p53 mutations occur in aggressive breast cancer.

Using a polymerase chain reaction-single strand conformation polymorphism approach we analyzed 96 human primary breast tumors for the presence of mutations in exons 2, 5, 6, 7, 8, and 9 of the p53 gene. These exons have been shown to comprise highly conserved sequences and the portion including exons 5 through 9 is believed to be the target for over 90% of the acquired mutations in human cancer. Eighteen tumors of the 96 (18.7%) tested showed reproducibly a variant band indicative of a mutation. Most (15 tumors) of the mutations were single nucleotide substitutions and G:C to A:T transitions were prevalent (6 tumors), G:C to T:A transversions came next (4 tumors), and guanines were always on the nontranscribed strand. Concomitant loss of the wild type allele and mutation of the other copy was observed in only 3 of 18 mutated cases; this is consistent with the heterogeneous cellular composition of breast tumors. Furthermore p53 mutations were correlated to estrogen and/or progesterone receptor negative tumors, thus indicating their relationships to aggressive breast cancer. No association could be observed with DNA amplification events in these tumors.

Mapping of DNA amplifications at 15 chromosomal localizations in 1875 breast tumors: definition of phenotypic groups.

DNA amplification is frequent in breast cancer and has been associated with specific clinicopathological parameters and/or worsened course of the disease. In the present work, we were interested in further defining the association linking the occurrence of DNA amplification to the emergence of specific breast tumor phenotype. To this aim, we studied by Southern blotting a total of 1875 breast tumor DNAs with 26 probes mapping at 15 distinct chromosomal localizations. Of the 26 loci tested, 11 loci showed elevated levels of amplification, 9 loci showed occasional and/or low level of DNA copy number increase, and 6 loci showed very rare or no variation. This allowed us to define six amplified domains mapping at 8p12, 8q24, 11q13, 12q13, 17q12, and 20q13.2, respectively. Over 60% of the tumors analyzed presented at least one amplification at one of these localizations. Amplifications often covered large regions of DNA and bore complex patterns involving coamplification of several colocalized markers. Statistical analysis revealed correlations associating DNA amplification with breast tumor phenotype, as well as sets of preferential coamplifications. Based on these correlations, we defined three subsets of breast cancer according to their patterns of DNA amplification. The first subset (group A) was organized around the amplifications at 11q13 and/or 8p12 and was predominantly composed of estrogen receptor-positive tumors and presented a large proportion of lobular cancers. The second subset (group B) was organized around the amplifications of ERBB2 and/or MYC. These tumors were mostly estrogen receptor-negative and of the ductal invasive type. The third subset (group C) corresponded to tumors in which no amplification was detected in the present screen. Tumors in this group were largely diploid and of low histopathological grading.

Genome-wide search for loss of heterozygosity shows extensive genetic diversity of human breast carcinomas.

Deletions of genomic regions involving tumor suppressor genes are thought to be important in the initiation and progression of breast cancer. We conducted a genome-wide search for deleted regions in a series of 75 human breast carcinomas by studying the allelic patterns of 184 microsatellite markers distributed over all chromosomes and looking for loss of heterozygosity (LOH). We identified 56 regions of consistent LOH. Strikingly, every tumor had a different set of deletions. To study this complexity, we applied a phylogenetic-like type of analysis. Each region was involved in a certain proportion of tumors, ranging from 20 to 62%; the most frequently involved regions were on chromosome arms 8p, 11q, 16q, and 17p. There was a correlation (P = 0.005) between the level of LOH and the size of the tumors. Tumors with a high level of LOH were also highly proliferative and had a high mitotic index.

Potential value of increased MYC but not ERBB2 RNA levels as a marker of high-risk mastopathies.

MYC and ERBB2 levels were measured in 38 benign breast diseases using a semiquantitative in situ hybridization technique. Mean levels of MYC and ERBB2 gene expression in benign tissues were similar to those measured in 15 breast cancers with no amplification at the loci concerned. Interestingly, MYC but not ERBB2 RNA levels were increased (t-test, P = 0.03) in benign mastopathies of patients with a first-degree (mother/sister) family history (FH) of breast cancer. Among patients without a first-degree FH, MYC RNA levels were significantly higher (t-test, P = 0.02) during the follicular (preovulatory) than the luteal (post-ovulatory) phase and also significantly higher than levels observed in patients with no menstrual cycle (peri- or postmenopausal) (P = 0.004), indicating an in vivo hormonal regulation of MYC. After exclusion of the first-degree FH patients a higher MYC expression was detected in atypia than in other histological types at the follicular but not at the luteal phase, suggesting an increased sensitivity of these high-risk lesions to estrogens. We propose that in addition to a family history and proliferative atypia, elevated MYC RNA levels during the post-ovulatory phase could potentially be used as a marker of the risk of developing breast cancer. The increase in MYC RNA in high-risk breast diseases also suggests that MYC deregulation might be involved in the early stages of mammary carcinogenesis.

