Synergistic tumor suppressor activity of BRCA2 and p53 in a conditional mouse model for breast cancer.

Inheritance of one defective BRCA2 allele predisposes humans to breast cancer. To establish a mouse model for BRCA2-associated breast cancer, we generated mouse conditional mutants with BRCA2 and/or p53 inactivated in various epithelial tissues, including mammary-gland epithelium. Although no tumors arose in mice carrying conditional Brca2 alleles, mammary and skin tumors developed frequently in females carrying conditional Brca2 and Trp53 alleles. The presence of one wildtype Brca2 allele resulted in a markedly delayed tumor formation; loss of the wildtype Brca2 allele occurred in a subset of these tumors. Our results show that inactivation of BRCA2 and of p53 combine to mediate mammary tumorigenesis, and indicate that disruption of the p53 pathway is pivotal in BRCA2-associated breast cancer.

The value of cytokeratin immunohistochemistry in the evaluation of axillary sentinel lymph nodes in patients with lobular breast carcinoma.

BACKGROUND: Cytokeratin immunohistochemistry (IHC) reveals a higher rate of occult lymph node metastases among lobular carcinomas than among ductal breast cancers. IHC is widely used but is seldom recommended for the evaluation of sentinel lymph nodes in breast cancer patients. OBJECTIVE: To assess the value of cytokeratin IHC for the detection of metastases in sentinel lymph nodes of patients with invasive lobular carcinoma. METHODS: The value of IHC, the types of metastasis found by this method, and the involvement of non-sentinel lymph nodes were analysed in a multi-institutional cohort of 449 patients with lobular breast carcinoma, staged by sentinel lymph node biopsy and routine assessment of the sentinel lymph nodes by IHC when multilevel haematoxylin and eosin staining revealed no metastasis. RESULTS: 189 patients (42%) had some type of sentinel node involvement, the frequency of this increasing with increasing tumour size. IHC was needed for identification of 65 of these cases: 17 of 19 isolated tumour cells, 40 of 64 micrometastases, and 8 of 106 larger metastases were detected by this means. Non-sentinel-node involvement was noted in 66 of 161 cases undergoing axillary dissection. Although isolated tumour cells were not associated with further lymph node involvement, sentinel node positivity detected by IHC was associated with further nodal metastases in 12 of 50 cases (0.24), a proportion that is higher than previously reported for breast cancer in general. CONCLUSIONS: IHC is recommended for the evaluation of sentinel nodes from patients with lobular breast carcinoma, as the micrometastases or larger metastases demonstrated by this method are often associated with a further metastatic nodal load.

Differences in breast cancer risk factors to neu (c-erbB-2) protein overexpression of the breast tumor.

To investigate whether overexpression of the neu protein in breast tumors differentiates risk factor patterns for breast cancer, neu protein overexpression was determined in 296 breast carcinomas of patients participating in an ongoing population-based case-control study. Risk factor information on these patients and 737 controls was obtained during home interviews. Most breast cancer risk factors showed similar associations with neu-positive and neu-negative tumors, but remarkable differences were found for breast-feeding and age at first full-term pregnancy. In contrast to the slightly protective effect of breast-feeding in the neu-negative group, the risk of neu-positive breast cancer was 4.2-fold increased in women who ever breast-fed. Increasing age at first full-term pregnancy was positively associated with both neu-positive and neu-negative breast cancer, but the association was about 2 times stronger for neu-positive tumors. We conclude that neu oncogene overexpression of the breast tumor seems to be associated with a distinct risk factor pattern.

Risk factors in breast-conservation therapy.

