Detection of metastases in sentinel lymph nodes of breast cancer patients by multiple mRNA markers.

Disseminated breast tumour cells in sentinel lymph nodes (SNs) were evaluated by quantitative real-time PCR and the sensitivity of this assay was compared to the routine histological analysis. First, several candidate marker genes were tested for their specificity in axillary lymph nodes (ALN) of 50 breast cancer patients and 43 women without breast cancer. The marker gene panel selected, designed to detect the mRNA of CK19, p1B, EGP2 and SBEM, was subsequently applied to detect metastases in 70 SNs that were free of metastases as determined by standard histological evaluation. Remarkably, seven negative SNs showed increased marker gene expression, suggesting the presence of (micro) metastases. Four of these seven SNs positive by real-time PCR proved to contain tumour deposits after careful review of the slides or further sectioning of the paraffin-embedded material. In three PCR positive SNs, however, no tumour cells were found by haematoxylin and eosin staining (H&E) and immunohistologically analysis. The quantitative real-time PCR assay with multiple mRNA markers for the detection of disseminated breast cancer cells in SNs thus resulted in an upstaging of SNs containing metastastic disease of 10% compared to the routine histological analysis. The application of this technique may be of clinical relevance, as it is suggested that micrometastatic disease in SNs are associated with further nodal non-SN metastases in breast cancer.

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.

Gangliocytic paraganglioma of the appendix.

AIMS: A case of gangliocytic paraganglioma is reported in the appendix which, to the best of our knowledge, is the first case at this particular site to be described in modern literature. METHODS AND RESULTS: A 47-year-old man with signs and symptoms of acute appendicitis underwent appendectomy. In the resected specimen a tumour with a diameter of 9 mm was found, which microscopically consisted of three different cell types: (a) epithelioid cells lying in a trabecular pattern and in formations reminiscent of 'Zellballen' as seen in paragangliomas (b) spindle cells and (c) ganglion-like cells. A diagnosis of 'gangliocytic paraganglioma' was made and confirmed by immunohistochemical and ultrastructural examination. CONCLUSION: Gangliocytic paragangliomas are rare tumours of uncertain histogenesis. More than 40 years ago a tumour in the appendix with features similar to our case was described by Masson as 'neuro-carcinoïde'. Concerning its origin, Masson, as well as other authors describing gangliocytic paragangliomas decades later, referred to the endodermal-neuroecto dermal complexes found by Van Campenhout. It is felt that the current finding of a gangliocytic paraganglioma in the appendix supports the hypothesis that gangliocytic paragangliomas arise from these embryonal structures.

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.

Pathological investigation of sentinel lymph nodes.

The sentinel lymph-node procedure enables selective targeting of the first draining lymph node, where the initial metastases will form. A negative sentinel node (SN) predicts the absence of tumour metastases in the other regional lymph nodes with high accuracy. This means that in the case of a negative SN, regional lymph-node dissection is no longer necessary. Besides saving costs, this will prevent many side-effects as a result of lymph-node dissection. The task of the pathologist is to screen SNs for metastases. To this end, several techniques are available such as standard histo- and cytopathological techniques, immunohistochemistry, flow cytometry, and molecular biological techniques. These methods are explained and their sensitivity for detecting SN metastases is discussed. Some of these techniques also appear to be useful for intra-operative evaluation of SNs. The standard protocol for detection of SN metastases consists of extensive histopathological investigation including step H&E stained sections and immunohistochemistry. Intra-operative frozen-section analysis of SNs has been shown to be reliable for breast-cancer axillary lymph nodes. In the intra-operative setting, imprint cytology can also be used but its additional value to frozen section analysis is not yet clear. Further studies are necessary to establish the role of sophisticated molecular biological techniques such as reverse transcription polymerase chain reaction (RT-PCR) in detecting SN metastases. The sensitivity of flow cytometry is too low for this purpose.

Local recurrence after breast conservation therapy for early stage breast carcinoma: detection, treatment, and outcome in 266 patients. Dutch Study Group on Local Recurrence after Breast Conservation (BORST).

