Ten-year follow-up of skin-sparing mastectomy followed by immediate breast reconstruction.

BACKGROUND: The oncological safety of skin-sparing mastectomy (SSM) followed by immediate breast reconstruction (IBR) is debated owing to a presumed compromise in the completeness of mastectomy. Current evidence is poor as it is based mostly on short-term follow-up data from highly selected patients. METHODS: A prospectively maintained institutional database was searched to identify patients who underwent SSM and IBR between 1995 and 2000. A retrospective review of medical records was carried out, including only patients with ductal carcinoma in situ and invasive breast cancer. During this time all patients treated with mastectomy were offered IBR regardless of tumour stage. RESULTS: Follow-up data from 253 consecutive patients with IBR were reviewed. Patients with incomplete follow-up data and those undergoing SSM for recurrent disease following previous lumpectomy were disregarded, leaving 207 for analysis. Offering IBR to all women requiring mastectomy resulted in a large proportion of patients with advanced disease. During a median follow-up of 119 months, 17 (8·2 per cent) locoregional, six (2·9 per cent) local and 22 (10·6 per cent) distant recurrences were detected; the overall recurrence rate was 39 (18·8 per cent). Overall recurrence rate was associated with axillary lymph node metastasis (P = 0·009), higher stage (P < 0·001) and higher tumour grade (P = 0·031). The breast cancer-specific survival rate was 90·8 per cent (19 of 207 women died from recurrence). CONCLUSION: Based on these long-term follow-up data, SSM combined with IBR is an oncologically safe treatment option regardless of tumour stage.

Impact of national guidelines on family history breast cancer surveillance.

The breast cancer risk of women already under family history surveillance was accurately assessed according to national guidelines in an attempt to rationalize the service. Women attending two breast units in Glasgow between November 2003 and February 2005 were included. One thousand and five women under annual surveillance were assessed and had their relatives diagnoses verified. Four hundred and ninety-seven women were at significantly increased risk and eligible for follow-up. Five hundred and eight (50%) women attending were not eligible for family history surveillance, and 498 (98%) of these women accepted discharge. In conclusion, national guidelines have helped to more clearly define women who should undergo surveillance. This avoids unnecessary and potentially harmful routine investigations, and the service has been improved.

A new pathological system for grading DCIS with improved prediction of local recurrence: results from the UKCCCR/ANZ DCIS trial.

BACKGROUND: There is no consensus agreement regarding optimal management of locally excised ductal carcinoma in situ (DCIS) or features of greatest assistance in predicting disease behaviour. Cases in the UKCCCR/ANZ DCIS trial have been histologically reviewed to determine the features of prognostic importance. METHOD: A total of 72% of 1694 cases entered into the UKCCCR/ANZ DCIS trial had full pathological review. A large number of histological features were assessed, blinded to outcome and compared regarding ability to predict ipsilateral recurrence, as either DCIS or progression to invasive carcinoma. RESULTS: Pathological features associated with ipsilateral recurrence in univariate analysis included high cytonuclear grade, larger lesion size, growth pattern, presence of necrosis or chronic inflammation, incompleteness (or uncertainty of completeness) of excision and smaller margin width. Receipt of post-operative radiotherapy was also a strong prognostic factor.We report a novel sub-division of the large group of high-grade lesions, which enables identification of a very poor prognosis sub-group; namely, DCIS that is of high cytonuclear grade, predominantly (>50%) solid architecture, bearing extensive comedo-type necrosis (>50% of ducts). In addition, we found little difference in ipsilateral recurrence rates between low- and intermediate-grade groups. Hazard ratios for low, intermediate, high and the new, very high, grade were 0.42, 0.33, 0.62 and 1.00, respectively, for ipsilateral in situ or invasive recurrence. CONCLUSION: We present a novel pathological classification for DCIS with substantially better prognostic discrimination for ipsilateral recurrence than the classical categorisation based on cytonuclear grade alone.

Characterization of extensive genetic alterations in ductal carcinoma in situ by fluorescence in situ hybridization and molecular analysis.