PURPOSE: To identify clinical and pathologic factors associated with an increased risk of local recurrence following breast-conservation therapy (BCT) to assess the safety of this procedure for all subgroups of patients. PATIENTS AND METHODS: The study population consisted of 1,026 patients with clinical stage I and II breast cancer treated between 1979 and 1988 at the Netherlands Cancer Institute. The BCT regimen consisted of local excision and axillary lymph node dissection (ALND) followed by whole-breast irradiation to a total dose of 50 Gy in 2-Gy fractions and boost irradiation (mostly by iridium implant) of 15 to 25 Gy. RESULTS: With a median follow-up duration of 66 months, the actuarial breast recurrence rate was 4% at 5 years, counting all breast recurrences. Univariate analysis showed seven factors to be associated with an increased risk of local recurrence; age, residual tumor at reexcision, histologic tumor type, presence of any carcinoma-in-situ component, vascular invasion, microscopic margin involvement, and whole-breast radiation dose. Three factors remained independently significant after proportional hazard regression analysis: age, margin involvement, and the presence of vascular invasion. When the analysis was repeated, but counting only those breast recurrences that occurred before regional or distant failures, only young age and vascular invasion were independent predictive factors. In the third analysis, factors predicting the necessity of local salvage treatment were analyzed. In this analysis, the possible bias in the former analysis caused by censoring actuarial methods was avoided. The results were the same as in the second analysis, showing young age and vascular invasion as the only independent predictive factors. Breast recurrence rates were 6% for patients less than 40 years of age and 8% for patients with tumors showing vascular invasion. In the absence of risk factors, the breast recurrence rate is only 1% at 5 years. CONCLUSION: Slightly higher recurrence rates were found in patients less than 40 years of age and in patients with tumors showing vascular invasion. The role of margin involvement is uncertain.

Prognostic factors for survival after breast conserving therapy for stage I and II breast cancer. The role of local recurrence.

Risk factors for local recurrence (LR) in a univariate analysis had a significant correlation with survival. Local and distant failure could not be regarded as independent events. We undertook a multivariate survival analysis to study the relation between LR and survival. In a retrospective study of 1026 patients treated with tumorectomy, axillary dissection and radiotherapy, factors associated with disease-specific survival (DSS) were analysed. Actuarial estimates for DSS are 91% at 5 years and 86% at 10 years. The multivariate analysis revealed five factors: clinical stage, number of affected axillary nodes, histological grade, degree of tubule formation and left-sided primary tumour. Controlling for these factors, LR appeared to be significantly correlated with DSS. The hazard rate of DSS was estimated to increase by a factor 8.8 (95% confidence interval 4.6-16.8) upon occurrence of a LR. Local recurrence per se, apart from the identified prognostic factors, is a risk factor for DSS. The exact mechanism by which LR has an influence on survival cannot be clarified from these data.

Can patient-, treatment- and pathology-related characteristics explain the high local recurrence rate following breast-conserving therapy in young patients?

The aim of this study was to identify patient-, tumour- or treatment-related factors associated with young age that might explain the higher risk of ipsilateral breast recurrence that occurs after breast-conserving therapy (BCT) in young breast cancer patients. In the 'boost versus no boost trial', 5569 early-stage breast cancer patients were entered. All patients underwent tumorectomy followed by whole breast irradiation of 50 Gy. Patients having a microscopically complete excision were randomised between receiving no boost or a 16-Gy boost, while patients with a microscopically incomplete excision were randomised between receiving a boost dose of 10 or 26 Gy. The 5-year local control rate was 82% for patients 60 years of age (P<0.0001). In young patients, the tumour was significantly larger and more often oestrogen and progesterone receptor-negative. Invasive carcinoma and the intraductal component were more often of a high grade. The intraductal component was more frequently incompletely resected in young patients. Re-excisions were performed more often (most probably due to a more frequent incomplete excision at the first attempt). The total volume of breast tissue removed at the tumorectomy was smaller in the younger patient group, even after including the volume removed during re-excision. When relating all these parameters (including age itself) to local control, the multivariate analysis stratified by treatment showed that age was the only independent prognostic factor for local control (P=0.0001). Including the boost treatment as a separate covariate, the analysis retained age and boost treatment as significant factors related to local control (P<0.0001). It was shown that the boost dose significantly reduced the 5-year local recurrence rate from 7 to 4% for patients with a complete excision (P<0.001). For patients 40 years of age or younger, the boost dose reduced the local recurrence rate from 20 to 10% (P=0.002). This large European Orgnaization for Research and Treatment of Cancer (EORTC) trial demonstrated an increased local recurrence rate in young patients. Although several associations between patient, tumour and treatment factors and age were found, that might explain the high local recurrence rate in the younger patients, it appears that age itself and the boost dose were the only factors that were independently related to local control.