Proto-oncogene amplification and human breast tumor phenotype.

Amplification of c-myc, c-erbB-2, hst and int-2 proto-oncogenes was investigated in two independently collected breast tumor series comprising 292 carcinomas. Differences in the frequencies of amplification could be observed between these two series for c-myc (9.3% vs. 20.8%) and hst/int-2 (21.5% vs. 15.6%) whereas similar values were found for c-erbB-2 (22.5% vs. 20.3%). Statistical correlations between amplification and disease parameters were also dependent on population sampling. Therefore we performed our statistical analysis on the pooled populations and focused on the 219 primary breast carcinomas from patients without therapy prior to surgery. Amplification of c-erbB-2 was strongly correlated to the absence of either estrogen (ER-, P = 0.003) or progesterone (PR-, P = 0.004) receptors. An amplified c-myc was significantly associated with PR- (P = 0.005) and was prevalent in high grade tumors. On the contrary, hst/int-2 amplification was correlated to PR+ tumors (P = 0.01) and was more frequent in ER+ and low grade tumors, and was also correlated with lymph node involvement (P = 0.04). Our data suggest that amplification of each of these proto-oncogenes could be representative of a particular subset of breast tumors. Therefore, proto-oncogene amplification may be helpful in characterizing new biological subclasses in human breast cancer.

Amplification of FGF-related genes in human tumors: possible involvement of HST in breast carcinomas.

In order to document a possible involvement of structural alterations of FGF (Fibroblast Growth Factor)-like genes in human oncogenesis, we have screened a large series of human tumors for amplification of five FGF-related genes (Basic-FGF, INT2, HST, FGF5 and FGF6). None of 37 hematopoietic neoplasms, one out of 13 melanomas (8%), three out of 43 bladder tumors (7%) and 41 out of 238 breast carcinomas (17%) contained amplified FGF-related sequences, namely HST and INT2. Only these two genes, both located on band q13 of chromosome 11 have been found amplified. In all cases they were co-amplified and in only one instance did amplification extend to the ETS1 locus at position 11q23. INT2 and HST RNA could be evidenced by RNA/RNA in situ hybridization in breast carcinomas. Our results indicate a correlation between RNA expression and gene amplification in the case of HST but not of INT2. Although evaluation of the clinical significance of HST amplification and expression must await long-term follow-up of the patients, we suggest that HST gene product could play a role in development and/or progression of human breast cancer.

Decrease of c-erbB-2 and c-myc RNA levels in tamoxifen-treated breast cancer.

The c-myc, c-erbB-2, hst and int-2 oncogenes are frequently amplified and/or overexpressed in human breast carcinomas. We studied the effect of tamoxifen on RNA levels of these oncogenes in 19 breast cancer patients treated for 3 weeks prior to surgery as compared with 22 control patients. RNA levels were measured by in situ hybridization coupled with computer-aided quantification. c-myc and c-erbB-2 expression was high in the control population (mean values: 23.4 and 29.1 grains/cell respectively) and significantly decreased in the tamoxifen-treated population (mean values: 14.6 and 7.4 grains/cell respectively) (P = 0.018, P = 0.003 respectively); hst and int-2 RNA levels were low (2-6 grains/cell) and not significantly altered by the treatment. There was a correlation between gene amplification and expression for c-erbB-2 (P = 0.0005) and hst (P = 0.02) in the control population. Elevated c-erbB-2 RNA level was correlated with the absence of estrogen (P = 0.02) or progesterone (P = 0.05) receptors. In the ER+ population, the tamoxifen-treated group had significantly lower c-myc expression levels than the control group (P = 0.04) which is in agreement with the estrogen induction of c-myc in ER+ T47D cell line and its inhibition by antiestrogens. Surprisingly, c-erbB-2 expression in the tamoxifen-treated group was significantly diminished in the ER- (P = 0.02) and PR- (P = 0.01) populations. This effect was not observed in the ER- BT474 cell line. These results suggest that in vivo tamoxifen decreases c-myc and c-erbB-2 RNA levels in breast cancer cells via two different mechanisms. To our knowledge this is the first evidence of in vivo down regulation of a gene by tamoxifen in ER- breast cancer cells.

