tumor types. Conclusion: Our analysis provides strong evidence that the 1100delC variant of CHEK2 confers a colorectal cancer risk in HNPCC/HNPCC-related families, supporting the hypothesis that CHEK2 is a multiorgan cancer susceptibility gene.

Purpose: Magnetic resonance imaging (MRI) screening enables early detection of breast cancers in women with an inherited predisposition. Interval cancers occurred in women with a BRCA1 mutation, possibly due to fast tumor growth. We investigated the effect of a BRCA1 or BRCA2 mutation and age on the growth rate of breast cancers, as this may influence the optimal screening frequency. Experimental Design: We reviewed the invasive cancers from the United Kingdom, Dutch, and Canadian MRI screening trials for women at hereditary risk, measuring tumor size at diagnosis and on preceding MRI and/or mammography. We could assess tumor volume doubling time (DT) in 100 cancers. Results: Tumor DT was estimated for 43 women with a BRCA1 mutation, 16 women with a BRCA2 mutation, and 41 women at high risk without an identified mutation. Growth rate slowed continuously with increasing age (P = 0.004). Growth was twice as fast in BRCA1 (P = 0.003) or BRCA2 (P = 0.03) patients as in high-risk patients of the same age. The mean DT for women with BRCA1/2 mutations diagnosed at ages ≤40, 41 to 50, and >50 years was 28, 68, and 81 days, respectively, and 83, 121, and 173 days, respectively, in the high-risk group. Pathologic tumor size decreased with increasing age (P = 0.001). Median size was 15 mm for patients ages ≤40 years compared with 9 mm in older patients (P = 0.003); tumors were largest in young women with BRCA1 mutations. Conclusion: Tumors grow quickly in women with BRCA1 mutations and in young women. Age and risk group should be taken into account in screening protocols.

Background: TIMP-1 protein is a prognostic factor for recurrence-free and overall survival (OS) time in breast cancer. We evaluated the prognostic value of TIMP1 mRNA and a novel TIMP1 mRNA splice variant in 1301 primary breast cancer patients.Methods: We measured mRNA transcripts of full-length TIMP1 (TIMP1-v1) and the novel splice variant lacking exon 2 (TIMP1-v2) by use of real-time RT-PCR in frozen primary tumor samples. Transcript concentrations are correlated with histomorphological and biological factors, TIMP-1 protein, and distant metastasis-free survival (MFS) and OS time.Results: TIMP1-v1 and TIMP1-v2 alone were not informative with respect to predicting prognosis. However, the PCR assay designed to measure the combination of v1 + v2 showed that high concentrations of this combination were associated with good prognosis. In Cox multivariate regression analysis, which also included the traditional prognostic factors, increasing concentrations were independently associated with prolonged MFS (P = 0.004) and OS (P = 0.048). Including TIMP-1 protein and TIMP1-v1+v2 mRNA together in the multivariate model revealed that protein and mRNA were both independently associated with prognosis, with hazard ratios pointing in opposite directions.Conclusion: High concentrations of TIMP1-v1+2 mRNA are associated with good prognosis in patients with primary breast cancer. Since high concentrations of TIMP-1 protein are associated with poor prognosis, the presence of possible posttranscriptional mechanisms requires further investigation.

Breast cancer exhibits familial aggregation, consistent with variation in genetic susceptibility to the disease. Known susceptibility genes account for less than 25% of the familial risk of breast cancer, and the residual genetic variance is likely to be due to variants conferring more moderate risks. To identify further susceptibility alleles, we conducted a two-stage genome-wide association study in 4,398 breast cancer cases and 4,316 controls, followed by a third stage in which 30 single nucleotide polymorphisms (SNPs) were tested for confirmation in 21,860 cases and 22,578 controls from 22 studies. We used 227,876 SNPs that were estimated to correlate with 77% of known common SNPs in Europeans at r2 > 0.5. SNPs in five novel independent loci exhibited strong and consistent evidence of association with breast cancer (P

Background BRCA1/2 mutation carriers and women from a hereditary breast(/ovarian) cancer family have a highly increased risk of developing breast cancer (BC). Prophylactic mastectomy (PM) results in the greatest BC risk reduction. Long-term data on the efficacy and sequels of PM are scarce. Methods From 358 high-risk women (including 236 BRCA1/2 carriers) undergoing PM between 1994 and 2004, relevant data on the occurrence of BC in relation to PM, complications in relation to breast reconstruction (BR), mutation status, age at PM and preoperative imaging examination results were extracted from the medical records, and analyzed separately for women without (unaffected, n = 177) and with a BC history (affected, n = 181). Results No primary BCs occurred after PM (median follow-up 4.5 years). In one previously unaffected woman, metastatic BC was detected almost 4 years after PM (primary BC not found). Median age at PM was younger in unaffected women (P < .001), affected women more frequently were 50% risk carriers (P < .001). Unexpected (pre)malignant changes at PM were found in 3% of the patients (in 5 affected, and 5 unaffected women, respectively). In 49.6% of the women opting for BR one or more complications were registered, totaling 215 complications, leading to 153 surgical interventions (71%). Complications were mainly related to cosmetic outcome (36%) and capsular formation (24%). Conclusions The risk of developing a primary BC after PM remains low after longer follow-up. Preoperative imaging and careful histological examination is warranted because of potential unexpected (pre)malignant findings. The high complication rate after breast reconstruction mainly concerns cosmetic issues.

textabstractBackground: Radiotherapy for breast cancer as delivered in the 1970s has been associated with increased risk of cardiovascular disease, but recent studies of associations with modern regimens have been inconclusive. Few data on long-term cardiovascular disease risk according to specific radiation fields are available, and interaction with known cardiovascular risk factors has not been examined. Methods: The studied treatment-specific incidence of cardiovascular disease in 4414 10-year survivors of breast cancer who were treated from 1970 through 1986. Risk of cardiovascular disease in these patients was compared with general population rates and evaluated in Cox proportional hazards regression models. All statistical tests were two-sided. Results: After a median follow-up of 18 years, 942 cardiovascular events were observed (standardized incidence ratio = 1.30, 95% confidence interval [CI] = 1.22 to 1.38; corresponding to 62.9 excess cases per 10 000 patient-years). Breast irradiation only was not associated with increased risk of cardiovascular disease. However, radiotherapy to either the left or right side of the internal mammary chain was associated with increased cardiovascular disease risk for the treatment period 1970-1979 (for myocardial infarction, hazard ratio [HR] = 2.55, 95% CI = 1.55 to 4.19; P

A single nucleotide polymorphism (SNP309T>G) in the intronic promoter of MDM2 was recently found to accelerate carcinogenesis in early-onset cancer cases. This cancer acceleration presumably was due to increased SP1 binding, resulting in enhanced MDM2 transcriptional activation by estrogens. We evaluated MDM2 SNP309 in 343 familial breast cancer cases with known mutation status for CHEK2 1100delC, BRCA1 and BRCA2. Cancer acceleration was indeed observed in early-onset familial breast cancer cases (diagnosed