Growth arrest associated changes of mRNA levels in breast cancer cells measured by semi-quantitative RT-PCR: potential early indicators of treatment response.

To find early and sensitive indicators of treatment response in breast cancer, we studied the mRNA levels of proliferation-related genes during growth arrest of the human breast cancer cell lines T47D and MCF7. A sensitive reverse transcriptase-PCR (RT-PCR) technique was used in order to monitor gene expression in small samples of cells. Estrogen-depletion and treatment with tamoxifen effectively induced a G1-arrest in both cell lines, accompanied by a decrease of the mRNA levels of histone H4, cyclin A, cyclin D1, and c-myc. Cyclin A expression decreased most strongly: up to 32-fold within 7 days. The expression of c-fos and WAF1 increased during growth arrest. In conclusion, significant changes of the levels of proliferation-related mRNAs, induced by growth arrest, can be measured in small samples of breast carcinoma cells using RT-PCR. Especially the decrease of the cyclin A mRNA level seems a potential early indicator of clinical response to tamoxifen therapy in breast cancer patients.

Limited prognostic value of cellular DNA content to classical and morphometrical parameters in invasive ductal breast cancer.

This retrospective study evaluates several prognostic factors in 63 patients with invasive ductal breast cancer. Special attention is paid to the additional prognostic value of cellular DNA content to the previously developed and evaluated quantitative features mitotic activity index (MAI) and multivariate morphometric prognostic index (MPI). Follow-up was monitored for at least 50 months (median survival, 78 months) and only patients who died of distant metastases were included. The results show that the MAI is the strongest prognostic factor of all single features (Mantel-Cox, P = 0.008). Although patients with a diploid or tetraploid tumor tended to have a better prognosis than those with an aneuploid cancer, the DNA index as a single parameter was a weak prognosticator in the univariate survival analysis (Mantel-Cox, P = 0.24). Within the diploid and tetraploid tumors the MAI could distinguish patients with a favorable and unfavorable prognosis prediction (chi-square, P = 0.01). For aneuploid tumors this was not possible. Analysis of combined features revealed that the MPI has a high prognostic value (Mantel-Cox, P = 0.0015), thus confirming other studies. A linear combination of the nuclear DNA index, MAI, nodal status, and mean nuclear area showed only a slight improvement in prognosis prediction compared with the MPI (Mantel-Cox, P = 0.0005); with this rule, the classification of the patients was more in agreement with the actual outcome in 4% of the cases. The gain was in the poor prognosis group. These results suggest that the additional prognostic value of nuclear DNA content is restricted when compared with the morphometric prognostic factors. Further studies on a larger number of patients are required to confirm these findings.

A method to monitor mRNA levels in human breast tumor cells obtained by fine-needle aspiration.

A method based on the reverse transcriptase-polymerase chain reaction (RT-PCR) was developed that allows the determination of relative mRNA expression levels in fine-needle aspirates from human tumors. The method was developed for the c-erbB-2 gene, using the porphobilinogen deaminase (PBGD) gene as an internal standard. It was validated for mRNA isolated from cell lines and for material obtained by fine-needle aspiration from human breast cancer. Gene expression levels were determined by measuring the activity of radiolabeled RT-PCR-amplified gene-specific bands with a phosphor imager. At least four points are measured on the log-linear part of the amplification cycle versus signal intensity curves, and subsequently the distance between the curves of the gene of interest and that of an internal standard gene is used to calculate the relative expression levels. The method worked equally well with the BRCA1 gene, illustrating that it can be generalized to other genes. The method is suitable to measure or monitor semiquantitively gene expression levels in accessible human tumors in situ.

