Ubiquitin pathway involvement in human lymphocyte gamma-irradiation-induced apoptosis.

Apoptosis (the classical type of programmed cell death) can be triggered in many cell types by widely diverse stimuli. gamma rays, at low doses, can induce apoptosis in vitro in interphase human lymphocytes. In this type of apoptosis induction, activated gene expression is necessary for the fulfillment of the death program. In this report, we present evidence for a relationship between ubiquitin gene expression or ubiquitination and gamma-irradiation-mediated apoptosis in normal circulating human lymphocytes. Using in vitro nuclear transcription assays (run-on), Northern (RNA) blot analysis, immunolocalization studies, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis after immunoprecipitation, we demonstrate that (i) the ubiquitin mRNA level is increased as a consequence of the activation of ubiquitin gene transcription 15 to 90 min after initiation of apoptosis; (ii) specifically in apoptotic cells, and not in all irradiated cells, nuclear proteins are highly ubiquitinated; and (iii) ubiquitin sequence-specific antisense oligonucleotide inhibition results in a decreased level of ubiquitinated nuclear proteins and considerably diminishes the proportion of cells exhibiting the apoptotic death pattern. Each of these results might be explained by different modifications occurring in irradiated cells. Their convergence strongly suggests that the ubiquitin gene is one of the genes with induced activity in the apoptotic death program and that ubiquitination of nuclear proteins might be involved in chromatin disorganization and oligonucleosomal fragmentation, which are among the key events occurring in apoptosis.

Correlation of pretreatment proliferative activity of breast cancer with the response to cytotoxic chemotherapy.

That most cytotoxic agents act specifically against actively proliferating cells is well-recognized. In this study, we attempted to correlate pretreatment S-phase fractions (SPF) measured on DNA histograms with regression of the tumor mass after the administration of neoadjuvant chemotherapy. Tumor cells were obtained from 60 previously untreated, premenopausal patients with no metastases and with noninflammatory disease by fine needle sampling without aspiration. We could evaluate DNA ploidy in all patients and SPF in 50 or 83% of them. Tumor responsiveness was significantly related to SPF. The 12 patients who had SPF of 10% or more showed demonstrable regression; six had complete responses. None of the other parameters tested, i.e., DNA ploidy, histopathologic grade, or hormone receptor content, correlated with response. We believe this information may prove valuable for clinicians as they make their decisions regarding patient therapy.

Enzyme immunoassay of estrogen receptors in fine needle aspirates of breast tumors.

Enzyme immunoassay of estrogen receptors (ER-EIA) was compared to radioligand assay (ER-RLA) in fine needle aspirates of breast tumors. Fine needle aspiration is a relatively atraumatic means of harvesting malignant cells from breast tumors. Fine needle aspiration provides a homogeneous suspension (about 90% malignant cells) with a sufficient amount of cellular material (10 to 50 micrograms DNA per sample) for single point radioligand assays of extractable estrogen (ER) and/or progesterone receptor (PR) in about 85% of primary adenocarcinomas at the time of diagnosis. Sixty-one different samples of malignant mammary cells were obtained by fine needle aspiration from 43 adenocarcinomas, 11 metastatic axillary nodes or 7 cutaneous nodules. Thirteen patients were under antiestrogen therapy (tamoxifen). ER-EIA was performed with Abbott's reagents, following the manufacturer's protocol. ER-RLA was a single saturation (5 nM) dextran-charcoal assay with [3H]R2858 as the labeled estrogen. The sensitivity of ER-EIA allowed dilution of the sample up to 10 times (according to sample cellularity and ER level) with less than 20% deviation from undiluted samples. Three levels of dilution of the samples (1/1, 1/2, and 1/10) allowed them to fall at least once into the range of the ER-EIA standard curve. Quantitative correlation between ER-EIA and ER-RLA was high (r = 0.86), and highest (r = 0.97) when samples from patients undergoing tamoxifen treatment were excluded. Major discrepancies between ER-EIA and ER-RLA appeared in those patients undergoing tamoxifen therapy; much higher values were obtained by ER-EIA. Eight of 13 of these patients were ER negative by ER-RLA but ER positive by ER-EIA. This preliminary observation indicates that in vivo ER modulation by hormones and antihormones should be reevaluated.

Relationship between multiple forms of plasminogen activator in human breast tumors and plasma and the presence of metastases in lymph nodes.