Contains fulltext : 49322.pdf (Publisher’s version ) (Closed access) BACKGROUND: The MRISC study is a screening study, in which women with an increased risk of hereditary breast cancer are screened by a yearly mammography and MRI, and half-yearly clinical breast examination. The sensitivity found in this study was 40% for mammography and 71% for MRI and the specificity was 95 and 90%, respectively. In the current subsequent study we investigated whether these results are influenced by age, a BRCA1/2 mutation, menopausal status and breast density. PATIENTS AND METHODS: From November 1999 to October 2003, 1909 eligible women were screened and 50 breast cancers were detected. For the current analysis, data of 4134 screening rounds and 45 detected breast cancers were used. For both imaging modalities, screening parameters, receiver operating characteristic (ROC) curves and uni- and multivariate odds ratios (ORs) were calculated. All analyses were separately performed for age at entry (< 40, 40-49, >/=50), mutation status, menopausal status and breast density. RESULTS: Sensitivity of MRI was decreased in women with high breast density (adjusted OR 0.08). False-positive rates of both mammography (OR(adj) 1.67) and MRI (OR(adj) 1.21) were increased by high breast density, that of MRI by pre-menopausal status (OR(adj) 1.70), young age (OR(adj) 1.58 for women 40-49 years versus women >/=50 years) and decreased in BRCA1/2 mutation carriers (OR(adj) 0.74). In all investigated subgroups the discriminating capacity (measured by the area under the ROC-curve) was higher for MRI than for mammography, with the largest differences for BRCA1/2 mutation carriers (0.237), for women between 40 and 49 years (0.227) and for women with a low breast density (0.237). CONCLUSIONS: This report supports the earlier recommendation that MRI should be a standard screening method for breast cancer in BRCA1/2 mutation carriers.

Purpose The biology of tumors relapsing to bone is poorly understood. In this study, we initiated a search for genes that are implicated in tumors relapsing to bone in breast cancer. Patients and Methods We analyzed 107 primary breast tumors in patients who were all lymph node negative at the time of diagnosis and all had experienced relapse. Total RNA isolated from frozen tumor samples was used to gather gene expression data using oligo microarrays. Results A panel of 69 genes was found significantly differentially expressed between patients who experienced relapse to bone versus those who experienced relapse elsewhere in the body. The most differentially expressed gene, TFF1, was confirmed by quantitative reverse transcriptase polymerase chain reaction in an independent cohort (n = 122; P = .0015). Our differentially expressed genes, combined with a recently reported gene set relevant to tumors relapsing to bone in an animal model system, pointed to the involvement of the fibroblast growth factor receptor signaling pathway in preference of tumor cells that relapse to bone. Given that patients who experience relapse to bone may benefit from bisphosphonate therapy, we developed a classifier of 31 genes, which in an independent validation set correctly predicts all tumors relapsing to bone with a specificity of 50%. Conclusion Our study identifies a panel of genes relevant to bone metastasis in breast cancer. The subsequently developed classifier of tumors relapsing to bone could, after thorough confirmation on an extended number of independent samples, and in combination with our previously developed high-risk profile, provide a diagnostic tool for the recommendation of adjuvant bisphosphonate therapy in addition to endocrine therapy or chemotherapy.

BACKGROUND. Within the Dutch MRI Screening (MRISC) study, a Dutch multicenter screening study for hereditary breast cancer, the authors investigated whether previously reported increased diagnostic accuracy of magnetic resonance imaging (MRI) compared with mammography would be maintained during subsequent screening rounds. METHODS. From November 1999 to October 2003, 1909 eligible women were included in the study. Screening parameters and tumor characteristics of different rounds were calculated and compared. The authors defined 3 different types of imaging screening rounds: first round in women never screened by imaging before, first round in women screened by imaging (mainly mammography) before, and subsequent rounds. RESULTS. The difference in sensitivity for invasive cancers between mammography and MRI was largest in the first round of women previously screened with mammography (20.0 vs. 93.3%; P = .003), but also in subsequent rounds, there was a significant difference in favor of MRI (29.4 vs. 76.5%; P = .02). The difference in false-positive rate between mammography and MRI was also largest in the first round of women previously screened with mammography (5.5 vs. 14.0%; P

Purpose To assess treatment-specific risk of cerebrovascular events in early breast cancer (BC) patients, accounting for cerebrovascular risk factors. Patients and Methods We studied the incidence of cerebrovascular accidents (CVA; stroke and transient ischemic attack [TIA]) in 10-year survivors of early BC (n = 4,414) treated from 1970 to 1986. Follow-up was 96% complete until January 2000. Treatment-specific incidence of CVA was evaluated by standardized incidence ratios (SIRs) based on comparison with general population rates and by Cox proportional hazards regression. Results After a median follow-up of 18 years, 164 strokes and 109 TIAs were observed, resulting in decreased SIRs of 0.8 (95% CI, 0.6 to 0.9) for stroke and 0.8 (95% CI, 0.7 to 1.0) for TIA. Significantly increased risk of stroke was found in women who had received hormonal treatment (HT; tamoxifen) and in women who had hypertension or hypercholesterolemia, with hazard ratios (HRs) of 1.9, 2.1, and 1.6, respectively. Patients irradiated on the supraclavicular area and/or internal mammary chain (IMC) did not experience a higher risk of stroke (HR = 1.0; 95% CI, 0.7 to 1.6) or TIA (HR = 1.4; 95% CI, 0.9 to 2.5) compared with patients who did not receive radiotherapy or who were irradiated on fields other than the supraclavicular area or IMC. Conclusion Long-term survivors of BC experience no increased risk of cerebrovascular events compared with the general population. HT is associated with an increased risk of stroke. Radiation fields including the carotid artery do not seem to increase the risk of stroke compared with other fields.

The p53 tumor suppressor gene is frequently mutated in breast cancer. Here, we used direct sequencing to screen the complete coding sequence of the p53 gene from 41 human breast cancer cell lines. We identified 32 cell lines (78%) with a p53 gene alteration that predicted a change in the encoded protein. Thirty-one of these mutations were accompanied by loss of the other p53 allele. All mutations but one were unique and 27 mutations had previously been identified in uncultured human cancers. Ten mutations were predicted to encode a truncated p53 protein and 22 missense mutations were identified. p53 transcript expression was analyzed by semi-quantitative RT-PCR and p53 protein expression was determined by Western blotting. Our analyses revealed three p53 expression patterns: wild-type p53 cell lines had normal transcript levels and low or no detectable protein expression; cell lines with a p53 truncating mutation had low transcript levels and low or no detectable protein expression; and cell lines with a p53 missense mutation had highly variable transcript and protein expression levels. As a whole, our data represent a p53 mutation profile in breast cancer cell lines, providing a model for structural, functional and pharmacological studies on p53 in human cancer.