Lymphedema-induced lymphangiosarcoma.

A series of eight patients with chronic lymphedema-related lymphangiosarcoma is presented. All but one case showed a typical rapid progression and fatal outcome, as has been reported in other series. In one patient the lymphangiosarcoma developed on the chest wall, the axilla and the arm where persisting lymphedema and fibrosis occurred after bilateral mastectomy and bilateral postoperative radiotherapy. In this patient an asymptomatic course and slow locoregional progression of lesions was seen. The clinical picture, the etiological considerations and the therapeutic options of the lymphedema-induced lymphangiosarcoma with regard to the literature are discussed.

The Multicenter Morphometric Mammary Carcinoma Project (MMMCP). A nationwide prospective study on reproducibility and prognostic power of routine quantitative assessments in The Netherlands.

The Multicenter Morphometric Mammary Carcinoma Project (MMMCP) has been set up to investigate prospectively the prognostic value and reproducibility of routine assessments of the morphometric Multivariate Prognostic Index (MPI) and other quantitative parameters in comparison with classical prognosticators and steroid receptors in breast cancer patients. In this project, 34 hospitals participate, divided over six geographically different regions. Of each patient entering in the study, multiple clinical and classical pathological parameters (including tumor size and lymph node status) as well as several quantitative parameters such as mean nuclear area, DNA index and mitotic activity index will be evaluated. Of all patients, the MPI will be assessed with tumour size, lymph node status and mitotic activity index. The quantitative assessments are performed in all consecutive breast cancers which enter the participating pathology laboratories, and all measurements are controlled in Amsterdam. The patient intake time will be from January 1, 1988 until January 1, 1990. It is expected that 3000 patients will enter in this study. Follow up data will be gathered up to 10 years. However, two to five years after the initiation of the Project, a first evaluation of the reproducibility and prognostic significance of routine MPI and other assessments in breast cancer patients will be possible. A detailed description of this project is given.

Glioblastoma multiforme in a dermoid cyst of the ovary. A case report.

Malignant change to glioblastoma multiforme in a mature cystic teratoma (dermoid cyst) is exceptionally rare. Besides a report on such a case we give a brief review of the literature on this subject and a comment on treatment. The reported case is about a 41-year-old woman with a mature cystic teratoma of the ovary with transformation to glioblastoma multiform. The tumor was limited to the ovary and completely excised by salpingo-oophorectomy. Three and a half years after surgery the patient is alive without evidence of recurrent disease. For limited stage Ia tumors we found support in the literature for expectant management after surgery, without adjuvant chemotherapy.

Tumor characteristics and detection method in the MRISC screening program for the early detection of hereditary breast cancer.

In the MRISC study, women with an inherited risk for breast cancer were screened by a 6-month clinical breast examination (CBE) and yearly MRI and mammography. We found that the MRISC screening scheme could facilitate early breast cancer diagnosis and that MRI was a more sensitive screening method than mammography, but less specific. In the current study we investigated the contribution of MRI in the early detection of breast cancer in relation to tumor characteristics. From November 1999 to October 2003, 1909 women were included and 50 breast cancers were detected, of which 45 were evaluable and included in the current study. We compared the characteristics of tumors detected by MRI-only with those of all other (non-palpable) screen-detected tumors. Further, we compared the sensitivity of mammography and MRI within subgroups according to different tumor characteristics. Twenty-two (49%) of the 45 breast cancers were detected by MRI and not visible at mammography, of which 20 (44%) were also not palpable (MRI-only detected tumors). MRI-only detected tumors were more often node-negative than other screen-detected cancers (94 vs. 59%; P=0.02) and tended to be more often

Future prospects for the staging of breast cancer.