BACKGROUND: The molecular genetic analysis of invasive breast cancer has identified breast cancer as a genetically complex disease. Ductal carcinoma in situ (DCIS) is thought to represent a preinvasive step in breast cancer progression, yet we know little about its biologic behavior or the genetic alterations present. Because of the increasing diagnosis of DCIS by mammography screening and the debate over how DCIS should be managed, there is a clear need to define the molecular events underlying the development of DCIS. PURPOSE: Our purpose was to identify patterns of genetic alterations in DCIS. METHODS: A group of 30 formalin-fixed, paraffin-embedded blocks of tissue collected from 1987 through 1989 from 21 patients with DCIS was studied. Chromosomal imbalances were determined by interphase cytogenetic analysis using the fluorescence in situ hybridization (FISH) technique. DNA probes were used that recognize chromosome-specific repetitive sequence loci at the centromeres of chromosomes 1, 3, 4, 6, 7, 8, 9, 10, 11, 16, 17, and 18. FISH was also used to detect ERBB2 gene amplification in DCIS. To complement the FISH studies, microsatellite analysis of markers near the BRCA1 region of chromosome 17 was done on tissue microdissected from multiple areas of DCIS. Chromosomal imbalances were determined by comparisons of chromosomal indices (total number of hybridization spots per total number of nuclei counted) of normal and DCIS tissue, using the two-sided Mann-Whitney test. RESULTS: Using FISH, we have identified patterns of DNA loss and gain of certain chromosome-specific centromeric markers in DCIS. We observed frequent gains of markers on chromosomes 3, 10, and 17 as well as loss of chromosome 18-specific centromeric sequences. ERBB2 gene amplification was detected in tumors from four of 15 patients studied and was clearly limited to the tumor cells within the ducts. Because of the availability of topologically distinct regions of tumors from individuals, we were able to show that paired tumor specimens from individuals share genetic alterations and also have unique ones, suggesting clonal diversity within tumors. The combination of FISH and microsatellite analyses suggested that alterations in chromosome 17 may be quite complex; three of five patients whose samples were analyzed had allelic imbalance at markers on the long arm of chromosome 17. CONCLUSIONS: FISH and microsatellite analyses are useful in detecting extensive genetic alterations in DCIS. Examinations of DCIS tissue using these techniques have identified chromosomes 1, 3, 10, 16, 17, and 18 as candidate sites worthy of immediate study. IMPLICATIONS: This approach may give direction to future research aimed at precisely mapping loci altered in DCIS and help in understanding the biologic events associated with tumor progression or recurrence.

Adjuvant cyclophosphamide, methotrexate, and fluorouracil in patients with axillary node-positive breast cancer: an update of the Guy's/Manchester trial.

Between 1976 and 1985, 391 patients (202 premenopausal, 189 postmenopausal) with operable breast cancer and positive axillary lymph nodes were randomized after total mastectomy and axillary clearance to receive cyclophosphamide, methotrexate, and fluorouracil (CMF) (n = 193) or no adjuvant therapy (n = 198). After a median follow-up of 8 years, both relapse-free survival (RFS) and survival (S) were significantly prolonged in premenopausal patients receiving CMF (RFS, P less than .001; S, P = .003). Treatment with CMF resulted in a significant improvement in RFS in premenopausal patients both with steroid receptor-positive and steroid receptor-negative tumors and also in subgroups of premenopausal patients defined by the number of axillary nodes involved. Premenopausal patients who developed permanent amenorrhea following CMF had a significantly better RFS than those who continued to menstruate. Induction of amenorrhea following CMF was related to age, with almost all patients over 40 years becoming amenorrheic. For patients less than or equal to 40 years, development of amenorrhea following CMF did not influence outcome. No difference was detected between control and CMF groups (RFS, P = .9; S, P = .9) in postmenopausal patients nor in any subgroup of these patients. The results of this trial of the efficacy of CMF for improving RFS and S have strengthened with longer follow-up.

Radiotherapy and tamoxifen in women with completely excised ductal carcinoma in situ of the breast in the UK, Australia, and New Zealand: randomised controlled trial.