Contains fulltext : 47763.pdf (Publisher’s version ) (Closed access) Imaging is often performed yearly for the surveillance of BRCA1/2 mutation carriers and women at high familial breast cancer risk. Growth of cancers in carriers may be faster as these tumours are predominantly high grade. Quantitative data on tumour growth rates in these 2 groups are lacking. Here, we have examined 80 high-risk women under surveillance for tumour size at diagnosis and preceding examinations at mammography and/or MRI. Tumour volume doubling time (DT) was assessed in 30 cancers in BRCA1/2 mutation carriers and 25 non-carriers. Impact of age and menopausal status were also evaluated. Mean DT of all invasive cancers was shorter in carriers (45 days CI: 26-73) than non-carriers (84 days CI: 58-131) (P = 0.048). Mean age at diagnosis was lower in carriers (40 years) than non-carriers (45 years) (P = 0.007). At multivariable analysis only age (P = 0.03), not risk-group (P = 0.26) nor menopause (P = 0.58) correlated significantly with DT. The mean growth rate slowed down to half in each successive 10 years-older group. In conclusion, age at detection indicated the growth rates of hereditary and familial breast cancers. It is recommended that the screening frequency should be adjusted according to a woman's age and a high-sensitive biannual test may be appropriate before the age of 40 years.

To understand the biological basis of resistance to endocrine therapy is of utmost importance in patients with steroid hormone receptor–positive breast cancer. Not only will this allow us prediction of therapy success, it may also lead to novel therapies for patients resistant to current endocrine therapy. DNA methylation in the promoter regions of genes is a prominent epigenetic gene silencing mechanism that contributes to breast cancer biology. In the current study, we investigated whether promoter DNA methylation could be associated with resistance to endocrine therapy in patients with recurrent breast cancer. Using a microarray-based technology, the promoter DNA methylation status of 117 candidate genes was studied in a cohort of 200 steroid hormone receptor–positive tumors of patients who received the antiestrogen tamoxifen as first-line treatment for recurrent breast cancer. Of the genes analyzed, the promoter DNA methylation status of 10 genes was significantly associated with clinical outcome of tamoxifen therapy. The association of the promoter hypermethylation of the strongest marker, phosphoserine aminotransferase (PSAT1) with favorable clinical outcome was confirmed by an independent quantitative DNA methylation detection method. Furthermore, the extent of DNA methylation of PSAT1 was inversely associated with its expression at the mRNA level. Finally, also at the mRNA level, PSAT1 was a predictor of tamoxifen therapy response. Concluding, our work indicates that promoter hypermethylation and mRNA expression of PSAT1 are indicators of response to tamoxifen-based endocrine therapy in steroid hormone receptor–positive patients with recurrent breast cancer.

Objective Women at high risk of ovarian cancer are currently offered two options: either surveillance or prophylactic bilateral salpingo-oophorectomy. The efficacy and outcome of surveillance remain unclear. Methods We performed a retrospective study. Between 1994 and 2000, we screened 383 high-risk women, of which 152 were BRCA1/2 mutation carriers. Surveillance consisted of annual gynecological examination, transvaginal ultrasound, and serum CA125 measurement. Exploratory or prophylactic surgery was performed in selected cases. Results There were no screen-detected primary ovarian cancers. Abnormal results at surveillance were observed in 74 (19.3%) of women; in 47 (63.5%), the abnormalities disappeared spontaneously. Exploratory surgery was performed in 20 (27.0%) women in whom one malignancy was found (metastatic breast cancer in the ovary). A rising CA125 value prompted further (non-surgical) evaluation in three women with a history of breast cancer: recurrent breast cancer was diagnosed in two women; in the third, a chondrosarcoma was found. 133 women opted for prophylactic bilateral salpingo-oophorectomy, whereby two unexpected malignancies were found (fallopian tube cancer and metastatic breast cancer). One interval primary ovarian cancer occurred, presenting as papillary serous carcinoma of the peritoneum 14 months after prophylactic bilateral salpingo-oophorectomy. Complications of prophylactic surgery were encountered in 15 (11.5%) women. Conclusions Ovarian cancer surveillance has limited sensitivity, and a high number of false positive findings. This can lead to unnecessary surgical interventions, possibly resulting in surgery-related complications. It is important to inform high-risk women of these limitations. For now, prophylactic bilateral salpingo-oophorectomy remains the optimal risk-reducing strategy for women at high risk.

Purpose: To investigate the proportion of breast cancers arising in patients with germ line BRCA1 and BRCA2 mutations expressing basal markers and developing predictive tests for identification of high-risk patients. Experimental Design: Histopathologic material from 182 tumors in BRCA1 mutation carriers, 63 BRCA2 carriers, and 109 controls, collected as part of the international Breast Cancer Linkage Consortium were immunohistochemically stained for CK14, CK5/6, CK17, epidermal growth factor receptor (EGFR), and osteonectin. Results: All five basal markers were commoner in BRCA1 tumors than in control tumors (CK14: 61% versus 12%; CK5/6: 58% versus 7%; CK17: 53% versus 10%; osteonectin: 43% versus 19%; EGFR: 67% versus 21%; P < 0.0001 in each case). In a multivariate analysis, CK14, CK5/6, and estrogen receptor (ER) remained significant predictors of BRCA1 carrier status. In contrast, the frequency of basal markers in BRCA2 tumors did not differ significant from controls. Conclusion: The use of cytokeratin staining in combination with ER and morphology provides a more accurate predictor of BRCA1 mutation status than previously available, that may be useful in selecting patients for BRCA1 mutation testing. The high percentage of BRCA1 cases positive for EGFR suggests that specific anti-tyrosine kinase therapy may be of potential benefit in these patients.

In this article we determined the course of psychological distress during a breast cancer surveillance program in women at increased risk of developing hereditary breast cancer (BC). The sample comprised of 357 unaffected women (mean age 40.5 years) adhering to a surveillance programme (MRISC-study). Before and after two successive biannual surveillance appointments, the Impact of Event Scale (BC-specific distress) and the Hospital Anxiety and Depression Scale (general distress) were administered, totalling four measurement moments. In general, psychological distress remained within normal limits and decreased significantly after a surveillance appointment, except for breast cancer specific distress after the second appointment. Scheduled imaging examinations were not significantly related to distress. The course of BC specific distress differed significantly for risk over-estimators and for young (

Purpose: BCAR1, the human homologue of the rat p130Cas protein, was identified in a functional screen for human breast cancer cell proliferation resistant to antiestrogen drugs. Here, we study the prognostic value of quantitative BCAR1 levels in a large series of breast cancer specimens. Experimental Design: A specific ELISA was developed to measure BCAR1 protein levels in 2593 primary breast tumor cytosols. Tumor levels of BCAR1 were correlated with relapse-free survival (RFS) and overall survival (OS) and compared with collected data on urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor 1 (PAI-1). Results: In tumor cytosols, BCAR1 protein levels varied between 0.02 and 23 ng/mg protein. BCAR1 levels exhibited a positive correlation with steroid hormone receptor levels, age and menopausal status, and uPA and PAI-1 levels. The level of BCAR1 (continuous or categorized as low, intermediate, or high) was inversely related with RFS and OS time. Multivariate analysis showed that BCAR1 levels contributed independently to a base model containing the traditional prognostic factors for both RFS and OS (both P < 0.0001). When added together with uPA and PAI-1 in the multivariate model, BCAR1 contributed independently of PAI-1 and was favored over uPA. Interaction tests allowed for additional analyses of BCAR1 protein levels in clinically relevant subgroups stratified by nodal and menopausal status. Conclusions: The quantitative BCAR1 protein level represents a prognostic factor for RFS and OS in primary breast cancer, independent of the traditional prognostic factors and the other novel marker PAI-1.