Risk factors for recurrence and metastasis after breast-conserving therapy for ductal carcinoma-in-situ: analysis of European Organization for Research and Treatment of Cancer Trial 10853.

PURPOSE: In view of the increasing number of patients treated with breast-conserving treatment (BCT) for ductal carcinoma-in-situ (DCIS), risk factors for recurrence and metastasis should be identified. PATIENTS AND METHODS: Clinical and pathologic characteristics from patients with DCIS in the European Organization for Research and Treatment of Cancer trial 10853 (excision with or without radiotherapy) were related to the risk of recurrence. Pathologic features were derived from a central review of 863 of the 1,010 randomized cases (85%). The median follow-up was 5.4 years. RESULTS: Factors associated with an increased risk of local recurrence in the multivariate analysis were young age (< or = 40 years) (hazard ratio, 2.14; P =.02), symptomatic detection of DCIS (hazard ratio, 1.80; P =.008), growth pattern (solid and cribriform) (hazard ratios, 2.67 and 2.69, respectively; P =.012), involved margins (hazard ratio, 2.07; P =.0008), and treatment by local excision alone (hazard ratio, 1.74; P =.009). The risk of invasive recurrence was not related to the histologic type of DCIS (P =.63), but the risk of distant metastasis was significantly higher in poorly differentiated DCIS compared with well-differentiated DCIS (hazard ratio, 6.57; P =.01). CONCLUSION: Patients with poorly differentiated DCIS have a high risk of distant metastasis after invasive local recurrence. Margin status is the most important factor in the success of BCT for DCIS; additionally, young age and symptomatic detection of DCIS have negative prognostic value.

Cathepsin D assay in primary breast cancer and lymph nodes: relationship with c-myc, c-erb-B-2 and int-2 oncogene amplification and node invasiveness.

In breast cancer, axillary lymph node invasiveness is the major prognostic factor in predicting relapse and metastasis. Nevertheless, since 30% of node-negative tumors also relapse, it is necessary to develop other independent prognostic factors. Oncogene amplification and the level of cathepsin D (cath-D), an acidic lysosomal protease produced and secreted in excess by breast cancer cells, have been proposed as additional prognostic factors. We have compared the cytosolic cath-D level and the amplification of three oncogenes: c-myc, neu-erb-B-2 and int-2 in 140 primary breast carcinomas and 64 axillary lymph nodes collected in 1987 and 1988 at the Cancer Center of Montpellier (Centre Paul Lamarque). None of the patients had previously received hormonal or chemotherapy. The cath-D concentration was measured with an immunoradiometric assay using monoclonal antibodies. DNA purified from the same samples was analyzed by a standard Southern blotting technique to estimate oncogene amplification. No correlation was found between the level of cath-D in the tumor and node invasiveness. Using a cut-off level of 60 pmol/mg protein, the status of cath-D was not correlated with neu-erb-B-2 and int-2 amplification and only correlated with c-myc amplification (P = 0.011). Both c-myc and cath-D are associated with cell proliferation, induced by estrogens in ER+ breast cancer, and constitutively produced in ER- breast cancer. The level of cath-D was significantly higher in the invaded lymph nodes (P = 0.04) than in the histologically non-invaded ones. Nevertheless, some non-invaded lymph nodes contained a high level of cath-D, as confirmed by immunoperoxidase staining. In conclusion, in breast cancer, a high cytosolic cath-D concentration is more frequent in tumors with c-myc amplification but is dissociated from neu-erb-B-2 or int-2 amplification, suggesting that the determination of these three markers will have an additional prognostic value.

Tumor progression and oxidant-antioxidant status.

We previously reported on a paradoxical oxidant-antioxidant status in breast cancer patients, more so in pre-menopausal than menopausal women. In this study, measurements were performed on 146 patients with various carcinomas. Vitamin E/total cholesterol increased and plasma malondialdehyde decreased with tumor size and progression. To investigate the difference between young pre-menopausal and aged menopausal breast cancer patients, the same measurements were performed in 365 breast cancer patients according to pathology, tumor size and estrogen receptors. The oxidant-antioxidant status varied with these prognosis factors in the same pattern, and was more pronounced in young than aged women.

Quantification of ERBB2 protein expression in breast cancer: three levels of expression defined by their clinico-pathological correlations.

