Augustinus A. M. Hart

Gene expression profiling predicts clinical outcome of breast cancer.

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.

High-throughput retroviral tagging to identify components of specific signaling pathways in cancer

Genetic screens carried out in lower organisms such as yeast, Drosophila melanogaster and Caenorhabditis elegans have revealed many signaling pathways. For example, components of the RAS signaling cascade were identified using a mutant eye phenotype in D. melanogaster as a readout. Screening is usually based on enhancing or suppressing a phenotype by way of a known mutation in a particular signaling pathway. Such in vivo screens have been difficult to carry out in mammals, however, owing to their relatively long generation times and the limited number of animals that can be screened. Here we describe an in vivo mammalian genetic screen used to identify components of pathways contributing to oncogenic transformation. We applied retroviral insertional mutagenesis in Myc transgenic (EμMyc) mice lacking expression of Pim1 and Pim2 to search for genes that can substitute for Pim1 and Pim2 in lymphomagenesis. We determined the chromosomal positions of 477 retroviral insertion sites (RISs) derived from 38 tumors from EμMyc Pim1−/− Pim2−/− mice and 27 tumors from EμMyc control mice using the Ensembl and Celera annotated mouse genome databases. There were 52 sites occupied by proviruses in more than one tumor. These common insertion sites (CISs) are likely to contain genes contributing to tumorigenesis. Comparison of the RISs in tumors of Pim-null mice with the RISs in tumors of EμMyc control mice indicated that 10 of the 52 CISs belong to the Pim complementation group. In addition, we found that Pim3 is selectively activated in Pim-null tumor cells, which supports the validity of our approach.

Risk factors in breast-conservation therapy.

PURPOSE To identify clinical and pathologic factors associated with an increased risk of local recurrence following breast-conservation therapy (BCT) to assess the safety of this procedure for all subgroups of patients. PATIENTS AND METHODS The study population consisted of 1,026 patients with clinical stage I and II breast cancer treated between 1979 and 1988 at the Netherlands Cancer Institute. The BCT regimen consisted of local excision and axillary lymph node dissection (ALND) followed by whole-breast irradiation to a total dose of 50 Gy in 2-Gy fractions and boost irradiation (mostly by iridium implant) of 15 to 25 Gy. RESULTS With a median follow-up duration of 66 months, the actuarial breast recurrence rate was 4% at 5 years, counting all breast recurrences. Univariate analysis showed seven factors to be associated with an increased risk of local recurrence; age, residual tumor at reexcision, histologic tumor type, presence of any carcinoma-in-situ component, vascular invasion, microscopic margin involvement, and whole-breast radiation dose. Three factors remained independently significant after proportional hazard regression analysis: age, margin involvement, and the presence of vascular invasion. When the analysis was repeated, but counting only those breast recurrences that occurred before regional or distant failures, only young age and vascular invasion were independent predictive factors. In the third analysis, factors predicting the necessity of local salvage treatment were analyzed. In this analysis, the possible bias in the former analysis caused by censoring actuarial methods was avoided. The results were the same as in the second analysis, showing young age and vascular invasion as the only independent predictive factors. Breast recurrence rates were 6% for patients less than 40 years of age and 8% for patients with tumors showing vascular invasion. In the absence of risk factors, the breast recurrence rate is only 1% at 5 years. CONCLUSION Slightly higher recurrence rates were found in patients less than 40 years of age and in patients with tumors showing vascular invasion. The role of margin involvement is uncertain.

Recombinant congenic strains — A new tool for analyzing genetic traits determined by more than one gene

Discovery of the H-2 system by Gorer (1936) and its further analysis (for review of the early work, see Klein 1975, Snell et al. 1976, Festenstein and Demant 1978) led to several unexpected findings concerning the biological effects of the histocompatibility genes or genes closely linked to the major histocompatibility complex (MHC). One of these findings was that the MHC genes in the mouse influence susceptibility to virally induced leukemia (Lilly et al. 1964); another finding was that susceptibility to a number of diseases is specifically associated with certain HLA haplotypes (for review, see Tiwari and Terasaki 1985). The control of these phenomena frequently involves, in addition to the MHC, several genes not linked to the MHC. Our interest in understanding the genetic context in which the action of the MHC genes takes place led us to evaluate the available genetic approaches used to analyze such traits controlled by multiple genes. We show here that in order to achieve sufficient resolution power to analyze such traits, a new genetic system, the recombinant congenic strains (RCS), is needed. We describe the main features of this new system and its advantages and disadvantages as compared with other genetic systems. This tool for genetic analysis will be discussed with respect to its application to tumor biology; however, it can be applied also to other types of multigenic quantitative polymorphisms.