The prognostic value of components of the urokinase-type plasminogen activator (uPA) system, its receptor uPAR (CD87), and plasminogen activator inhibitors PAI-1 and PAI-2 is well established. We studied the predictive value of these proteolytic factors by evaluating the association of their tumor expression level and the efficacy of tamoxifen therapy in patients with recurrent breast cancer. The antigen levels of the four factors were determined by ELISA in cytosols prepared from estrogen receptor-positive primary breast tumors of 691 hormone-naive breast cancer patients with recurrent disease and treated with tamoxifen as first-line systemic therapy. High tumor levels of uPA (P < 0.001), uPAR (P < 0.01), and PAI-1 (P = 0.01) were associated with a lower efficacy of tamoxifen therapy. In the multivariable analysis, uPA (P < 0.001) provided additional information independent of the traditional predictive factors to predict benefit from tamoxifen therapy. High levels of uPA, uPAR, and PAI-1 predicted a shorter progression-free survival (PFS) on tamoxifen in an analysis of the first 9 months of therapy. However in the analysis during the total follow-up period, high PAI-2 levels (P = 0.01) showed a longer response to tamoxifen. In conclusion, uPA, uPAR, and PAI-1, components of the urokinase system, are predictive for the efficacy of tamoxifen therapy in patients treated for recurrent breast cancer. Knowledge of their tumor expression levels might be helpful for future individualized therapy protocols, including possible new-targeted therapies based on the interference in the urokinase system.

Purpose: In the present study, we investigated the association between tumor tissue levels of tissue inhibitor of metalloproteinase-1 (TIMP-1) and prognosis in patients with primary breast cancer and analyzed whether TIMP-1 may be useful as a prognostic marker in combination with urokinase plasminogen activator (uPA) and plasminogen activator inhibitor type-1 (PAI-1).Experimental Design: In cytosolic extracts of 2984 primary breast tumors, total levels of TIMP-1 were determined using an established, validated ELISA. Levels of uPA and PAI-1 have previously been determined in the extracts.Results: Univariate survival analysis showed a significant relationship between higher levels of TIMP-1 (continuous log-transformed variable) and poor prognosis [recurrence-free survival (RFS), overall survival (OS); P < 0.001]. Performing isotonic regression analysis, we identified a cut point to classify tumors as TIMP-1-low or TIMP-1-high. Using this cut point, high levels of TIMP-1 were significantly associated with shorter survival in univariate analysis, both in the total patient group (RFS, OS; P < 0.001), in the node-negative subgroup (RFS, hazard ratio = 1.28, P = 0.006), and in the node-positive subgroup (RFS, hazard ratio = 1.43, P < 0.001). In multivariate analysis, including uPA and PAI-1, TIMP-1 was significantly associated with shorter RFS, both when included as a continuous log-transformed (P = 0.03) and as a dichotomized variable (P = 0.002).Conclusions: This study validates previous findings that tumor tissue levels of TIMP-1 are associated with prognosis in patients with primary breast cancer. It confirms that TIMP-1 may be useful as a prognostic marker in combination with uPA/PAI-1 and adds substantial positive information on the use of TIMP-1 as a prognostic marker in breast cancer.

Contains fulltext : 58587.pdf (Publisher’s version ) (Open Access) BACKGROUND: The value of regular surveillance for breast cancer in women with a genetic or familial predisposition to breast cancer is currently unproven. We compared the efficacy of magnetic resonance imaging (MRI) with that of mammography for screening in this group of high-risk women. METHODS: Women who had a cumulative lifetime risk of breast cancer of 15 percent or more were screened every six months with a clinical breast examination and once a year by mammography and MRI, with independent readings. The characteristics of the cancers that were detected were compared with the characteristics of those in two different age-matched control groups. RESULTS: We screened 1909 eligible women, including 358 carriers of germ-line mutations. Within a median follow-up period of 2.9 years, 51 tumors (44 invasive cancers, 6 ductal carcinomas in situ, and 1 lymphoma) and 1 lobular carcinoma in situ were detected. The sensitivity of clinical breast examination, mammography, and MRI for detecting invasive breast cancer was 17.9 percent, 33.3 percent, and 79.5 percent, respectively, and the specificity was 98.1 percent, 95.0 percent, and 89.8 percent, respectively. The overall discriminating capacity of MRI was significantly better than that of mammography (P

Purpose: Germline mutations in the BRCA1 and BRCA2 genes confer increased susceptibility to ovarian cancer. There is evidence that tumors in carriers may exhibit a distinct distribution of pathological features, but previous studies on the pathology of such tumors have been small. Our aim was to evaluate the morphologies and immunophenotypes in a large cohort of patients with familial ovarian cancer. Experimental Design: We performed a systematic review of ovarian tumors from 178 BRCA1 mutation carriers, 29 BRCA2 mutation carriers, and 235 controls with a similar age distribution. Tumors were evaluated by four pathologists blinded to mutation status. Both morphological features and immunochemical staining for p53 and HER2 were evaluated. Results: Tumors in BRCA1 mutation carriers were more likely than tumors in age-matched controls to be invasive serous adenocarcinomas (odds ratio, 1.84; 95% confidence interval, 1.21–2.79) and unlikely to be borderline or mucinous tumors. Tumors in BRCA1 carriers were of higher grade (P < 0.0001), had a higher percentage solid component (P = 0.001), and were more likely to stain strongly for p53 (P = 0.018). The distribution of pathological features in BRCA2 carriers was similar to that in BRCA1 carriers. Conclusions: Use of pathological features can substantially improve the targeting of predictive genetic testing. Results also suggest that BRCA1 and BRCA2 tumors are relatively aggressive and may be expected to have poor prognosis, although this may be treatment dependent.