Rapidly growing knowledge about the nature and behaviour of breast cancer has led to many treatment modalities. Consequently, the possibilities of individualizing the treatment of breast cancer increase. The major tool for the determination of an optimal treatment plan is the estimation of the extent of the disease: in other words, staging. As a consequence, together with the expected result of the treatment, the stage of the disease gives information on the prognosis of the patient. Current staging systems insufficiently describe the clinically important features of breast cancer with respect to management and outcome: local and regional extent, invasiveness, aggressiveness, the state of dissemination, and the effectiveness of different treatment modalities. For staging of the local and regional extent, histology plays a prominent role and should be incorporated in future staging systems. Histological workup therefore needs standardisation. Histological parameters as tumour size, grade, nodal status, and vascular invasion are also the most important prognostic factors. Many so-called biological prognostic factors are related to the invasiveness and aggressiveness (metastatic potential) of the tumour, and therefore to the prognosis of the patient. However, these factors do not necessarily predict the effectiveness of certain systemic treatments. Only if the biological foundation of a prognostic factor is completely clarified can treatment be based on this knowledge, and the factor will become a predictor for the treatment effect. Many "biological" prognostic factors do not fulfil this main criterion and are therefore not useful for clinical decision making. A clinically useful staging system covers three primary aims: (1) to guide locoregional treatment, (2) to prognosticate the chance of survival, and (3) to indicate who needs what kind of adjuvant treatment. For the conception of a new staging system the following steps should be taken: standardization of all aspects of histology, identification of regional nodal involvement, and validation of prognostic factors with respect to their predictive value to treatment outcome.

Angiosarcoma of the breast after lumpectomy and radiation therapy for adenocarcinoma.

Three cases of angiosarcoma of the breast after lumpectomy and radiation therapy for adenocarcinoma are presented. Only two similar cases have been documented. The role of radiation therapy and chronic lymphedema is discussed. Although the overall survival is usually less than 22 months, two of these patients are still alive after 2 years.

Clinical presentation, endoscopic features, and treatment of gastric metastases from breast carcinoma.

BACKGROUND: Breast carcinoma is the most common malignancy in women. Metastatic involvement of the stomach is not well known. METHODS: Endoscopic features and treatment options were evaluated retrospectively for 51 patients with gastric metastases of breast carcinoma. RESULTS: The presenting sites of metastases were skeleton (43%), stomach (27%), lung (8%), and liver (4%). Diagnosis of gastric involvement was histologically confirmed in 41 patients and based on endoscopic features, despite negative biopsies in 10 patients. Six patients (12%) presented with nonfatal hemorrhage; in the others, symptoms were nonspecific: anorexia (71%), epigastric pain (53%), and vomiting (41%). Endoscopy showed 3 patterns: 18% localized lesions, 57% diffuse infiltration, and 25% external compression at the cardia or pylorus. Histology showed mainly lobular breast carcinoma (n = 36) compared with ductal carcinoma (n = 10) and other types (n = 5), contrary to the usual distribution. The overall response to systemic therapy was 46% (17 of 37 treated patients). Median survival from detection of gastric metastases was 10 months, with a 2-year survival rate of 23%. CONCLUSIONS: Gastric metastases usually derive from lobular rather than ductal breast carcinoma. Endoscopy revealed mainly a diffuse linitis plastica-like infiltration. Chemotherapy or hormonal treatment may result in fair palliation in selected patients, although many patients are heavily pretreated.

The spectrum of gastrointestinal metastases of breast carcinoma: I. Stomach.

In a 15-year period at the Netherlands Cancer Institute, 27 patients were found with breast carcinoma metastatic to the stomach. Presenting symptoms were non-specific, mainly nausea, vomiting, dysphagia, epigastric pain, and melena. Endoscopy, performed in 22 of these patients, yielded a correct diagnosis in 13. Lobular rather than ductal breast carcinoma was the predominant source of gastric metastases in this series. Non-surgical treatment was rewarded by a favorable, palliative response in 32% of cases.

The spectrum of gastrointestinal metastases of breast carcinoma: II. The colon and rectum.