Predicting a local recurrence after breast-conserving therapy by gene expression profiling

IntroductionTo tailor local treatment in breast cancer patients there is a need for predicting ipsilateral recurrences after breast-conserving therapy. After adequate treatment (excision with free margins and radiotherapy), young age and incompletely excised extensive intraductal component are predictors for local recurrence, but many local recurrences can still not be predicted. Here we have used gene expression profiling by microarray analysis to identify gene expression profiles that can help to predict local recurrence in individual patients.MethodsBy using previously established gene expression profiles with proven value in predicting metastasis-free and overall survival (wound-response signature, 70-gene prognosis profile and hypoxia-induced profile) and training towards an optimal prediction of local recurrences in a training series, we establish a classifier for local recurrence after breast-conserving therapy.ResultsValidation of the different gene lists shows that the wound-response signature is able to separate patients with a high (29%) or low (5%) risk of a local recurrence at 10 years (sensitivity 87.5%, specificity 75%). In multivariable analysis the classifier is an independent predictor for local recurrence.ConclusionOur findings indicate that gene expression profiling can identify subgroups of patients at increased risk of developing a local recurrence after breast-conserving therapy.

Impact of Pathological Characteristics on Local Relapse After Breast-Conserving Therapy: A Subgroup Analysis of the EORTC Boost Versus No Boost Trial

PURPOSE To investigate the long-term impact of pathologic characteristics and an extra boost dose of 16 Gy on local relapse, for stage I and II invasive breast cancer patients treated with breast conserving therapy (BCT). PATIENTS AND METHODS In the European Organisation for Research and Treatment of Cancer boost versus no boost trial, after whole breast irradiation, patients with microscopically complete excision of invasive tumor, were randomly assigned to receive or not an extra boost dose of 16 Gy. For a subset of 1,616 patients central pathology review was performed. RESULTS The 10-year cumulative risk of local breast cancer relapse as a first event was not significantly influenced if the margin was scored negative, close or positive for invasive tumor or ductal carcinoma in situ according to central pathology review (log-rank P = .45 and P = .57, respectively). In multivariate analysis, high-grade invasive ductal carcinoma was associated with an increased risk of local relapse (P = .026; hazard ratio [HR], 1.67), as was age younger than 50 years (P < .0001; HR, 2.38). The boost dose of 16 Gy significantly reduced the local relapse rate (P = .0006; HR, 0.47). For patients younger than 50 years old and in patients with high grade invasive ductal carcinoma, the boost dose reduced the local relapse from 19.4% to 11.4% (P = .0046; HR, 0.51) and from 18.9% to 8.6% (P = .01; HR, 0.42), respectively. CONCLUSION Young age and high-grade invasive ductal cancer were the most important risk factors for local relapse, while margin status had no significant influence. A boost dose of 16 Gy significantly reduced the negative effects of both young age and high-grade invasive cancer.

Maximizing setup accuracy using portal images as applied to a conformal boost technique for prostatic cancer

A design procedure of a patient setup verification protocol based upon frequent digital acquisition of portal images is demonstrated with an application for conformal prostatic boost fields. The protocol aims at the elimination of large systematic deviations in the patient setup and includes decision rules which indicate when correction of the patient setup is needed. The decision rules were derived from the results of a theoretical and quantitative analysis of patient setup variations measured in three pelvic fields (one anterior-posterior and two lateral fields) of 105 fractions for nine patients. Deviations in the patient positioning, derived from one field, were quantified as two-dimensional (2-D) displacement vectors in the plane perpendicular to the beam axis by alignment of anatomical features in the portal and the simulator image. The magnitude of the overall setup variations along the anterior-posterior, superior-inferior and lateral directions varied between 2.6 and 3 mm (1 S.D.). Inter- and intra-treatment variations could be separated, both having equal magnitudes of 1.7 to 2.2 mm (1 S.D.). In addition, intra-treatment variations appeared to be predictable which was a prerequisite for the development of the decision rules. The 2-D setup deviations, measured in the three fields of one fraction were strongly correlated and a 3-D displacement vector was calculated. Utilization of this 3-D vector in a setup verification protocol may lead to an early detection of systematic setup deviations.