Heterozygous loss of relatively large chromosomal regions is a hallmark of the inactivation of tumour suppressor genes. Searching for deletions in cancer genomes therefore provides an attractive option to identify new tumour suppressor genes. Here, we have performed a genome-wide survey for regions exhibiting allelic loss in 24 commercially available breast cancer cell lines and four breast cancer xenografts, using microsatellite analysis. The assembled allelotype revealed an average fractional allelic loss of 0.34. A total of 19 arms had low allelic loss frequencies (

Using array analysis for screening RNA from BRCA1-mutated and sporadic breast tumors, we observed that AIGF/FGF-8 expression was lost in BRCA1-mutated breast tumors. Since this growth factor is induced by androgens, we studied the androgen receptor (AR) expression in BRCA-mutated tumors and in matched sporadic breast tumors.Paraffin embedded breast tumors of carriers of a BRCA1 mutation (n=41, median age of patients at time of surgery was 41 years [range 28-59 years]) or a BRCA2 mutation (n=14, median age 41 years [range 31-85 years]) were analyzed for the presence of ER-alpha, PR, P53 and AR using standard immunohistochemical techniques. All statistical tests used, Pearson chi2 and Fisher exact, were two-sided.The AR was only present in 12% of BRCA1-mutated tumors, with mutations located at the C-terminal half of the BRCA1-gene. The AR expression was significantly more prevalent, however, in a series of 61 sporadic breast tumors (80%) and in BRCA2-mutated tumors (50%). In contrast to an increased percentage of p53 positive cells, in 66% of the BRCA1-mutated tumors, the ER-alpha expression was observed only in 25% and the PR in 13% of these specimens. The three steroid hormone receptors were expressed in about half of the BRCA2-mutated specimens studied.Our data add to the emerging evidence that the biological phenotype of BRCA1-associated tumors may be different from BRCA2 and non-hereditary cases. The loss of the AR expression, as shown by immunohistochemistry, together with the observed loss of other steroid hormone receptors in BRCA1-mutated tumors may lead to a hormone-independent growth or to anti-hormone resistant growth of these tumors.

Purpose: To explore long-term psychosocial consequences of carrying a BRCA1/2 mutation and to identify possible risk factors for long-term psychological distress. Patients and Methods: Five years after genetic test disclosure, 65 female participants (23 carriers, 42 noncarriers) of our psychological follow-up study completed a questionnaire and 51 participants were interviewed. We assessed general and hereditary cancer-related distress, risk perception, openness to discuss the test result with relatives, body image and sexual functioning. Results: Carriers did not differ from noncarriers on several distress measures and both groups showed a significant increase in anxiety and depression from 1 to 5 years follow-up. Carriers having undergone prophylactic surgery (21 of 23 carriers) had a less favorable body image than noncarriers and 70% reported changes in the sexual relationship. A major psychological benefit of prophylactic surgery was a reduction in the fear of developing cancer. Predictors of long-term distress were hereditary cancer-related distress at blood sampling, having young children, and having lost a relative to breast/ovarian cancer. Long-term distress was also associated with less open communication about the test result within the family, changes in relationships with relatives, doubting about the validity of the test result, and higher risk perception. Conclusion: Our findings support the emerging consensus that genetic predisposition testing for BRCA1/2 does not pose major mental health risks, but our findings also show that the impact of prophylactic surgery on aspects such as body image and sexuality should not be underestimated, and that some women are at risk for high distress, and as a result, need more attentive care.

SummaryAge is an important factor in the development and spread of breast cancer. Stromal cells also contribute to breast cancer growth and metastasis through the production of extracellular matrix (ECM) modifiers such as urokinase type plasminogen activator (uPA), its receptor (uPAR), its inhibitors (PAI-1 and PAI-2), matrix metalloproteinases (MMPs), and growth factors, including the fibroblast and insulin-like growth factors (FGF’s and IGF’s). In the present study we have investigated whether breast fibroblasts aged in vitro through passage in culture display altered levels of the plasminogen activator system and growth factors that are known to modulate that system.With real-time RT-PCR we found that during passage human breast fibroblasts, whether derived from the tumour burden or from matched adjacent normal breast tissue, exhibited a consistent increase in PAI-1 and FGF-1 and a decrease in MMP-2 mRNA expression. In addition, in 5 out of 7 fibroblast strains we observed an induction of uPA expression in combination with a reduced IGF-1 expression. Interestingly, while during aging MMP-2 protein increased in all tumour-derived fibroblast strains, these protein levels were reduced in all normal-tissue-derived fibroblasts. No other clear-cut age-dependent alterations were found in the all-together 25 factors investigated. We furthermore demonstrate in one tumour-derived fibroblast strain that the increases in uPA and PAI-1 mRNA and MMP-2 protein production are inversely related to the telomere length. Artificially increasing telomere length in this fibroblast strain by expressing human telomerase reverse transcriptase (hTERT) prevented senescence and resulted in late passage cultures with early passage uPA, PAI-1 and MMP-2 levels.Our results show that aging accompanied by telomere loss induces PAI-1 and FGF-1 mRNA expression in all breast fibroblast strains, increases uPA and decreases IGF-1 mRNA expression in a subset, and increases MMP-2 protein expression only in tumour-derived breast fibroblasts. These age-induced levels of PAI-1, FGF-1, uPA and MMP-2 in stromal breast fibroblast could contribute to breast cancer progression.

SummaryIn breast stroma urokinase plasminogen activator (uPA) is predominantly expressed by fibroblasts located in the near vicinity of tumor cells, and fibroblast-derived insulin-like growth factor-1 (IGF-1) may be involved in inhibiting the expression of uPA in these fibroblasts. To investigate a possible role for fibroblast growth factors (FGFs), we evaluated the expression of components of the PA system and the IGF system in normal and tumor-tissue-derived human breast fibroblasts exposed to various FGFs in vitro. mRNA analysis revealed that FGF-1, FGF-2 and FGF-4 induced the mRNA expression levels of uPA, tPA, uPAR, PAI-1 and PAI-2, and reduced those of IGF-1, IGF-1R, IGF-2R and IGFBP-4, without significantly affecting the levels of IGFBP-3, IGFBP-5 and IGFBP-6 mRNA. Concerning the expression of IGF-2 mRNA, the effects mediated by FGF-1, FGF-2 and FGF-4 were divergent. In general, the effects elicited by FGF-1 on the various mRNA levels studied were rapid and short-term. Those mediated by FGF-2 overall lagged behind but were longer-lasting. For FGF-4 an in between pattern was observed. Blocking transcription and translation demonstrated that a) both the FGF-1 and FGF-2 induced effects were the result of altered gene transcription or mRNA stability, b) the short-term effects mediated by FGF-1 and FGF-2 required de novo protein synthesis, and c) the long-term effects elicited by FGF-2 did not depend on de novo protein synthesis during the first 24 h, but were triggered by proteins produced or made available thereafter. The data presented propose that of the FGFs studied (FGF-1, -2, -4, -5, and -7), FGF-2 is the most attractive target for therapeutical strategies aimed at diminishing the contribution of stromal fibroblasts in the PA-directed breast tumor proteolysis.