BACKGROUND: As a consequence of mammographic breast screening programmes, ductal carcinoma in situ is diagnosed with increasing frequency. Mastectomy for localised ductal carcinoma in situ is thought to be an over-treatment by many physicians, but there is much controversy as to whether complete local excision alone is sufficient. We aimed to assess the effectiveness of adjuvant radiotherapy and tamoxifen. METHODS: We used a 2x2 factorial design in a randomised controlled trial. Between May, 1990, and August, 1998, 1701 patients recruited from screening programmes were randomised to both treatments in combination or singly, or to none, or to either one (eg, radiotherapy) with an elective decision to give or to withhold the other (ie, in this case tamoxifen). Patients had complete surgical excision of the lesion confirmed by specimen radiography and histology. Patients have been followed up at least once a year. Median follow-up was 52.6 (range 2.4-118.3) months. Our primary endpoint was the incidence of ipsilateral invasive disease. FINDINGS: Ipsilateral invasive disease was not reduced by tamoxifen but recurrence of overall ductal carcinoma in situ was decreased (hazard ratio 0.68 [0.49-0.96]; p=0.03). Radiotherapy reduced the incidence of ipsilateral invasive disease (0.45 [0.24-0.85]; p=0.01) and ipsilateral ductal carcinoma in situ (0.36 [0.19-0.66]; p=0.0004), but there was no effect on the occurrence of contralateral disease. There was no evidence of interaction between radiotherapy and tamoxifen. INTERPRETATION: Radiotherapy can be recommended for patients with ductal carcinoma in situ treated by complete local excision; however, there is little evidence for the use of tamoxifen in these women.

Axillary recurrence in breast cancer.

AIM: To determine whether axillary recurrence reflects inadequate axillary treatment or adverse pathological features. METHODS: The case-records were reviewed of 2122 women aged under 75 years, treated for invasive breast cancer during the time-period 1/1/86-31/12/91 in a geographically defined area. Data were abstracted on operations performed, pathological features, post-operative treatments and details of axillary recurrence. The risk of axillary recurrence was examined by pathological, treatment and patient factors. RESULTS: Axillary recurrence was more than twice as likely after inadequate compared to adequate treatment of the axilla (adequate staging or axillary radiotherapy or clearance). Delayed treatment of the axilla was not as successful as adequate primary treatment: multiple axillary recurrences were twice as common, one third of which were uncontrolled at time of death. Inadequate surgical treatment was associated with increased rates of recurrence despite endocrine therapy, chemotherapy or radiotherapy. Lymphoedema was twice as common if axillary radiotherapy was combined with any axillary surgical procedure. CONCLUSIONS: Axillary recurrence is more common in tumours with adverse pathology but may also result from inadequate axillary treatment. In order to minimise axillary recurrence, optimal treatment of the axilla entails adequate staging (sampling of four or more nodes) and treatment (axillary clearance or radiotherapy and endocrine therapy) in all women.

Flow cytometric analysis of tumour-draining lymph nodes in breast cancer patients.

The phenotype and activation status of lymphocytes from the peripheral blood and axillary lymph nodes of 40 patients with breast cancer were analysed using flow cytometry and compared with lymphocytes from the blood and lymph nodes of 7 control subjects. There was little difference in the overall proportions of T and B lymphocytes but there was a much larger population of B cells bearing surface IgG and a greater number of CD4+ helper T cells, particularly in the regional nodes, in the breast cancer patients. Many more T cells in the cancer patients were found to be carrying the HLA DR and Tac antigens. The axillary lymph nodes were the major site of B cells and CD4+ T cells whilst the primary tumour was the source of the CD8+ suppressor/cytotoxic T cells. Any immune response appeared to be largely loco-regional and may therefore destroyed by conventional surgery or radiotherapy.

Haemostatic abnormalities and outcome in patients with operable breast cancer.