Plasminogen activators (PAs), a family of proteases active in blood coagulation, may play an important role in cancer. Indeed, blood coagulation disorders, such as altered fibrinogen and fibrin metabolism and increased incidence of vascular thrombosis, are common in patients with advanced malignant disease. Different types of human tumors are known to contain high levels of PA. The isoelectric focusing patterns of the PAs present in tumors and plasma from patients with breast cancer were compared with those of purified human urokinase and melanoma tissue PA. The pattern of isoelectric molecular forms of PA active at pH 8 showed two groups of several bands: in plasma from tumor-bearing patients and controls, these groups were in the pl ranges of 6.6 to 6.8 and 8.0 to 8.5; in mammary adenocarcinoma tissue, the ranges were 6.8 to 7.9 and 9.0 to 9.4. These patterns were different from those obtained with purified markers; the latter were 5.8 to 9.4 and 5.9 to 7.6 for purified human urokinase and melanoma plasminogen tissue activator, respectively. PA activity in tumor-bearing patients was very high in malignant tissue and, on the contrary, very decreased in plasma; this latter decrease was correlated with the presence of metastases in the axillary lymph nodes. These results suggest that the high PA activity in the tumor tissue might participate in the destruction of the peritumoral tissue, thus allowing its invasion by tumor cells, whereas the low activity of PA in the plasma might increase plasma fibrin, reflecting thus an early disorder in blood coagulation which would enhance the formation of metastases.

New approach for visualizing estrogen receptors in target cells using inherently fluorescent ligands and image intensification.

Four fluorescent estrogen ligands were investigated as agents for visualization of estrogen receptors in cells: 2-(2,4-dihydroxyphenyl)-6-hydroxy-3-benzofurancarboxylic acid delta-lactone (coumestrol) and 9(11)-dehydro-12-oxoestradiol [12-oxo-1,3,5-(10),9(11)-estratetraene-3, 17 beta-diol] (12-oxoestradiol), which are inherently fluorescent compounds; and tamoxifen [Z)-1-[4-(2-dimethylaminoethoxy)phenyl]-1,2-diphenyl-1-butene) and 4-hydroxytamoxifen [Z)-1-[4-(2-dimethylaminoethoxy) phenyl]-1-(4-hydroxyphenyl)-2-phenyl-1-butene), which become maximally fluorescent only after ultraviolet irradiation. By conventional fluorescence techniques, these agents can be detected down to 10(-8) M in water, but only to 10(-6) to 10(-7) M in protein solutions; however, by photon-counting spectrofluorimetry, coumestrol and 12-oxoestradiol can be detected in protein solutions down to 5 X 10(-10) M. Three of these compounds have good affinity for the estrogen receptor: coumestrol (20%); 12-oxoestradiol (12%); and 4-hydroxytamoxifen (37%), relative to estradiol (100%). Under conditions where autoradiographic controls indicate that most of the estrogen receptor of MCF-7 human breast cancer cells is in the nucleus, we could demonstrate nuclear fluorescence using 10(-9) M concentrations of coumestrol, 12-oxoestradiol, and 4-hydroxytamoxifen. This nuclear fluorescence was abolished by a 200-fold excess of diethystilbestrol and could only be observed through a fluorescence microscope equipped with a microchannel image intensifier and a video camera detector that together provide a sensitivity enhancement of approximately 10(4). These studies indicate that the estrogen receptor in breast cancer cells can be visualized by fluorescence techniques, provided that the visualizing ligands have adequate affinity and specificity for the receptor and appropriate fluorescence characteristics, and provided that the fluorescence instrument has adequate sensitivity to observe fluorescence emission from cells treated with nM concentrations of the fluorescent agents.

Presence of villin, a tissue-specific cytoskeletal protein, in sera of patients and an initial clinical evaluation of its value for the diagnosis and follow-up of colorectal cancers.

Villin is an actin-binding protein found in a few normal adult epithelia, namely epithelial cells in the digestive and urogenital tracts. Moreover, villin production is maintained in malignant cells. We assumed that cell lysis and necrosis of solid tumors producing villin might result in villin release into blood. We analyzed the villin content of sera from 788 patients and controls using an enzyme-linked immunosorbent assay. Patients and controls were classified into healthy donors, patients with benign diseases of the gastrointestinal tract, patients with colorectal cancers, and patients with malignant nondigestive diseases. In the panel of sera analyzed, the sensitivity of the assay for colorectal cancers was 50.5%, and its overall specificity for malignant digestive tumors was 94.5%. Results were statistically analyzed comparing each group of sera with each other. We conclude that the presence of villin is indicative of a pathological state in the gastrointestinal tract (P less than 0.001). Finally, we followed villin levels after tumor resections (60 patients). We found that the villin level in sera remains low in remissions but is raised in recurrences. We suggest that the villin assay may have clinical utility as a diagnostic adjunct for adenocarcinoma of the gastrointestinal tract. It may also have some value in monitoring patients with advancing colorectal carcinomas after resection of these tumors.