The product of the Bcar1/p130Cas (breast cancer resistance/p130Crk-associated substrate) gene causes resistance to antiestrogen drugs in human breast cancer cells in vitro. To investigate its role in clinical breast cancer, we determined the levels of Bcar1/p130Cas protein in a large series of primary breast carcinomas.We measured Bcar1/p130Cas protein in cytosol extracts from 937 primary breast carcinomas by western blot analysis. The levels of Bcar1/p130Cas protein were tested for associations and trends against clinicopathologic and patient characteristics, the lengths of relapse-free survival and overall survival (n = 775), and the efficacy of first-line treatment with tamoxifen for recurrent or metastatic disease (n = 268).Bcar1/p130Cas levels in primary tumors were associated with age/menopausal status and the levels of estrogen receptor and progesterone receptor. In univariate survival analysis, higher Bcar1/p130Cas levels were associated with poor relapse-free survival and overall survival (both two-sided P =.04; log-rank test for trend). In multivariate analysis, a high level of Bcar1/p130Cas was independently associated with poor relapse-free survival and overall survival. The response to tamoxifen therapy in patients with recurrent disease was reduced in patients with primary tumors that expressed high levels of Bcar1/p130Cas. In multivariate analysis for response, Bcar1/p130Cas was independent of classical predictive factors, such as estrogen receptor status, age/menopausal status, disease-free interval, and dominant site of relapse.Patients with primary breast tumors expressing a high level of Bcar1/p130Cas protein appear to experience more rapid disease recurrence and have a greater risk of (intrinsic) resistance to tamoxifen therapy. Thus, measurement of Bcar1/p130Cas may provide useful prognostic information for patients with primary or metastatic breast cancer.

CD44 is a family of cell surface transmembrane glycoproteins members which differ in the extracellular part by sequences derived by alternative splicing of 10 variant exons (v1– v10). CD44 proteins containing such variant sequences have been implicated in tumor metastasis formation. Here, we have evaluated the expression of CD44 variants by immuno-histochemistry in primary breast cancer samples of 237 node-negative and 230 node-positive patients. For the analysis of samples derived from node-negative patients, the exon-specific antibodies used were DIII, vff7 and vff18 (v6), vff17 (v7/v8), fw11.24 (v9) and vff16 (v10). With the different antibodies which recognize v6 epitopes, the majority of tumors were positively stained (≥65% of the tumors) with varying intensities. Thirty-nine percent of the tumors were positively stained with the antibody vff16, and approximately half of the tumors with the antibodies vff17 and fw11.24. The expression of CD44 v6 epitopes in tumors from node-negative patients was associated with a favorable prognosis, both upon univariate and multivariate analysis. The expression of CD44 v7/8, v9 or v10 epitopes was not significantly related with relapse-free survival. Samples from node-positive patients were only examined with the antibodies vff7, vff17 and vff18. The staining with none of these antibodies was correlated with the length of relapse-free survival of the patients. Our data suggest that, generally, the usefulness of knowledge of CD44 variant expression is of limited value for assessing the risk of relapse in patients with primary breast cancer. However, the expression of exon v6 of CD44 may be a marker to identify patients with a relatively favorable prognosis in node-negative patients. Int. J. Cancer (Pred. Oncol.) 84:209–215, 1999. © 1999 Wiley-Liss, Inc.

Human breast fibroblasts have been shown to express urokinase-type plasminogen activator (uPA). This suggests that fibroblasts are actively involved in the process of uPA-directed breast tumor proteolysis. To investigate a possible role for the insulin-like growth factors (IGFs) in regulating uPA expression in human breast fibroblasts, we correlated the expression of uPA with the expression of IGF-1 and IGF-2 in a paired panel of normal and tumor tissue-derived human breast fibroblasts in vitro. Analysis of reverse transcribed polymerase chain reaction (RT–PCR) amplified mRNA revealed that the tumor-derived fibroblast strain expressed significantly more basal uPA mRNA and significantly less IGF-1 mRNA when compared to their normal counterpart. The expression of basal IGF-2 mRNA did not differ between these cultures. For both normal and tumor tissue-derived fibroblasts, cytokine- and growth factor-induced steady-state levels of uPA and IGF-1 mRNA were inversely related. No such correlation was found for uPA and IGF-2 mRNA. While exogenously added IGF-1 decreased the amount of uPA mRNA transcripts similarly in both normal and tumoral fibroblasts, exogenously added uPA decreased the amount of IGF-1 mRNA transcripts only in tumor tissue-derived fibroblasts. These data suggest that in human breast fibroblasts IGF-1 controls the expression of uPA and that, possibly due to an altered sensitivity to uPA, tumor-associated fibroblasts have escaped this local control mechanism.

It has been shown that, in breast stroma, urokinase-type plasminogen activator (uPA) mRNA is predominantly expressed by myofibroblasts located at the invasive areas of the tumor. To examine which factors present in a tumor environment are candidates responsible for the induction of these uPA-producing myofibroblasts, we studied in vitro the capacity of a paired panel of normal and tumor-derived human breast fibroblasts to produce uPA protein and the myofibroblast marker alpha-smooth-muscle-actin (alpha-SMA) in response to various cytokines implicated in the process of tissue-remodeling during malignant transformation. We found that fibroblasts produced increased amounts of uPA protein after exposure to a-FGF, b-FGF, EGF, PDGF-BB, and IFN-gamma, were unaffected in this respect by IL-6, M-CSF, GM-CSF and Oncostatin M, and produced decreased amounts of uPA protein after exposure to IL-1alpha, TNF-alpha, IGF-I, and IGF-II. None of these cytokines were able to induce a striking increase in the fraction of alpha-SMA-positive fibroblasts. On the other hand, 25 pM TGFbeta1 increased the fraction of alpha-SMA-positive fibroblasts 5-fold in both normal and tumor-tissue-derived fibroblasts. Nonetheless, the normal-derived fibroblasts were unaffected in their uPA-producing capacity by TGFbeta1, and the tumor-derived fibroblasts produced decreased amounts of uPA protein after exposure to this cytokine, implying that at least in vitro the myofibroblast phenotype is not a prerequisite for the production of uPA by human breast fibroblasts. In addition, we established that the basal-uPA-production of both normal and tumor-derived fibroblasts was increased by autocrinely produced b-FGF-like activity, and that the basal-uPA-production of at least the normal-derived fibroblasts was decreased by autocrinely produced IGF-like activity. Altogether, our data suggest an active role for fibroblasts in the process of uPA-directed breast tumor proteolysis.