In a 15-year period at the Netherlands Cancer Institute, 17 patients were found with breast carcinoma metastatic to the colon or rectum or both. The presenting symptoms and signs were non-specific and included diarrhea, crampy pain, vomiting, and palpable tumor. Endoscopic examination, possible in only 10 of the 17 patients because of luminal obstruction, yielded a correct diagnosis in seven cases. Biopsy was confirmatory in five cases. Lesions metastatic to the colorectum originated predominantly in lobular carcinoma of the breast. Systemic hormonal or chemotherapy or x-irradiation, either alone or as an adjunct to surgery, produced a favorable response in over half the patients so treated.

Immunomagnetic purification of human breast carcinoma cells allows tumor-specific detection of multidrug resistance gene 1-mRNA by reverse transcriptase polymerase chain reaction in fine-needle aspirates.

BACKGROUND: The heterogenous composition of tumors is a major obstacle for the measurement of mRNA levels in cancer cells. We report here a combination of immunomagnetic purification of cancer cells and reverse transcriptase polymerase chain reaction (RT-PCR) that enables highly sensitive detection of multidrug resistance gene 1 (MDR1)-mRNA levels in human breast carcinoma cells obtained from fine needle aspirates (FNA). EXPERIMENTAL DESIGN: Murine mAb 115D8 directed against episialin (MUC1/MAM6, epithelial membrane Ag) was used in combination with goat anti-mouse-coated magnetic microbeads to purify human T47D breast carcinoma cells (115D8+, MDR1-) from different mixtures with COLO320 human colon carcinoma cells (115D8-, MDR1+) and to purify carcinoma cells from FNA taken from axillary lymph node metastases in breast cancer patients. The efficacy of the purification was determined by FACS-analysis and by measurement of MDR1-mRNA levels by semiquantitative RT-PCR. RESULTS: FACS-analysis demonstrated that T47D cells could be purified up to 99.8% from mixtures with COLO320 cells ranging from 3:1 to 1:3. The MDR1-mRNA level in these enriched mixtures, as detected by RT-PCR, was reduced 250-fold. It was demonstrated that MDR1 expression present in an FNA from a lymph node metastasis of breast carcinoma could be attributed completely to the leukocytes present in this FNA, because MDR1 expression was no longer detectable after purification of the tumor cells. CONCLUSION: The combination of immunomagnetic purification of breast carcinoma cells and RT-PCR enables the measurement of cancer-specific MDR1 mRNA levels in small cell samples obtained by FNA.

p53 protein overexpression in relation to risk factors for breast cancer.

To investigate whether breast tumors developing through a pathway with p53 protein overexpression (p53+) show different risk factor associations compared with breast tumors without p53 overexpression (p53-), the authors determined p53 overexpression in tissue sections of 528 patients with invasive breast cancer by using immunohistochemistry. These patients and 918 healthy controls aged 20-54 years participated in a Netherlands population-based case-control study on oral contraceptives in 1986-1989. A total of 142 tumors (27%) demonstrated clear p53 overexpression (p53+). Most risk factors did not show different associations with p53+ and p53- tumors. However use of oral contraceptives for 9 or more years was associated with a 2.5-fold increase in the risk of p53+ tumors (95% confidence interval 1.4-4.4; test for trend with months of use, p = 0.01), whereas such use increase the risk of p53- tumors only 1.4-fold (95% confidence interval 0.9-2.1; test for trend p = 0.06). Prolonged lactation > or = 25 weeks) was associated with a 40% reduction in risk of p53+ tumors (odds ratio = 0.6; 95%, confidence interval 0.3-1.0; test for trend with weeks of lactation, p = 0.09), whereas the risk of p53- tumors was not associated with lactation. The authors conclude that p53+ and p53- breast tumors are not associated with very distinct risk profiles but that the stronger associations of p53+ tumors with oral contraceptive use and lactation suggest differences in risks that deserve further investigation. If these findings can be confirmed and possible molecular mechanisms explored, this may help to elucidate the associations between these risk factors and breast cancer in general.