A new member of the ETS family fused to EWS in Ewing tumors.

As a result of chromosome translocations, the EWS gene is fused to a variety of transcription factors in human solid neoplasia. In Ewing tumors EWS can be fused to four different members of the ETS family, namely FLI-1, ERG, ETV1 and E1AF. We have identified a new member of the ETS family, called FEV, which is fused to EWS in a subset of Ewing tumors. FEV encodes a 238 amino acid protein which contains an ETS DNA binding domain closely related to that of FLI-1 and ERG. However, the N-terminal portion of FEV is only 42 amino acids long which suggests that FEV is lacking important transcription regulatory domains contained in FLI-1 and ERG N-terminal parts. The C-terminal end of FEV is rich in alanine residues which may indicate that FEV is a transcription repressor. The FEV gene is encoded by three exons and is located on chromosome 2. FEV expression was only detected in adult prostate and small intestine but not in other adult nor in fetal tissues, thus indicating that FEV has a restricted expression pattern. Following a scheme similar to previously described translocations in Ewing tumors, a t(2;22) chromosome translocation fuses the N-terminal domain of EWS to the ETS DNA binding domain of FEV.

Presence of tumor cells in bone marrow but not in blood is associated with adverse prognosis in patients with Ewing's tumor. Société Française d'Oncologie Pédiatrique.

PURPOSE: Gene fusions that result from the chromosome translocations observed in Ewing's tumor (ET) provide tumor-specific markers that can be used to detect the presence of tumor cells in peripheral blood (PB), bone marrow (BM), and stem cell collection (SCC). These markers were used to evaluate, at diagnosis, a series of 67 ET patients. PATIENTS AND METHODS: RNA was extracted from nucleated cells from PB and BM and a nested reverse-transcriptase polymerase chain reaction (RT-PCR) was performed to search for EWS-FLI-1 or EWS-ERG fusion transcripts that resulted from the t(11;22) or t(21;22) translocations, respectively. RESULTS: At diagnosis, 16 of 62 (26%) patients had circulating tumor cells. This was not correlated with any clinical parameter. In contrast, Ewing's cells were detected by RT-PCR in BM in 14 of 43 (33%) patients and were associated with the presence of clinically detectable metastases and a statistically significant unfavorable outcome in univariate analysis. There was no correlation between the RT-PCR results in PB and in BM. CONCLUSION: These results suggested that the monitoring of BM but not of PB by RT-PCR might constitute an important criterion for the staging, at diagnosis, of patients with ET. Further studies should appreciate the relationship or independence of this marker toward other classical prognostic factors in ET, particularly to the presence of clinically detectable metastases.

Familial invasive breast cancers: worse outcome related to BRCA1 mutations.

PURPOSE: Although all studies confirm that BRCA1 tumors are highly proliferative and poorly differentiated, their outcomes remain controversial. We propose to examine, through a cohort study, the pathologic characteristics, overall survival, local recurrence, and metastasis-free intervals of 40 patients with BRCA1 breast cancer. PATIENTS AND METHODS: A cohort of 183 patients with invasive breast cancer, treated at the Institut Curie and presenting with a familial history of breast and/or ovarian cancer, were tested for BRCA1 germ-line mutation. Tumor characteristics and clinical events were extracted from our prospectively registered database. RESULTS: Forty BRCA1 mutations were found among the 183 patients (22%). Median follow-up was 58 months. BRCA1 tumors were larger in size (P =.03), had a higher rate of grade 3 histoprognostic factors (P =.002), and had a higher frequency of negative estrogen (P =.003) and progesterone receptors (P =.002) compared with non-BRCA1 tumors. Overall survival was poorer for carriers than for noncarriers (5-year rate, 80% v 91%, P =.002). Because a long time interval between cancer diagnosis and genetic counseling artificially increases survival time due to unrecorded deaths, the analysis was limited to the 110 patients whose diagnosis-to-counseling interval was less than 36 months (19 BRCA1 patients and 91 non-BRCA1 patients). The differences between the BRCA1 and non-BRCA1 groups regarding overall survival and metastasis-free interval were dramatically increased (49% v 85% and 18% v 84%, respectively). Multivariate analysis showed that BRCA1 mutation was an independent prognostic factor. CONCLUSION: Our results strongly support that among patients with familial breast cancer, those who have a BRCA1 mutation have a worse outcome than those who do not.