The urokinase-type plasminogen activator (uPA) may be considered as a key enzyme in the processes of cancer cell invasion and metastasis. Evidence has been presented that, in breast stroma, uPA is expressed predominantly by myofibroblasts located at the invasive areas of the tumor. To examine whether transforming growth factor type-1 (TGF beta(1)) produced by breast-carcinoma cells is a candidate responsible for the induction of uPA-producing myofibroblasts, we studied in vitro the capacity of normal and tumor-derived human breast fibroblasts to express uPA and the myofibroblast marker alpha-smooth-muscle actin in response to TGF beta(1). Next, we compared these influences with those elicited by factor(s) released by epithelial-cancer cells. In all 8 fibroblast strains tested, TGF beta(1) induced a similar concentration-dependent increase in the fraction of alpha-smooth-muscle-actin-positive fibroblasts. While uPA expression was decreased by TGF beta(1) in most of the fibroblast strains, 2 strains were relatively insensitive to TGF beta(1) in this respect. Although factors present in media conditioned by non-uPA-producing epithelial-tumor cells could trigger fibroblasts to become potent producers of uPA, the TGF beta(1) content of the conditioned media were linked to the differential effects of externally added TGF beta(1) with respect to uPA expression. The data demonstrate that, although fibroblasts may utilize TGF beta(1) secreted by tumor cells to differentiate into myofibroblasts, tumor cells secrete factor(s) other than TGF beta(1) ultimately responsible for the generation of powerful uPA-producing fibroblasts.

As in many human malignancies, TP53 mutations are the most common genetic alterations in malignant human ovarian tumours. An approach often used in the determination of TP53 status is immunohistochemical staining of the protein. Non-missense mutations, especially those of the null type, causing premature termination codons and resulting in truncated proteins, may often not be detectable by immunohistochemistry. Therefore, current estimates of TP53 alterations in ovarian cancer may be inaccurate. By using polymerase chain reaction-single strand conformation polymorphism analysis and sequencing techniques, we have found a high prevalence of TP53 non-missense mutations in exons 5-8 in ovarian tumour specimens from patients from the southwestern part of The Netherlands. Twenty-nine of 64 tumours showed mutations, of which 10 were non-missense mutations. The majority (9 of 10) of these non-missense mutations, including 7 nonsense mutations and 2 frameshift deletions, were null type mutations and could not be detected by immunohistochemical staining. Five of the 7 nonsense mutations were mutations at codon 213 (Arg-->Stop). The nature of the high prevalence of this nonsense mutation in our series of ovarian carcinomas remains unknown. In addition to the 9 null type mutations, a splice junction mutation was encountered. In conclusion, we have observed a high prevalence (13%) of ovarian tumours with null type mutations in exons 5-8 that did not result in immunostaining. Our data suggest that, especially in ovarian cancer, immunological assessment of TP53 is not an adequate tool to study TP53 alteration. A frequent nonsense mutation at codon 213 in 5 (8%) of 64 tumour specimens represents an important finding.

Insulin-like growth factors are involved in the paracrine growth regulation of human breast tumor cells. IGF2 is imprinted in most tissues, and shows expression of the paternal allele only. To investigate whether disruption of this monoallelic IGF2 expression is involved in breast cancer development, a series of primary tumors and adjacent, histologically normal, breast tissue samples, as well as matched primary in vitro fibroblast cultures were studied. Biallelic expression (partial) of IGF2 was found in the majority of in vivo samples, and corresponding fibroblast cultures, while monoallelic expression was found in a normal breast sample. In contrast, H19, a closely apposed, but reciprocally imprinted gene, assumed to be regulated by a common control element, showed retention of monoallelic H19 expression in all in vivo and in the majority of in vitro samples. These data indicate that IGF2, but not H19, is prone to loss of imprinting in breast cancer.

To date, more than 300 distinct small deletions, insertions and point mutations, mostly leading to premature termination of translation, have been reported in the breast/ovarian-cancer susceptibility gene BRCA1. The elevated frequencies of some mutations in certain ethnic subpopulations are caused by founder effects, rather than by mutation hotspots. Here we report that the currently available mutation spectrum of BRCA1 has been biased by PCR-based mutation-screening methods, such as SSCP, the protein truncation test (PTT) and direct sequencing, using genomic DNA as template. Three large genomic deletions that are not detected by these approaches comprise 36% of all BRCA1 mutations found in Dutch breast-cancer families to date. A 510-bp Alu-mediated deletion comprising exon 22 was found in 8 of 170 breast-cancer families recruited for research purposes and in 6 of 49 probands referred to the Amsterdam Family Cancer Clinic for genetic counselling. In addition, a 3,835-bp Alu-mediated deletion encompassing exon 13 was detected in 4 of 170 research families, while an deletion of approximately 14 kb was detected in a single family [corrected]. Haplotype analyses indicated that each recurrent deletion had a single common ancestor.

Thymidine kinase (TK) and thymidylate synthase (TS) play a key role in, respectively, the salvage and the de novo DNA synthesis pathways. TS is a crucial target for 5-fluoroura-cil(5-FU) and may also influence methotrexate(MTX) efficiency. Tyrosine kinase(TPK) has been associated with the cytoplasmic domain of growth factor receptors as well as oncoproteins. We investigated whether TK, TS and TPK are predictive factors for drug sensitivity evaluated in terms of relapse-free improvement in breast-cancer patients receiving adjuvant chemotherapy. TK, TS and TPK activities were determined in the cytosols of 154 node-positive primary breast cancers. All patients received 5-FU containing adjuvant chemotherapy. Measurements were performed using radioenzymatic methods. The levels of TK were correlated with those of TS and TPK. The levels of TS and TPK were less strongly correlated with each other. High TK levels were more often found in larger tumours, and the levels of both TK and TPK were negatively correlated with those of PgR. Patients whose tumours contained high levels of TK had increased risks of relapse and death. TS was not of prognostic value, while a high level of TPK was associated with early death. In Cox analysis, TK and TPK retained their independent prognostic value. While target enzyme activities on the de novo DNA synthesis pathway could determine response to anti-metabolics mainly inhibiting this pathway, high activities on the alternative salvage pathway could circumvent induced growth inhibition.

The authors report a prospective study comparing magnetic resonance imaging (MRI) with mammography for screening in women having an estimated cumulative lifetime risk of breast cancer of at least 15% because of a familial or genetic predisposition. A clinical breast examination was done every 6 months and imaging studies (with independent readings) every 12 months. The study group included 1909 eligible women 25 to 70 years of age, 358 of whom were carriers of germline mutations. A BRCA1 mutation was by far the most common pathogenic mutation. A total of 51 tumors were found during a median follow up of 2.9 years. There were 44 invasive cancers, 6 ductal carcinomas in situ, and 1 lymphoma. An additional woman had a lobular carcinoma in situ. The overall detection rate for all breast cancers was 9.5 per 1000 woman-years at risk. Twenty-two of the 32 cancers found by magnetic resonance imaging (MRI) were not found on mammograms. Eight of 13 cancers missed by MRI were visible on mammograms. Sensitivity figures for all breast cancers were 18% for clinical breast examination, 40% for mammography, and 71% for MRI. Twenty-two of 41 cancers found by screening were detected at the first imaging screening. Specificity figures for invasive breast cancer were 98% for clinical beast examination, 95% for mammography, and 90% for MRI. The overall discriminating ability of MRI significantly exceeded that of mammography. Invasive cancers measuring 10 mm or less in diameter were significantly more frequent in women under surveillance than in control women. More than half of control women but only approximately one fifth of screened women had positive axillary lymph nodes and/or micrometastases. These findings indicate that MRI is more sensitive than mammography for detecting beast cancers in women at increased risk because of inherited susceptibility.