Activation of the ERBB2 oncogene seems to be an early event in breast cancer progression and prevalent in in situ carcinomas. However, its prognosis value, albeit recognized for node-positive patients, remains controversial for those without apparent nodal involvement. One possible reason for this problem is likely to be the difficulty of defining threshold levels for ERBB2 protein overexpression. ERBB2 protein expression was therefore analyzed in primary invasive breast tumors. Quantification of the gene product by a commercial ELISA test was compared to results obtained by immunohistochemistry and western blotting, as well as to gene amplification status determined by Southern blotting. Correlations between results obtained by the different techniques were highly significant (P value < 10(-6)). Nevertheless, ELISA permitted us to determine three levels of protein expression corresponding to distinct tumor subsets. 1) Tumors with p185/ERBB2 expression levels exceeding 10 U/microgram exhibited in most cases amplification of the gene (83% of cases), DNA aneuploidy (81%) and absence of estrogen receptor (ER) (44%). Such high levels of protein expression were exclusively observed in invasive ductal carcinomas and were prevalent in those showing a significant in situ component. 2) "Intermediate" levels of expression (3-10 U/micrograms) were rarely observed in tumors exhibiting gene amplification (9%), but were preferentially found in cancers of more favorable prognosis (only 49% were aneuploid and 9% estrogen receptor negative). 3) Levels of p185/ERBB2 below 3 U/micrograms were detected in benign mastopathies and, thus, carcinomas presenting such levels were scored ERBB2 negative. Interestingly, invasive lobular carcinomas were rarely ERBB2 positive, and if so, only at intermediate levels. Moreover, our data show a complex interrelationship between p185/ERBB2 expression and ER levels. Indeed, tumors with more than 10 U/micrograms of p185 were prevalently ER, whereas those with p185 ranging from 3 to 10 U presented elevated levels of ER.

Tumor cell membrane as a potential target for methyl-beta-cyclodextrin.

The purpose of this work was to determine the role of methyl-beta-cyclodextrin (MEBCD) in combination with doxorubicin (DOX) on DOX intracellular accumulation and efflux, in comparison to verapamil in a sensitive parental and multidrug-resistant human cancer cell line (HL-60 S and HL-60 R). Moreover, cell membrane and nuclear modifications induced by MEBCD were investigated. At concentration of 10 mumol for 10(6) cells, MEBCD combined with doxorubicin (DOX), was able to significantly enhance the intracellular concentration of DOX in HL-60 S and HL-60 R cell lines during the period of exposure. In the resistant subline, MEBCD activity was higher than that of verapamil. Moreover, treatment of cells with MEBCD resulted in a modification in cell membrane integrity and cell morphology, but had no own activity in the distribution of the cells within cell cycle.

[Primary angiosarcoma of the breast. Apropos of 2 cases]. / Les angiosarcomes primitifs du sein. A propos de 2 cas.

We report here 2 cases of breast angiosarcoma observed at Centre Val-d' Aurelle of Montpellier over a period of 14 years (1980 to 1994). Based on a review of literature, we analyze the epidemiological, pathological, clinical, diagnostic and treatment aspects of this rare type of breast cancer. The difficulties of histological diagnosis are underlined. Mastectomy is the treatment of reference. Breast angiosarcoma has the worst prognosis of all mammary malignancies, with a mean survival of 24 months. The low histologic grade and an early diagnosis are the most important factors of good prognosis. The benefit of irradiation and chemotherapy as adjuvant therapy remains to be demonstrated.

[2002 Standards, Options and Recommendations: good practice for the management and shipment of histological and cytopathological cancer specimens]. / Standards, Options et Recommandations 2002 bonnes pratiques de l'acheminement et de la prise en charge initiale d'un prélèvement en anatomie et cytologie pathologiques en cancérologie (rapport intégral).

CONTEXT: The Standards, Options and Recommendations (SOR) collaborative project was initiated in 1993 by the Federation of the French Cancer Centres (FNCLCC), with the 20 French Regional Cancer Centres, several French public university and general hospitals, as well as private clinics and medical speciality societies. Its main objective is the development of serviceable clinical practice guidelines in order to improve the quality of health care and the outcome of cancer patients. The methodology is based on a literature review, followed by critical appraisal by a multidisciplinary group of experts. Draft guidelines are produced, then validated by specialists in cancer care delivery. OBJECTIVES: Produce clinical practice guidelines for the management and shipment of histological and cytopathological cancer specimens using the methodology developed by the Standards, Options and Recommendations project. METHODS: The FNCLCC designated the group of experts. Available data were collected by a search of Medline and lists selected by experts in the group. A first draft of the guidelines was written, then validated by independent reviewers. RESULTS: The main recommendations are: 1/ High-quality transmission of information between professionals is essential to the management of cancer specimens in order to assure high-quality diagnosis and evaluation of prognostic factors; 2/ Written procedures concerning sample shipment, handling, storage, registration, tracking and fixation exist; these procedures, as well as the necessary shipping material, will be sent to all clinical services involved; 3/ When possible, fresh, unfractionated, oriented surgical samples will be submitted to the same histological and cytopathological laboratory; 4/ Samples collected for extemporaneous examination, freezing or cell culture must be shipped immediately under appropriate storage conditions; 5/ Once frozen, samples can be stored in a deep freezer at temperatures of -80 degrees C or below, or kept in liquid nitrogen; 6/ Fixing tissues shortly after sample collection is essential to prevent cell lysis; 7/ Computerised systems will be used to assure correct specimen registration and tracking in histological and cytopathological laboratories.