Biological and clinical significance of concurrent p53 gene alterations, MDR1 gene expression, and S-phase fraction analyses in breast cancer patients treated with primary chemotherapy or radiotherapy.

We investigated the interrelationship between p53 gene alterations, MDR1 gene expression, and S-phase fraction (SPF) in breast carcinomas treated primarily with chemotherapy or radiotherapy and correlated the results with patient outcome to determine the potential clinical significance of these factors. In a consecutive series of 64 fine-needle samplings of breast cancer patients who underwent either neoadjuvant chemotherapy (n = 53) or radiotherapy (n = 11), p53 (exons 5-9) gene alterations by denaturating gradient gel electrophoresis and subsequent direct sequencing, MDR1 gene expression by semiquantitative reverse transcription-PCR, and SPF by DNA flow cytometry were determined. Our results show that p53 mutations (n = 20) were significantly associated (P = 0.01) with high SPF but not with de novo MDR1 gene expression. Most patients with wild-type p53 tumors were found to be resistant to neoadjuvant chemotherapy. No correlation was observed between p53 mutations and the induction of MDR1 gene expression during treatment. Although a significant correlation between shorter distant disease-free survival and high (>/=5%) SPF (P = 0.016) was found, no correlation between distant disease-free survival and p53 status or intrinsic MDR1 gene expression was found. Poor overall survival was observed in patients with tumors with high SPF (P < 0.0004) or lacking MDR1 gene expression (P = 0.03) before treatment, but not with p53 alterations. These data suggest that SPF remains the most relevant biological factor for breast cancer patients treated by primary chemotherapy or radiotherapy and that p53 and MDR1 status may identify a small subset of patients that may resist therapy or pursue an aggressive course.

Detection of MUC1-expressing mammary carcinoma cells in the peripheral blood of breast cancer patients by real-time polymerase chain reaction.

We have prospectively analyzed blood samples of 122 patients with breast disease for the presence of circulating expressing MUC1 cells before and after treatment. Among them, 28 patients had histologically confirmed benign breast disease (group 1), 34 patients had operable breast cancer (group 2), and 60 patients had advanced breast cancer (group 3). Circulating epithelial cells were isolated with BerEP4-coated immunomagnetic beads. Total RNA was extracted and reverse transcribed before analysis by real-time PCR of a MUC1-specific cDNA sequence. The sensitivity of the reverse transcription-PCR tested with blood spiked with MCF7 cells was one cell in 5 ml of blood. The immunomagnetic separation step was mandatory to obtain the maximum specificity. Control samples from healthy donors never displayed cycle threshold (Ct) values for MUC1 lower than 38. Circulating cells (Ct, <38) were detected in 3 of 28 (11%) cases in group 1, in 8 of 34 (24%) cases in group 2, and in 27 of 60 cases (45%) in group 3. A semiquantitative estimate of blood-borne cells could be derived from the Ct value when below 32 (the lowest was 28) or by the number of positive aliquots of the same blood sample. Thus, immunomagnetic separation, followed by MUC1-specific RT-PCR, allows the semiquantitative detection of circulating mammary cells. A significant correlation between the presence of MUC1-positive cells and the group of breast tumors was observed. The clinical significance of blood-borne cells in breast cancer, especially at the operable stage, may be investigated by following these patients.

[A new therapy with bortezomib, an oncologic medicinal product of the year 2004]. / Une nouvelle thérapie ciblée avec le bortézomib, médicament oncologique de l'année 2004.

Proteasome-mediated proteolysis is a mechanism for mediating important regulatory proteins within the cell. Proteins that have been targeted for degradation by the proteasome are convalently tagged with a poly-ubiquitin protein chain prior to be recognized by the 19S subunit of proteasome. This degradation system controls the expression of a wide variety of cellular targets including tumor suppressors such as p53, inhibitor of nuclear factor NFkappaB, cyclin-dependent kinase inhibitors such as p21 and p27. Because of these functions, the proteasome has become a new target for cancer treatment. The potent and selective proteasome inhibitor, PS-341 or Velcade was approved in the United States and launched in may 2003 for the treatment of multiple myeloma patients who have received at least two prior therapies. On April 2004, the European commission granted marketing authorization for Velcade with the same indication. The same year 2004, the Nobel Prize in chemistry was awarded to three researchers "for the discovery of ubitiquin-mediated protein degradation", a regulated process by which proteins are cleaved into peptides inside cells.

