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.

Tumor necrosis factor (TNF) may be a major endogenous mediator of sepsis-induced acute organ injury. We proposed that treatment of septic pigs with the combined agents ibuprofen, a cyclooxygenase inhibitor, and histamine receptor antagonists, cimetidine (H 2 antagonist) and diphenhydramine (H 1 antagonist) would result in lower circulating levels of TNF and decreased parameters of sepsis-induced injury in these animals. To test this, plasma TNF activity, cardiac index, systemic and pulmonary arterial pressures, arterial Po 2 and bronchoalveolar lavage protein content were monitored for 300 min in four groups of anesthetized pigs: saline-infused control pigs (n = 4); pigs infused for 60 min with Pseudomonas aeruginosa (5 x 10 8 organisms/mL,.3 mL/20 kg/min) (n = 5) and pigs infused for 60 min with P. aeruginosa plus ibuprofen (12.5 mg/kg) alone (n = 4) or ibuprofen plus cimetidine (150 mg) and diphenhydramine (30 mg/kg) at 0 and 120 min (CID, n = 4). Within 60 min, pigs infused with P. aeruginosa exhibited increased plasma TNF activity (>8-fold increase in ng/mL TNF; L929 cytolysis assay) and showed alterations in all hemodynamic and pulmonary parameters. Ibuprofen or CID administration in the septic pigs decreased peak TNF activity by 4.6 and 10.2 ng/mL, respectively, and CID treatment was correlated with better attenuation of certain sepsis-induced alterations. These results show that CID treatment attenuates sepsis-induced injury and that this is correlated with reduced plasma TNF activity in a porcine model of sepsis-induced acute organ injury.

Histamine receptor antagonists, cyclooxygenase blockade, and tumor necrosis factor during acute septic insult

Despite few laboratory studies concerning the effects of sensitized lymphocytes on experimental tumor growth, interest in the use of such cells for the treatment of human neoplasms is increasing. The present study presents findings concerning the effects of sensitized isologous, allogeneic, and xenogeneic lymphocytes on transplanted small (5 mm) methylcholanthrene (MC)‐induced and spontaneous mammary tumors in isogeneic hosts and on the growth of cells from such tumors inoculated into appropriate syngeneic recipients. Such lymphocytes were either those circulating in blood and lymph or those obtained from lymph nodes, spleen, or thymuses of tumor‐sensitized animals. In a number of studies, zymosan was employed alone and with lymphocytes. Despite a variety of therapeutic regimens, these studies have been disappointing in that they have failed to convincingly demonstrate that the employment of such cells in the model systems utilized have influenced tumor growth either of an inoculum of cells or of a small‐growing tumor. Of the many experiments carried out, only one using sensitized isologous circulating lymphocytes in animals harboring MC‐induced tumor cell inocula was effective in this regard. Allogeneic and xenogeneic cells were uniformly ineffectual. Consideration is given to these findings in relation to those of other investigators, and it is concluded that more experimental evaluation of the use of immune lymphocytes is in order prior to the extensive application of their clinical use.

Experiences with lymphocyte immunotherapy in experimental tumor systems.

Tumor transplantation by the subcutaneous inoculation of cell suspensions or implants is one of the oldest and most extensively employed techniques in experimental cancer research. Little information, however, is available relative to the time and extent of dissemination of such cells from their transplantation site, and even less has been reported regarding the influence of local factors upon this process. Such knowledge would seem fundamental for the proper interpretation of results obtained from investigations concerning tumor growth at the point of inoculation and the occurrence of distant metastases. The present study was conducted for the following reasons: (1) to provide quantitative information relative to the egress of labeled tumor cells from their injection site; (2) to compare the rapidity of tumor cell dissemination and development of lung metastases following inoculation of cell suspension with that occurring after insertion of tumor plugs; (3) to evaluate the effect of physical factors such

The Dissemination of Subcutaneously Inoculated Tumor Cell Suspensions

Cytoplasmic liver cell inclusions following arterialization in the dog.

Summary The incidence of hepatic metastases in rats subjected to intraportal injection of 5,000 Walker 256 carcinosarcoma cells was similar in rats receiving cortisone and their pair-fed controls. This result was obtained whether cortisone was given prior to, after, or preceding and following the injection of tumor cells. Both cortisone-treated and pairfed control rats exhibited significantly fewer metastases than did controls allowed food ad libitum and demonstrating a gain in body weight. The number of metastatic lesions in each positive instance was similar in each group. The relationship of these findings to liver composition following cortisone administration is discussed. The increase of hepatic glycogen in cortisone-treated rats (when compared with pair-fed controls) was apparently without effect on the incidence of such metastases. Adrenalectomy similarly failed to influence the incidence of hepatic metastases as compared with that observed in controls. The increased incidence of hepatic metastases observed following hepatic manipulation was not dependent upon intact adrenals. It was concluded that adrenal function played little specific role in the development of hepatic metastases in the experimental model employed.

Bernard Fisher

Papers

Histamine receptor antagonists, cyclooxygenase blockade, and tumor necrosis factor during acute septic insult

Experiences with lymphocyte immunotherapy in experimental tumor systems.

The Dissemination of Subcutaneously Inoculated Tumor Cell Suspensions

Cytoplasmic liver cell inclusions following arterialization in the dog.

Experimental studies of factors influencing hepatic metastases. V. Effect of cortisone and adrenalectomy.