The predictive value for cancer recurrence of five measurements of haemostatic activity was studied in 89 patients with operable breast cancer. Neither preoperative nor sequential measurements up to 9 months postoperatively of fibrinopeptide A, fibrin fragment B beta 15-42, fibrinogen and serum fibrin(ogen) degradation products nor the fibrin plate lysis assay correlated with early recurrent disease. B beta 15-42 values were higher preoperatively in patients with oestrogen receptor positive tumours (P = 0.017). Mean B beta 15-42 values rose postoperatively (P = 0.003), largely because of an increase in patients with oestrogen receptor negative tumours.

Aneuploid subpopulations in tumour-invaded lymph nodes from breast cancer patients.

Fresh, paired primary tumours and lymph node metastases from breast cancer patients were compared by DNA flow cytometry. Although 65% of primary tumours were aneuploid, the detection of aneuploid peaks in corresponding nodal metastases was rare (only 6 cases out of 25) in single-parameter DNA analysis. Detection of aneuploid subpopulations in lymph nodes was greatly improved in dual-parameter DNA analysis using an anticytokeratin (CK) antibody which allowed ploidy determination on CK+ epithelial cells alone. Examination of 12 lymph nodes for CK+ cells revealed the presence of both diploid and aneuploid tumour cells in tumour invaded nodes. In patients with multiploid primary tumours, a subpopulation of the primary aneuploid cells was dominant in the nodal metastases. This suggests that aneuploidy is an integral property of metastatic cells and that within a primary tumour a subpopulation may have a higher metastatic potential.

Co-amplification of erbB2, topoisomerase II alpha and retinoic acid receptor alpha genes in breast cancer and allelic loss at topoisomerase I on chromosome 20.

The DNA topoisomerase enzymes are targets for the cytotoxic effects of a number of anticancer agents termed topoisomerase inhibitors. We have analysed breast cancer biopsy specimens for genetic alterations at and around topoisomerase loci in order to obtain molecular insight into factors which may determine how tumours respond to chemotherapy. We show that of 50 tumours examined, the topoisomerase II alpha locus is co-amplified in 3 cases out of 6 with erbB2 amplification and that amplification can be accompanied by high expression of topoisomerase II alpha. In our attempts to distinguish amplification from aneuploidy and define the limits of amplification, we also observed co-amplification of the retinoic acid-alpha receptor with erbB2 and topoisomerase II alpha in the same three samples. At the topoisomerase I locus on chromosome 20, we observed allelic loss in two out of 17 samples. Genetics abberations at topoisomerase loci, therefore, appear to be relatively common in breast cancer.

Long-term prognostic significance of thymidine labelling index in primary breast cancer.

Tumour growth rates, as measured by incorporation of tritiated thymidine, have been reported as being of prognostic importance in breast cancer. We have measured the thymidine labelling index (TLI) of 185 early breast cancers, followed-up for a minimum of 8 years. Above median TLI was associated with higher tumour grade, but not with other prognostic factors. TLI was not predictive of survival in either univariate or multivariate analysis. The inter- and intra-observer reproducibilities of TLI measurements were poor, which may be a factor limiting its usefulness as a prognostic indicator in breast cancer.

In vivo measurements of fibrin formation and fibrinolysis in operable breast cancer.

Fibrin formation and fibrinolysis were estimated in 89 breast cancer patients by measurement in plasma of Fibrin Fragment B beta 15-42 and Fibrinopeptide A (FPA), serum Fibrin(ogen) Degradation Products (FDPs) and plasminogen activator by Fibrin Plate Lysis Assay. Results were compared with (a) 26 patients with benign breast diseases; and (b) 45 healthy factory workers. FPA, FDP and B beta 15-42 levels were elevated in both breast cancer patients and benign disease patients, but there were no significant differences between these two groups. Cancer stage, patient age and smoking habits did not affect these results, but Oestrogen Receptor (ER) positive patients had higher B beta 15-42 values than ER negative patients (p = 0.017). These results show that fibrin formation is enhanced preoperatively in patients with either benign or malignant breast disease. The fibrinolytic response to activated coagulation may be relatively deficient in breast cancer. The roles of malignancy, stress and other factors in the causation of these abnormalities require further assessment.