Phytoestrogens after breast cancer.

The current extension of the indications for adjuvant chemotherapy, which predisposes to early menopause, and the media coverage of the benefits of hormone replacement therapy (HRT) have led patients with a history of breast cancer to seek treatments for estrogen deprivation. In breast cancer survivors, most physicians avoid HRT because of concern regarding the potential promotion of growth of occult malignant cells by estrogens, due to the estrogen dependence of breast cancer. Soy phytoestrogens are being promoted as the 'natural alternative' to HRT and have been available without restrictions for several years as nutritional supplements. In this paper, data on the complex mammary effects of phytoestrogens in epidemiological studies, in in vitro studies, as well as in in vivo studies on animal carcinogenesis are reviewed. The potential benefits and risks of phytoestrogens are analyzed, and the prescription of phytoestrogens to postmenopausal women after breast cancer and the coprescription with the anti-estrogen tamoxifen are discussed. The absence of controlled trials and technical checking of extraction and titration in these preparations on 'free sale' raise a new problem in terms of public health and justify close reasoning and a cautious attitude of physicians, as well as straight information given to women, especially after breast cancer.

Validation of real-time RT-PCR for analysis of human breast cancer cell lines resistant or sensitive to treatment with antiestrogens.

Using a quantitative real-time RT-PCR technique we have compared the expression of a number of genes in two different human breast cancer model systems for development of acquired resistance to antiestrogens. The model system developed at the Danish Cancer Society comprises the cell lines MCF-7, MCF-7/TAMR-1, MCF-7/182R-6 and MCF-7/182R-7, and the model system developed at the Lombardi Cancer Research Center consists of the cell lines MCF-7/LCC1, MCF-7/LCC2 and MCF-7/LCC9. The findings on the well-known parameters estrogen receptor (ER)alpha, progesterone receptor (PR) and epidermal growth factor receptor (EGFR) are in good agreement with previous reports, thus documenting the usefulness of the real-time RT-PCR technique for multiparametric RNA analysis. The gene expression levels in the two model systems were found to be quite similar in relation to ERalpha, AIB1 (amplified in breast cancer-1), breast cancer antiestrogen resistance gene 1 (BCAR1) and ErbB-2 mRNA expression, whereas significant differences were observed on the expression of ERbeta, multidrug resistance gene 1 (MDR1), PR and EGFR. Furthermore, the presented data suggest that ERbeta, AIB1, BCAR1, CYP19 and MDR1 are unlikely to be causally involved in development of antiestrogen resistance in these breast cancer cell lines.

Inter-laboratory quality control for hormone-dependent gene expression in human breast tumors using real-time reverse transcription-polymerase chain reaction.

Quantitative reverse transcription-polymerase chain reaction (RT-PCR) used to detect minor changes in specific mRNA concentrations may be associated with poor reproducibility. Stringent quality control is therefore essential at each step of the protocol, including the PCR procedure. We performed inter-laboratory quality control of quantitative PCR between two independent laboratories, using in-house RT-PCR assays on a series of hormone-related target genes in a retrospective consecutive series of 79 breast tumors. Total RNA was reverse transcribed in a single center. Calibration curves were performed for five target genes (estrogen receptor (ER)alpha, ERbeta, progesterone receptor (PR), CYP19 (aromatase) and Ki 67) and for two reference genes (human acidic ribosomal phosphoprotein PO (RPLPO) and TATA box-binding protein (TBP)). Amplification efficiencies of the calibrator were determined for each run and used to calculate mRNA expression. Correlation coefficients were evaluated for each target and each reference gene. A good correlation was observed for all target and reference genes in both centers using their own protocols and kits (P < 0.0001). The correlation coefficients ranged from 0.90 to 0.98 for the various target genes in the two centers. A good correlation was observed between the level of expression of the ERalpha and the PR transcripts (P < 0.001). A weak inverse correlation was observed in both centers between ERalpha and ERbeta levels, but only when TBP was the reference gene. No other correlation was observed with other parameters. Real-time PCR assays allow convenient quantification of target mRNA transcripts and quantification of target-derived nucleic acids in clinical specimens. This study addresses the importance of inter-laboratory quality controls for the use of a panel of real-time PCR assays devoted to clinical samples and protocols and to ensure their appropriate accuracy. This can also facilitate exchanges and multicenter comparison of data.

