The purpose of this study was to classify breast carcinomas based on variations in gene expression patterns derived from cDNA microarrays and to correlate tumor characteristics to clinical outcome. A total of 85 cDNA microarray experiments representing 78 cancers, three fibroadenomas, and four normal breast tissues were analyzed by hierarchical clustering. As reported previously, the cancers could be classified into a basal epithelial-like group, an ERBB2-overexpressing group and a normal breast-like group based on variations in gene expression. A novel finding was that the previously characterized luminal epithelial/estrogen receptor-positive group could be divided into at least two subgroups, each with a distinctive expression profile. These subtypes proved to be reasonably robust by clustering using two different gene sets: first, a set of 456 cDNA clones previously selected to reflect intrinsic properties of the tumors and, second, a gene set that highly correlated with patient outcome. Survival analyses on a subcohort of patients with locally advanced breast cancer uniformly treated in a prospective study showed significantly different outcomes for the patients belonging to the various groups, including a poor prognosis for the basal-like subtype and a significant difference in outcome for the two estrogen receptor-positive groups.

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Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications

Breast cancer patients with the same stage of disease can have markedly different treatment responses and overall outcome. The strongest predictors for metastases (for example, lymph node status and histological grade) fail to classify accurately breast tumours according to their clinical behaviour. Chemotherapy or hormonal therapy reduces the risk of distant metastases by approximately one-third; however, 70-80% of patients receiving this treatment would have survived without it. None of the signatures of breast cancer gene expression reported to date allow for patient-tailored therapy strategies. Here we used DNA microarray analysis on primary breast tumours of 117 young patients, and applied supervised classification to identify a gene expression signature strongly predictive of a short interval to distant metastases ('poor prognosis' signature) in patients without tumour cells in local lymph nodes at diagnosis (lymph node negative). In addition, we established a signature that identifies tumours of BRCA1 carriers. The poor prognosis signature consists of genes regulating cell cycle, invasion, metastasis and angiogenesis. This gene expression profile will outperform all currently used clinical parameters in predicting disease outcome. Our findings provide a strategy to select patients who would benefit from adjuvant therapy.

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Gene expression profiling predicts clinical outcome of breast cancer

Background A more accurate means of prognostication in breast cancer will improve the selection of patients for adjuvant systemic therapy. Methods Using microarray analysis to evaluate our previously established 70-gene prognosis profile, we classified a series of 295 consecutive patients with primary breast carcinomas as having a gene-expression signature associated with either a poor prognosis or a good prognosis. All patients had stage I or II breast cancer and were younger than 53 years old; 151 had lymph-node–negative disease, and 144 had lymph-node–positive disease. We evaluated the predictive power of the prognosis profile using univariable and multivariable statistical analyses. Results Among the 295 patients, 180 had a poor-prognosis signature and 115 had a good-prognosis signature, and the mean (±SE) overall 10-year survival rates were 54.6±4.4 percent and 94.5±2.6 percent, respectively. At 10 years, the probability of remaining free of distant metastases was 50.6±4.5 percent in the group with a...

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A Gene-Expression Signature as a Predictor of Survival in Breast Cancer

background The likelihood of distant recurrence in patients with breast cancer who have no involved lymph nodes and estrogen-receptor–positive tumors is poorly defined by clinical and histopathological measures. methods We tested whether the results of a reverse-transcriptase–polymerase-chain-reaction (RT-PCR) assay of 21 prospectively selected genes in paraffin-embedded tumor tissue would correlate with the likelihood of distant recurrence in patients with node-negative, tamoxifen-treated breast cancer who were enrolled in the National Surgical Adjuvant Breast and Bowel Project clinical trial B-14. The levels of expression of 16 cancerrelated genes and 5 reference genes were used in a prospectively defined algorithm to calculate a recurrence score and to determine a risk group (low, intermediate, or high) for each patient. results Adequate RT-PCR profiles were obtained in 668 of 675 tumor blocks. The proportions of patients categorized as having a low, intermediate, or high risk by the RT-PCR assay were 51, 22, and 27 percent, respectively. The Kaplan–Meier estimates of the rates of distant recurrence at 10 years in the low-risk, intermediate-risk, and high-risk groups were 6.8 percent (95 percent confidence interval, 4.0 to 9.6), 14.3 percent (95 percent confidence interval, 8.3 to 20.3), and 30.5 percent (95 percent confidence interval, 23.6 to 37.4). The rate in the low-risk group was significantly lower than that in the high-risk group (P<0.001). In a multivariate Cox model, the recurrence score provided significant predictive power that was independent of age and tumor size (P<0.001). The recurrence score was also predictive of overall survival (P<0.001) and could be used as a continuous function to predict distant recurrence in individual patients. conclusions The recurrence score has been validated as quantifying the likelihood of distant recurrence in tamoxifen-treated patients with node-negative, estrogen-receptor–positive breast cancer.