Altered lymphocyte populations in tumour invaded nodes of breast cancer patients.

Lymphocytes from matched pairs of tumour-invaded and tumour-free lymph nodes from 22 stage II breast cancer patients have been analysed for expression of phenotypic and activation markers by flow cytometry. Although the relative proportions of T and B lymphocytes were similar in the two nodes, significant differences in the distribution of T cell subsets were observed between nodes that were invaded and those that were not. The CD4/CD8 ratio was markedly depressed in tumour invaded nodes (P < 0.001). This was due to an increase in the number of CD8+ T cells (P < 0.001) and a decrease in the CD4+ T cell population (P = 0.008) in invaded nodes in comparison with tumour-free nodes. The percentage of CD8+ T cells expressing HLA DR (P = 0.023) and IL-2 receptors (Tac) (P = 0.029) was significantly higher in invaded nodes and, while CD4+ T cells expressing HLA DR (P = 0.036) were also in a higher proportion of Tac expressing CD4+ T cells failed to reach significance. Although invaded nodes in a few patients were found to have a higher percentage of IgG-expressing B cells, no significant differences were observed between the two groups of nodes. These results suggest that the presence of metastatic tumour cells in a lymph node is associated with specific alterations in the T cell population.

Analysis of clonality of tumour infiltrating lymphocytes in breast cancer and uveal melanoma.

Fresh tumour infiltrating lymphocytes (TILs) from 6 uveal melanomas and 4 breast cancers were analysed by flow cytometry with a panel of 6 monoclonal antibodies to V beta regions of the T cell receptor (V betas 5a, 5b, 5c, 6, 8a and 12a). With a single exception where one TIL sample lacked V beta 12a, lymphocytes from both tumour and blood contained cells reactive with all 6 probes, and no probe was highly dominant or missing. The proportions of reactive cells differed between tumour and blood within each patient. The data indicate that while tumour infiltrating lymphocytes have a capacity to locate selectively within the tumour they nonetheless comprise a population expressing a diversity of TCR V beta genes.

T cell receptor gamma/delta expression on lymphocyte populations of breast cancer patients.

The quantitative distribution and phenotype of gamma/delta lymphocytes in the peripheral blood (PBL), tumour draining lymph node (LNL) and tumour infiltrating lymphocytes (TIL) from breast carcinoma patients were determined by one- and two-colour flow cytometry. The TCR-gamma/delta + cells generally expressed the T cell lineage antigen CD3. The proportions of such cells were variable but generally small from all the three sources. Phenotypic analysis revealed that the CD8 marker was consistently and predominantly observed on gamma/delta + CD3+ cells in the tumour infiltrate, whereas CD4 expression, while generally low, was noted on a significant percentage (median 10%) of LNL gamma/delta + lymphocytes. In both PBL and LNL the predominant gamma/delta cell population was CD4-8-.

Flow cytometric analysis of tumour infiltrating lymphocyte activation and tumour cell MHC class I and II expression in breast cancer patients.

The primary tumour cells and tumour infiltrating lymphocytes (TILs) of 31 breast cancer patients have been analysed by dual colour flow cytometry to determine whether the phenotype and/or activation status of the TILs bears any relationship to the expression of MHC antigens on the tumour cells. The phenotype and activation status of 5000 TILs were studied using Mabs to CD4, CD8, HLA DR, CD25 (the low affinity inducible IL-2 receptor) and the transferrin receptor and related to Class I and Class II MHC expression on 5000 primary tumour cells. On the tumour cells, Class I MHC expression ranged from 1-74%, averaging 12.9%. HLA DR expression ranged from 1-69% averaging 14.3%. When the phenotypic proportions of the lymphocytic infiltrate were analysed there was found to be a correlation between tumour expression of Class I MHC and the proportion of both CD4+ (P less than 0.05) and CD8+ (P less than 0.02) T cells within the tumour. No such relationship was found with the MHC Class II antigen. When TIL activation markers were analysed, the percentage of CD8+ TILs positive for HLA DR expression correlated strongly with the expression of Class I (P less than 0.001) and Class II (P less than 0.001) antigens on the tumour cells. The percentage of CD4+ TILs positive for HLA DR expression also correlated significantly, but less strongly with the expression of Class I (P less than 0.01) and Class II (P less than 0.02) antigen expression on the tumour cells. The percentage of CD4+ TILs positive for CD25 expression correlated with both Class I (P less than 0.05) and Class II (P less than 0.03) expression on the tumour cells while the percentage of CD8+ TILs positive for CD25 did not. The percentage of TILs bearing the transferrin receptor showed no measurable correlation with the expression of either class of MHC antigen on the tumour. The data suggest that MHC expression on the tumour cells has a selective effect on the response capacity of different parts of the immune system.