Elevated levels of expression of the urokinase-type plasminogen activator (uPA) and its inhibitor PAI-1 have shown to be related to poor prognosis in a variety of cancer types. In the present study, cytosolic levels of uPA and PAI-1 were determined with enzyme-linked immunosorbent assays in cytosols prepared from 244 human ovarian tissues of different histological sub-types. Both uPA and PAI- I were significantly associated with the malignant progression of ovarian tissues; the levels were increased going from normal tissue, via benign and borderline adenomas, to primary and metastatic adenocarcinomas. For the 90 patients (34 early-stage and 56 patients with advanced disease) from whom the primary adenocarcinoma tissues were examined, uPA and PAI- I levels were evaluated for their association with clinicopathological parameters and with progression-free and overall survival. Neither uPA nor PAI-1 were significantly associated with the age of the patient, FIGO stage, tumor grade, tumor rest, the presence of ascites, or with progression-free or overall survival. On the other hand, age, FIGO stage/tumor rest and the presence of ascites, were significantly related to the length of both progression-free and overall survival in univariate analyses. Tumor grade was of prognostic significance in the analysis for progression-free survival, but not for overall survival. After adjustment for FIGO stage/tumor rest, only age retained its prognostic significance, in the analysis for progression-free survival and in that for overall survival.

We intended to establish the frequency of exon-specific TP53 gene alterations and the relation to patient and tumor characteristics and clinical outcome of patients with breast cancer. By using polymerase chain reaction-single-strand conformation polymorphism analysis (PCR-SSCP) and sequencing techniques, TP53 gene alterations were found in 59 (32%) of the 187 samples studied. Most of the TP53 changes (37%) were observed in exon 7. In patients with known follow up (median, 107 months), there was no significant association of the frequency of TP53 mutation with menopausal or nodal status, tumor size, or progesterone receptor status. TP53 gene alterations were more frequently present in estrogen receptor (ER)-negative (ER-) tumors (P = 0.04) and in tumors with an amplified HER2/NEU oncogene (P = 0.03). Univariate analysis showed that patients with a mutated TP53 in their primary tumors had shorter relapse-free (P = 0.01) and overall (P = 0.03) survival. Patients with a TP53 gene mutation in exon 8 may be identified as having a particularly rapid rate of relapse. In Cox multivariate regression analysis, which included age, menopausal status, lymph node status, tumor size, steroid-hormone-receptor status, and oncogene amplifications, both TP53 gene alteration and MYC amplification independently predicted poor prognosis, with relative hazard rates for TP53 and MYC of 1.8 and 1.6, respectively, in analysis for relapse-free survival and of 1.7 and 1.6, respectively, in analysis for overall survival.

In a previous short-term study (4 months) we found that Sandostatin, when administered prophylactically, inhibited growth of putative pre-neoplastic ductular lesions induced in hamster pancreas by N-nitrosobis(2-oxopropyl)amine (BOP), but not of acinar lesions induced in rat pancreas by azaserine. The present long-term (12 months) study was carried out to investigate the effects of Sandostatin (3 μg/day), alone and in combination with orchiectomy, on pancreatic carcinogenesis in azaserine-treated rats and BOP-treated hamsters. In order to mimic therapy in humans, treatment of the animals started 4 months after the last injection with carcinogen, when (pre)neoplastic lesions had already developed. After treatment with Sandostatin for 8 months, rats developed fewer pancreatic atypical acinar cell nodules and tumours than those not treated with Sandostatin. Moreover, multiplicity of (pre)neoplastic acinar lesions was also lower in orchiectomized rats than in intact rats. However, Sandostatin treatment did not enhance the inhibitory effect of surgical castration on pancreatic carcinogenesis in rats. In hamsters that were both orchiectomized and treated with Sandostatin, the development of borderline lesions was significantly inhibited, whereas such an effect was not present in hamsters that were either surgically castrated or treated with Sandostitin alone. In Sandostatin-treated hamsters a significantly lower number of microcarcinomas was found than in hamsters not treated with Sandostatin. The present findings suggest that Sandostatin, particularly in combination with surgical castration, might be of therapeutic value for treatment of ductular pancreatic tumours. Chemicals/CAS: Anticarcinogenic Agents; Azaserine, 115-02-6; Carcinogens; Nitrosamines; nitrosobis(2-oxopropyl)amine, 60599-38-4; Octreotide, 83150-76-9

A stromal fibroblast-mediated paracrine regulation of epithelial tumor cell proliferation and differentiation plays an important role in the development and progression of breast tumors. We have studied the paracrine growth regulation of various phenotypically different breast cancer cell lines using conditioned serum-free media (C-SFM) from primary breast fibroblasts. Fibroblast cultures were established from malignant primary tumors and adjacent normal breast tissue, benign fibroadenomas, cosmetic reduction mammoplasties and breast skin tissues. All fibroblast-conditioned media were shown to stimulate the proliferation of breast cancer cell lines. However, the C-SFM-induced MCF-7 proliferative response was shown to be significantly higher than the proliferative response observed with any of the other cell lines tested. More importantly, the MCF-7 proliferative response obtained with malignant tumor tissue fibroblast C-SFM was shown to be significantly higher than the response to C-SFM from paired (and unpaired) normal adjacent breast tissue fibroblasts. The MCF-7 proliferative response to fibroblast C-SFM from normal tissue (adjacent to the tumor) was further shown to be comparable to the MCF-7 response using benign or reduction mammoplastic tissue fibroblast C-SFM. In addition, we show that IGFs are only partly responsible for the observed proliferative effect of the C-SFMs, while EGF, TGF alpha and basic-FGF are shown not to be involved. We conclude that stromal fibroblasts can differentially regulate breast cancer cell proliferation. Both the fibroblast's tissue source as well as the target tumor cell's phenotype will determine the extent of the proliferative response.

We have analyzed, by a combination of mutation and linkage analysis, the genetic basis of 22 breast cancer families in which at least 4 cases of either breast cancer diagnosed under the age of 60 or ovarian cancer had occurred. Chain-terminating mutations in BRCA1 were evidenced in 6 families, and posterior probabilities of > 0.90 of being linked to BRCA1 in 3. The breast versus ovarian cancer ratio in these 9 families was approximately 2:1. Among the remaining 13 families, significant linkage to markers flanking BRCA2 was established in the admixture test with a maximum multipoint lod score of 3.38, but there was no statistical evidence for genetic heterogeneity. The breast:ovarian cancer ratio in these families was 7:1, suggesting BRCA2 confers a much lower risk for ovarian cancer than does BRCA1. These results suggest that BRCA2 will explain a significant proportion of hereditary breast cancer in the Netherlands, and, together with BRCA1, account for the majority of all high-risk families