[Recommendations for the immunohistochemical evaluation of hormone receptors on paraffin sections of breast cancer. Study Group on Hormone Receptors using Immunohistochemistry FNCLCC/AFAQAP. National Federation of Centres to Combat Cancer/French Association for Quality Assurance in Pathology]. / Recommandations pour l'évaluation immunohistochimique des récepteurs hormonaux sur coupes en paraffine dans les carcinomes mammaires. Groupe d'Etude des Récepteurs Hormonaux par Immunohistochimie FNCLCC/AFAQAP. Fédération Nationale des Centres de Lutte Contre le Cancer Association Française d'Assuransce de Qualité en Pathologie.

These recommendations regard the immunohistochemical evaluation of estrogen and progesterone receptors in paraffin sections of breast cancers. All the components of the procedure are dealt with: fixation, antigen retrieval, antibodies, controls, analysis and interpretation of immunostaining, report and quality assurance parameters. The purpose of these guidelines is to serve as a basis for standardization of techniques and results and to improve quality control.

[HER2 gene amplification assay: is CISH an alternative to FISH?]. / Recherche de l'amplification du gène HER2: la CISH est-elle une alternative à la technique FISH?

The HER2 proto-oncogene encodes a transmembrane protein, which is considered to function as a growth factor receptor. Overexpression of this protein found by immunohistochemistry in about 20% of infiltrating breast carcinomas, has a predictive value of response to treatment by trastuzumab, an anti-HER2 humanized monoclonal antibody. Search for HER2 gene amplification is necessary to adapt the immunohistochemical technique quality and also in the cases of delicate analysis or weak overexpression. It is usually carried out by Fluorescence In Situ Hybridization (FISH). A more recent hybridization technique, named CISH because of its chromogenic revelation is an alternative method, which gives highly correlated results with FISH. We present details of this technique, which may be more familiar for the pathologists than FISH, because reading analysis is similar to that of immunohistochemical staining.

[Appendiceal carcinoid tumor of systematic detection. When to propose right hemi-colectomy and how to monitor? Thoughts apropos of a case and review of the literature]. / Carcinoïde appendiculaire de découverte systématique. Quand proposer une hémi-colectomie droite et comment surveiller? Réflexions à propos d'un cas et revue de la littérature.

In relation to a case of carcinoid tumor of the appendix discovered after appendectomy and already metastatic, we discuss the prognostic factors of these lesions. Size is the most important one. For tumors less than two centimetres diameter, site, mesoappendix and lymph node involvement do not fully summarize the aggressiveness of the disease. We would like to emphasize the poor prognosis of perineural involvement, especially for young patients, in order to discuss the indication for salvage right colectomy. A positive reaction to Neuron-Specific Enolase allows this enzyme to be proposed as a postoperative serum marker.

[Positive sentinel node biopsy in breast cancer: is axillary surgery necessary in all cases?]. / Biopsie du ganglion sentinelle positive dans le cancer du sein: place du curage axillaire de complément?

Our retrospective study analyzes various factors to evaluate the risk of invasion of the not sentinel node when the sentinel node biopsy is positive in the infiltrated breast cancers. We compared in single varied then multivaried analysis, various parameters between two groups: positive not sentinel nodes and negative not sentinel nodes among 180 cases of positive sentinel node biopsy between 2001 and 2004. At the time of the single varied analysis, seem to be risk factors of non sentinel node involvement: the histopronostic SBRIII rank, positive a HER2neu status, the presence of extracapsulal node extension and infiltration of the sentinel node by a macrometastasis. The tumoral embol, the absence of hormonal receivers, a tumoral size > 10 mm and the number of sentinel node taken appear at the limit of the significativity. In multivaried analysis, SBRIII rank and the presence of an extracapsular node extension remain related to non sentinel node involvement. The histological type, association with a CIS, the size of the sentinel nodes, the number of positive sentinel nodes and the year of surgery are nonsignificant. Additional axillairy clearing out at the time of a positive node sentinel biopsy should be discussed according to different criteria determined by a precise histological analysis.