Dual colour flow cytometry of p53 and c-erbB-2 expression related to DNA aneuploidy in primary and metastatic breast cancer.

DNA aneuploidy and p53 or c-erbB-2 expression were simultaneously measured in 29 breast tumours by two-colour flow cytometry. (i) The majority of tumours had some cells expressing either p53 (5-68%) or c-erbB-2 (1-56%). (ii) Expression of p53 and c-erbB-2 was observed mainly in the aneuploid population of mixed aneuploid and diploid tumours but there was no significant correlation with a specific DNA index. Aneuploid tumours contained higher percentages of c-erbB-2 positive cells (average 25%) than purely diploid tumours (average 15%) but this just failed to reach significance (P = 0.074). No relevant trends were noted for p53 expression. (iii) Significantly increased c-erbB-2 expression was observed in stage 2 tumours (26%) compared to stage 1 tumours (12%) (P = 0.001) with no trend evident for p53 expression. (iv) The metastatic tumour in the axillary node contained similar or slightly higher percentages of positive cells than the matched primary tumour.

Semiquantitative analyses of fine needle aspirates from benign and malignant breast lesions.

A scoring system for the assessment of fine needle aspirates of benign and malignant breast lesions was devised which showed a positive correlation (r = 0.67) between the scores obtained from the fine needle aspirates from ductal carcinomas and the Bloom and Richardson-type scores for their paired excision biopsy specimens. This system permitted grades II and III ductal breast carcinoma to be distinguished reliably from grade I tumours but no correlation with the lymph node state of patients with breast carcinoma was shown. Some overlap between the scores for grade I ductal carcinomas and some benign lesions was found, and this underlines a need for caution in the reporting of such equivocal aspirates. No cytological features that distinguished reliably ductal from lobular carcinoma were identified but the same spectrum of severity of cytological abnormality in the ductal and lobular carcinoma aspirates was seen. This system may be of prognostic value in the assessment of lobular carcinoma which has hitherto defied histological grading.

Detection of intraoperative tumor cell dissemination in patients with breast cancer by use of reverse transcription and polymerase chain reaction.

BACKGROUND: Animal studies have shown that malignant cells are shed into the blood stream during surgical resection of a primary tumor and that this may enhance the development of metastases. The evidence for tumor cell dissemination during surgical manipulation of human cancer is unclear. We have applied the technique of reverse transcription and polymerase chain reaction to detect circulating tumor cells in peripheral venous blood of patients with breast cancer perioperatively. METHODS: To target breast-specific gene transcription complementary DNA was prepared by reverse transcription of blood messenger RNA with oligonucleotide primers unique to CK18 and DF3 antigens. Preliminary assessment of specificity showed that the DF3 antigen was more suitable than CK18 for the purpose of this study. Assessment of sensitivity showed that as few as 10 tumor cells per 5 ml blood could be identified by this method. Peripheral blood samples were obtained by venepuncture from patients before, during, and 24 hours after breast surgery (nine malignant and three benign). RESULTS: In the group of patients with malignant disease, tumor cells were detected in one patient before operation and four patients during operation. No tumor cells were detected in the postoperative samples nor in any of the samples of patients with benign disease. CONCLUSIONS: These findings suggest that tumor manipulation during operation encourages tumor cell dissemination.