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A Multigene Assay to Predict Recurrence of Tamoxifen-Treated, Node-Negative Breast Cancer

Morphological assessment of the degree of differentiation has been shown in numerous studies to provide useful prognostic information in breast cancer, but until recently histological grading has not been accepted as a routine procedure, mainly because of perceived problems with reproducibility and consistency. In the Nottingham/Tenovus Primary Breast Cancer Study the most commonly used method, described by Bloom & Richardson, has been modified in order to make the criteria more objective. The revised technique involves semiquantitative evaluation of three morphological features--the percentage of tubule formation, the degree of nuclear pleomorphism and an accurate mitotic count using a defined field area. A numerical scoring system is used and the overall grade is derived from a summation of individual scores for the three variables: three grades of differentiation are used. Since 1973, over 2200 patients with primary operable breast cancer have been entered into a study of multiple prognostic factors. Histological grade, assessed in 1831 patients, shows a very strong correlation with prognosis; patients with grade I tumours have a significantly better survival than those with grade II and III tumours (P less than 0.0001). These results demonstrate that this method for histological grading provides important prognostic information and, if the grading protocol is followed consistently, reproducible results can be obtained. Histological grade forms part of the multifactorial Nottingham prognostic index, together with tumour size and lymph node stage, which is used to stratify individual patients for appropriate therapy.

Pathological prognostic factors in breast cancer. I. The value of histological grade in breast cancer: experience from a large study with long-term follow-up.

SummaryDuring the production and reversion of profound hypothermia with extracorporeal perfusion temperatures were recorded from various sites throughout the heart. In all animals studied thermal gradients were observed. In some instances during cooling temperature variations as marked as 10°C were recorded. During rewarming similar differences were noted. From these studies it is concluded that thermal gradients alone are not responsible for hypothermic ventricular fibrillation.

Cardiac temperature gradients during profound hypothermia with extracorporeal perfusion.

Beginning in 1958, the first clinical trial of the National Surgical Adjuvant Breast Project compared a brief postoperative course of low dose triethylenethiophosphoramide (TSPA) with no adjuvant therapy or placebo in 826 patients who underwent radical mastectomy for breast cancer. After 5 years, one subset of patients made up of premenopausal women with 4 or more positive axillary lymph nodes had a significantly higher survival rate and longer disease free interval following TSPA therapy than after placebo therapy. After 10 years, these significant differences in survival and incidence of treatment failure between the TSPA-treated patients and the controls persisted. 
In 1972, the NSABP initiated a trial comparing prolonged postoperative chemotherapy with 1-phenylalanine mustard (L-PAM) with placebo in patients with positive nodes who had undergone radical mastectomy. By 1975 it appeared that premenopausal women who received L-PAM had a significantly lower incidence of treatment failure than those who received placebo. L-PAM therapy produced little alteration in the well-being of the patients. 
Between February 1975 and March 1976, 700 patients were entered into NSABP Protocol #7 which compares 2 regimens of prolonged postoperative chemotherapy, L-PAM alone and L-PAM plus 5-fluorouracil (5-FU). In June 1976, NSABP Protocol #8 was initiated to compare another 2 regimens of prolonged postoperative chemotherapy, L-PAM plus 5-FU and L-PAM plus 5-FU plus methotrexate. It is too early to evaluate the results of these studies. It seems likely that the many trials of adjuvant chemotherapy being conducted throughout the world will provide significant improvement in the curability of breast cancer, provided these trials are carefully planned and thoroughly executed.

United States trials of adjuvant therapy.

The two greatest deterrents to a more general acceptance of hypothermia as a clinical modality have been (1) the fear of ventricular fibrillation 1,2 and (2) the fear that hypothermia and rewarming will result in profound pathologic 3-5 and metabolic 6-8 changes which are difficult to correct or are irreversible. It has been felt by us that if, in spite of the control of fibrillation, the latter anxiety is justified, then serious consideration to the abandonment of clinical hypothermia is warranted. A systematic evaluation of the effects of short and prolonged body cooling followed by rewarming has been made during the last three years by this laboratory. Information concerning the liver, 9 cardiovascular systems, 10,11 general pathology and histochemistry, 12 and various metabolic parameters 13-15 has been reported. From these experimental findings and a critical evaluation of the literature it is believed that there is no conclusive evidence to suggest

Prevention of fibrillation during hypothermia by pericardial space perfusion.

Earlier reports from this laboratory have demonstrated that prolonged survival of skin homografts in adult rats could be accomplished when animals were made pyridoxine-deficient either by diet or by administration of the specific B6 antagonist deoxypyridoxine (DB6), and that “takes” persisted even after animals had returned to a pyridoxine-containing diet and were no longer clinically deficient. In the present investigation the effect of pyridoxine deficiency upon the RES has been evaluated. It was observed that: (1) ungrafted animals maintained on a B6-free diet for varying periods of time demonstrated a marked depression in phagocytic activity which persisted after return to a normal diet, and that DB6 had a similar effect which was discernable for as long as 3 months after its administration; (2) following application of a skin homograft the RES-suppressing effect of the vitamin deficiency noted in ungrafted animals was not observed; (3) no difference in RES activity was present in animals which rejected their grafts and those whose grafts survived. It is concluded that: (a) because of the ability of a skin homograft to provide a sufficient stimulus to overcome even the most profound RES suppression, the concept that blockade of the RES prolongs graft survival remains to be proven; and (b) the prolongation of skin graft survival following pyridoxine deficiency is not related to the RES depression observed under such circumstances in non-grafted animals. It is considered that the prolongation of survival may be due to grafts becoming less antigenic or less responsive to antibody with the passage of time.

Bernard Fisher

Papers

Cardiac temperature gradients during profound hypothermia with extracorporeal perfusion.

United States trials of adjuvant therapy.

Prevention of fibrillation during hypothermia by pericardial space perfusion.

Tissue transplantation and the reticuloendothelial system. ii. effect of vitamin b6 deficiency on phagocytic activity.

Chemotherapy and Tamoxifen for Breast Cancer