Meningeal carcinomatosis in breast cancer. Prognostic factors and influence of treatment

In 58 breast cancer patients with meningeal carcinomatosis (MC) pretreatment characteristics, clinical course, and response to treatment were evaluated. Forty‐four patients were uniformly treated with intraventricular chemotherapy. Fourteen patients did not receive intraventricular treatment. In the intraventricularly treated group the median survival was 12 weeks. Multivariate analysis of the pretreatment characteristics of the intraventricularly treated patients demonstrated a prognostic significance with respect to survival for age older than 55 years, lung metastases, cranial nerve involvement, cerebrospinal fluid (CSF) glucose < 2.5 mmol/l, and CSF protein 0.51 to 1.0 g/l. Based on the significance of these predicting factors a prognostic index (PI) identified four groups of patients with a median survival of 43 weeks, 22 weeks, 11 weeks, and 3 weeks, respectively. After 6 weeks of intraventricular treatment 22 patients showed a neurologic improvement or stabilization, and nine patients showed a worsening of the neurologic signs, whereas 13 patients (30%) had already died. The responders had a median additional survival of 5 months versus 1 month for nonresponders. No relation was found between survival and intensity of the intraventricular treatment after the first 6 weeks of treatment. Almost all long survivors had also received systemic treatment for systemic disease, whereas most patients who died within 6 months did not receive systemic therapy. Radiation therapy had no influence on the survival time. Early death due to the intensive treatment occurred in three patients. In 11 of the 17 patients who survived more than 4 months an often seriously debilitating late neurotoxicity developed. The survival curve of the nonintraventricularly treated patients appeared to be essentially the same as the curve of the intraventricularly treated patients. Using the same PI the predicted survival time was also the same as in the intraventricularly treated group. It is concluded that survival in MC from breast carcinoma may be more dependent on some pretreatment characteristics than on treatment intensity. On the basis of these pretreatment characteristics the survival time seems to be predictable. Finally, late neurotoxicity due to aggressive treatment leads to impairment of the quality of life in more than 50% of the long survivors. The exact value of intraventricular and systemic therapy in patients with MC still has to be determined.

Bone mineral density after adjuvant chemotherapy for premenopausal breast cancer.

Lumbar bone mineral density (BMD) determination by dual photon absorptiometry was used to study the influence of adjuvant chemotherapy for premenopausal breast cancer on the risk of premature osteoporosis. Six cycles of combination chemotherapy caused ovarian failure in 31 of 44 (71%) women, amenorrhoea mostly already beginning during treatment. In contrast, only seven of 44 (16%) women, who were pair-matched for age and year of breast cancer surgery and had not been treated with chemotherapy, were post-menopausal at the time of measurement. The mean interval after breast surgery was 3.5 years. The significantly decreased BMD in the treated group (1.17 compared to 1.29 g cm-2) could only be explained by the high incidence of menopause in these women, which on average occurred 10 years prematurely. Extrapolation of these findings suggests that adjuvant chemotherapy may precipitate osteoporotic fractures by some 10 years in a considerable proportion of women cured of premenopausal breast cancer.

Brain metastases in breast cancer; natural history, prognostic factors and outcome

SummaryOne hundred and thirty seven breast cancer patients with CT scan documented brain metastasis (BM) were reviewed. Occurrence of brain as first site of relapse was associated with adjuvant systemic therapy of the primary tumor. Multivariate analysis showed significantly longer survival in patients without manifest systemic disease, in patients with a solitary BM, in those with neurologic symptoms present for more than 4 weeks prior to diagnosis, and in those treated with chemotherapy after diagnosis. When controlling for prognostic factors no significant difference in survival was found between surgery and radiotherapy (RT) as treatment of a solitary lesion. Tumor size, tumor necrosis and mass effect had no demonstrable influence on survival. Overall median survival was 16 weeks and 19% survived one year. Neurologic disease was the cause of death or a major contributing factor to it in 68% of the patients, indicating the need for improvement of the treatment of BM itself. These results warrant further studies on the value of surgery, RT and chemotherapy in solitary as well as multiple BM from breast carcinoma.