BACKGROUND: Controversy exists concerning the roles of mammography and physical examination in the detection of local recurrence after conservation therapy for breast carcinoma. In addition, the prognostic factors for and optimal treatment of patients with local recurrence are uncertain. METHODS: At eight radiotherapy institutes, two cancer institutes, and one surgical clinic in the Netherlands, regular follow-up of patients who underwent breast conservation therapy between 1980 and 1992 identified 266 patients with local recurrence in the breast. These patients exhibited no clinical signs of distant metastases at the time of diagnosis of the local recurrence. Data on the method of detection were available for 189 patients (72%). Local recurrence was diagnosed by mammography alone in 47 cases (25%). Of all patients, 85% underwent salvage mastectomy, 8% underwent local excision, 4% received systemic treatment only, and 3% remained untreated. Specimens of the primary tumor were available for review from 238 of the 266 patients (89%). RESULTS: Local recurrences detected by mammography alone were smaller than those detected by physical examination (P = 0.04). At 5 years from the date of salvage treatment, the overall survival rate for all 266 patients was 61% (95% confidence interval [CI], 55-67%), and the distant recurrence free survival rate was 47% (95% CI, 40-53%). For the 25 patients with noninvasive recurrence, these figures were 95% and 94%, respectively. Skin involvement, the extent of recurrence (< or = 10 mm vs. >10 mm), and both lymph node status and histologic grade of the primary disease were strong predictors for distant metastases in patients with invasive recurrence. CONCLUSIONS: Patients with invasive local recurrence more than 1 cm in size are at a substantial risk for distant disease. The better distant disease free survival for patients with recurrence measuring 1 cm or less may indicate that early detection can improve the treatment outcome. Recurrence with skin involvement should be considered generalized disease.

Angiosarcoma of the breast after conservation therapy for invasive cancer, the incidence and outcome. An unforseen sequela.

PURPOSE: In the past 15 years breast conserving therapy (BCT) has become an important treatment option for primary breast cancer. Thirty three angiosarcomas (AS) after BCT have been described in a total of 20 published reports. Limited follow-up data and the lack of information on incidence of AS prompted the authors to review the comprehensive experience in the Netherlands. METHODS: Between 1987 and 1995 twenty-one patients with BCT-associated AS were diagnosed in the Netherlands. Follow-up after diagnosis of AS ranged from 6 to 82 months with a median of 24 months. Information on the total number of patients treated with BCT and on the numbers of angiosarcoma in the breast was obtained. RESULTS: The median interval between BCT and AS was 74 months (range: 29-106) and appeared to decrease with higher age. Detection of skin changes followed by incisional biopsy provided the diagnosis. Two year overall (OS) and disease free survivals were 72% (s.e. 10.9) and 35% (s.e. 10.7), respectively. Two year OS after initial complete surgical resection was 86% (s.e. 9.3) compared to 0% after incomplete resection of the AS (P = 0.04). The estimated incidence of AS after BCT is 0.16%. CONCLUSIONS: BCT-associated AS arises after a relatively short interval. Although the incidence of AS is low, the absolute number of patients at risk is increasing. This calls for vigilance concerning skin changes occurring after BCT. An incisional biopsy provides the only reliable diagnosis. The prognosis appears to be related to the completeness of surgical resection.

Prognostic implications of different cell cycle analysis models of flow cytometric DNA histograms of 1,301 breast cancer patients: results from the Multicenter Morphometric Mammary Carcinoma Project (MMMCP).

