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.

Summary The administration of a saturating dose of either India ink or Thorotrast prior to or within 24 hours after the intraportal injection of Walker 256 carcinoma cells resulted in an increased incidence and size of artificially induced hepatic metastases. The enhancing effect of reticuloendothelial interference on tumor “takes” may be explained as being due to a trapping of injected tumor cells as a result of marked swelling of Kupffer cells with sinusoidal occlusion and hepatic parenchymal damage which was observed after the administration of both Thorotrast and India ink. These mechanisms abrogate the necessity of relating such an effect to an inhibitory action of these agents on a specific activity of the reticuloendothelial system associated with tumor resistance. Established hepatic metastases of Walker 256 carcinoma fail to influence the phagocytic activity of the reticuloendothelial system as disclosed by a normal blood clearance of carbon particles. Similarly carbon clearance was normal when observed in animals 14 days after tumor cell injection but in whom hepatic metastases were not demonstrable.

Experimental studies of factors influencing hepatic metastases. VII. Effect of reticuloendothelial interference.

Breast cancer, Pathologic discriminants breast cancer, Clinical discriminants breast cancer. Spread breast cancer, Lymph nodes breast cancer, Metastases breast cancer. The conventional view regarding the natural history of breast cancer indicates that the primary tumor spreads to the regional lymph nodes at some given time(s) and subsequently disseminates systemically with further time. The significance of the time factor in this scheme at present is polemical. This concept also ascribes a primary role to lymphatic extension in the spread of the disease. Experimental studies which have demonstrated that tumor cells traverse lymph nodes’ and thus gain access into the general circulation tend to support such a contention. Yet, it is surprising that little attention has been directed to the phenomenon of direct neoplastic venous extension. That such an event occurs might be deduced from the, albeit rare, histopathological evidence of intravascular extension’* as well as from the presence of tumor cells in venous tributaries draining breast cancers in about 0.25 of a small series of cases.” This latter phenomenon was noted with equal frequency in both pathologic Stage I and II cases. Nevertheless, it has been demonstrated repeatedly that regional nodal metastases represent an ominous prognostic discriminant for breast cancer. Indeed, a prevalent view regards nodal metastases as an index of systemic spread. It readily becomes apparent that identification of factors which might influence or reflect the nodal status of patients with breast cancer might provide some insight into the mechanism as well as significance of such an event. Accumulating evidence of decreasing treatment failure rates with the use of adjuvant chemotherapy in patients with Stage II disease indicates the pragmatic value of such information.5*9.” In this communication we shall attempt to identify those pathological and clinical characteristics that were found to be associated with lymph node metastases and treatment failure rates [% failure/(average months followed)] in patients with invasive, operable mammary cancer (clinical Stages I and II) entered in the National Surgical Breast Project, protocol No. 4. These women were randomized prospectively so that those with clinically negative axillae were treated by radical mastectomy or total (simple) mastectomy with or without postoperative irradiation. Those with clinically positive axillae also were randomized and received either radical mastectomy or total mastectomy and irradiation. Information relating to nodal involvement is based upon the 386 patients of the first 1000 entered that were subjected to radical mastectomy regardless of clinical nodal status. These patients were categorized according to whether pathological examination of their axillary nodes revealed 1-3, 4 or more, or no nodal metastases. This stratification was derived from previous studies which indicated that the number rather than proportion of involved nodes were significantly related to survival.” Associations with treatment failure rates were compiled from information obtained from all arms of the study constituting 1665 patients. Treatment failure is defined as the occurrence of local or regional recurrence or systemic metastases or death resulting from breast cancer. The average period of

Relationship of pathologic and some clinical discriniinants to the spread of breast cancer

Antilymphocyte serum (ALS) administration was observed to abrogate preexisting immunity to C3H tumors, permitting them to grow equally as well as, or slightly better than, in nonimmunized animals. Metastases from spontaneous C3H tumors were observed in ALS-treated mice but not in controls, indicating the possible importance of concomitant immunity in the phenomenon of metastases. This was not related to effects of ALS on the tumor cells per se as revealed by the behavior of transplants from treated animals in syngeneic hosts as well as their unaltered subcellular appearance. ALS failed to influence the latent period of 3-methylcholanthrene-induced tumors. These findings are considered in relation to the observations of others on the mechanism of 3-methylcholanthrene tumor induction. Incubation in vitro of tumor cells in ALS was without effect on their subsequent growth in syngeneic hosts. However, such incubation resulted in complete abrogation of the tumor growth-inhibitory effect observed in F1 hybrids (C3H × DBA). The amount of ALS used in the incubation was insufficient to influence tumor growth when administered at a site remote from that utilized for tumor incubation. Prior incubation in ALS allowed C3H tumor cells to grow in allogeneic DBA mice. When F1 hybrid spleen cells or sensitized lymphocytes were incubated with tumor cells before their inoculation into syngeneic hosts, a retardation of tumor growth occurred. Addition of ALS to the incubate eliminated this inhibitory effect. Inoculation of tumor cells alone resulted in a depression of circulating lymphocytes. When cells were injected in ALS or the latter was inoculated at a separate site, depression of lymphocytes was greater and more sustained than after the administration of either alone. Such findings minimize the importance of lymphopenia in accounting for these results, particularly in the F1 hybrid. Considerations pertinent to these observations strongly indicate the nonimmunological nature of allogeneic inhibition but, also as discussed, tend to minimize the role of the latter as a surveillance mechanism for autochthonous tumors.

https://cancerres.aacrjournals.org/content/canres/30/7/2035.full.pdf

Bernard Fisher

Papers

Experimental studies of factors influencing hepatic metastases. VII. Effect of reticuloendothelial interference.

Relationship of pathologic and some clinical discriniinants to the spread of breast cancer

Further observations concerning effects of antilymphocyte serum on tumor growth: with special reference to allogeneic inhibition.

Thyroid and parathyroid implantation: an experimental re-evaluation.

Rewarming following hypothermia of two to twelve hours. I. Cardiovascular effects.