Conflicting prognostic results with regard to DNA flow cytometric cell cycle variables have been reported for breast cancer patients. An important reason for this may be related to differences in the interpretation of DNA histograms. Several computer programs based on different cell cycle fitting models are available resulting in significant variations in percent S-phase and other cell cycle variables. Our present study evaluated the prognostic value of percent S-phase cells obtained using 5 different cell cycle analysis models. Flow cytometric DNA histograms obtained from 1,301 fresh frozen breast cancer samples were interpreted with 5 different cell cycle analysis models using a commercially available computer program. Model 1 used the zero order S-phase calculation and "sliced nuclei" debris correction, model 2 added fixed G2/M- to G0/G1-phase ratio, and model 3 added correction for aggregates. Model 4 applied the first-order S-phase calculation and sliced debris correction. Model 5 fixed the coefficients of variation CVs of the G0/G1- and G2/M-phases in addition to applying the sliced nuclei debris correction and zero order S-phase calculation. The different models yielded clearly different prognostic results. The average percent S-phase cells of the aggregate correction model (model 3) provided the best prognostic value in all cases for overall survival (OS) as well as disease-free survival (DFS) (OS: p < 0.0001; DFS: p < 0.0001), in lymph node-positive cases (OS: p < 0.0001; DFS: p = 0.004) and in DNA-diploid subgroups (OS: p = 0.004; DFS: p = 0.001). For the lymph node negative and DNA-non-diploid subgroups, the percent S-phase of the second cell cycle reached slightly better prognostic significance than the average percent S-phase cells. In multivariate analysis, the average percent S-phase of the aggregate correction model had the best additional prognostic value to tumor size and lymph node status. In conclusion, different cell cycle analysis models yield clearly different prognostic results for invasive breast cancer patients. The most important prognostic percent S-phase variable was the average percent S-phase cells when aggregate correction was included in cell cycle analysis.

Cyclin D1 expression in invasive breast cancer. Correlations and prognostic value.

Cyclin D1 overexpression, detected by standard immunohistochemistry, was correlated with other prognostic variables and its prognostic value was evaluated in a group of 148 invasive breast cancers with long-term follow-up. Overexpression of cyclin D1 (59% of cases) was negatively correlated (chi 2 test) with histological grade (P = 0.0001), mean nuclear area (P = 0.004), mean nuclear volume (P = 0.02), and mitotic activity (P = 0.03) and positively correlated with estrogen receptor (P = 0.0001). There was a strong correlation between cyclin D1 overexpression and histological type (P = 0.0001). Positive cyclin D1 staining was seen in 11 of 13 tubular carcinomas, 3 of 3 mucinous carcinomas, 4 of 4 invasive cribriform carcinomas, and 17 of 20 lobular carcinomas. Of 102 ductal cancers, 52 were positive, and all 6 medullary carcinomas were negative. There were no significant correlations with lymph node status, tumor size, or DNA ploidy. In survival analysis, cyclin D1 overexpression did not provide significant univariate or multivariate prognostic value. In conclusion, cyclin D1 is mainly overexpressed in the well differentiated and lobular types of invasive breast cancer and is strongly associated with estrogen receptor positivity. It is negatively correlated with the proliferation marker mitoses count and with the differentiation markers nuclear area and nuclear volume. However, cyclin D1 overexpression does not seem to have prognostic value in invasive breast cancer when no adjuvant treatment is given.

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.

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.

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.

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.

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.

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.

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.

Influence of age and comorbidity on treatment choice and survival in elderly patients with breast cancer.

To determine the effect of age and comorbid diseases on treatment choice and survival, the medical records of 300 breast cancer patients of 55 years and older were reviewed. All patients were admitted to the Netherlands Cancer Institute (NKI) for first treatment between 1980 and 1987. Patients were classified according to severity level of comorbid diseases. Physicians were found to treat women of 75 years and older less often with adjuvant radiotherapy after a mastectomy, and more often to employ only primary endocrine treatment for local stage disease, as compared with younger patients. According to the treatment guidelines of the institute, the study sample was divided into patients who received standard vs. non-standard treatment. The treatment of 38 women (13.1%) did not correspond with the guidelines. Of these, 84% were 75 years and older and 50% had a severe comorbidity status. Logistic regression analysis indicated that advanced age, per se, was a better indicator of the risk of not being treated according to protocol than the comorbidity status. Cox multivariate analyses demonstrated that neither the severity of the comorbidity status nor the differences in treatment between younger and older patients had a significant effect on the risk of dying from breast cancer or on the risk of developing recurrences. In this analysis, age 75 years or more proved to be a significant and independent predictor of a worse overall and disease-specific survival as compared to age between 65-74 years.