Steroid receptor distribution in 47,892 breast cancers. A collaborative study of 7 European laboratories. The EORTC Receptor Study Group.

Seven laboratories of the EORTC Receptor Study Group reported the distribution of oestrogen (ER) and progesterone receptors (PR) routinely assayed in breast cancer cytosols. A low interlaboratory variability was demonstrated for the median values, and for the frequency of positive tumours as measured by enzyme immunoassay (EIA). Larger variations were found for the frequency of positive tumours, as measured by radioligand binding assay (RLA). They are probably due to differences in the cut-off levels and in the sensitivity of the assay. Analysis of the variability over time clearly demonstrated that the ER-EIA values initially increased compared with RLA. A possible source of variations could be the calibration drift in the ER-EIA kit. In conclusion, quality assessment of steroid receptors should be monitored by comparison of both common standards and distributions routinely obtained in each laboratory. In-house analysis over time is also essential for reagent survey.

S-phase fractions of breast cancer predict overall and post-relapse survival.

We studied the correlation of S-phase fraction (SPF) with clinical outcome in 127 pre- or perimenopausal patients with breast cancers treated by neoadjuvant chemotherapy from October 1986 to June 1990. When the patients were analysed using the median value of the SPF as a threshold, there was a small but non-significant difference in favour of low SPF tumours for metastasis-free survival. SPF was the only parameter predicting overall survival in multivariate analysis (P < 0.002) which included T, N, histopathological grade and steroid hormone receptors. The results of metastasis-free survival contrasted with previous analyses with shorter follow-up, so we tested the time-dependent influence of SPF on prognosis. It was thus shown that SPF significantly predicts metastasis-free survival only during the first 30 months, whereas the relative risk of cancer-related death according to SPF remains significant for 56 months. In order to find an explanation for the difference in predictivity between metastasis-free survival and overall survival, we studied the post-relapse survival. Significantly shorter survival (median 12 months) was associated with tumours presenting pre-treatment high SPF values, compared to the low SPF group for which 60% of the patients were still alive after 30 months of metastasis phase (P = 0.002). Our current results, in a homogeneous series with a median follow-up of over 5 years, emphasise the importance of proliferation-related parameters for breast cancer management.

A multiplex PCR assay for routine evaluation of deletion of the short arm of chromosome 1 in neuroblastoma.

Deletions of the short arm of chromosome 1 (1p) are frequent alterations in neuroblastoma. Although a consensus region of deletion has been mapped to chromosome subband 1p36, recent studies suggest that several distinct loci on this chromosome may be involved in neuroblastoma. Moreover, different patterns of deletion might be associated with different clinical and biological characteristics of the tumours. These findings emphasise the importance of assessing the localisation and the extent of the deletions in neuroblastoma. We developed a technique which allows analysis of loss of heterozygosity at multiple loci on 1p in a single step, making use of a multiplex PCR method. Primers specific for six microsatellite loci mapped in the different regions of interest on 1p were used for simultaneous amplification of DNA, and loss of heterozygosity was determined after separation of the alleles by denaturing polyacrylamide gel electrophoresis. This technique enables a simple analysis of the position and extent of 1p deletions, and can be used for routine evaluation of 1p status in neuroblastoma.

Cell cycle modifications of breast cancers during neoadjuvant chemotherapy: a flow cytometry study on fine needle aspirates.

Breast cancer cells from 92 patients were obtained by repeated fine needle sampling and analysed by flow cytometry for cell cycle modifications during neoadjuvant chemotherapy. Modifications of the histograms were observed for 47 of the 71 informative cases (66%), the most frequent concerning S-phase (increase or decrease) and G2M accumulation. These modifications correlated well with the efficacy of cytotoxic chemotherapy (P < 0.0001). A significant relationship between clinical regression and pretreatment proliferative activity was also observed, with 31/35 (89%) responders in the high proliferation group (S-phase fraction > 5% or BrdU labelling index > 3.3%) compared to 20/36 (56%) in the low proliferation group (P < 0.002). For patients undergoing chemotherapy including doxorubicin, a high incidence of G2M accumulation was observed (33%), a modification which was rare (4.5%) for a regimen with no anthracycline, for which S-phase was the most frequently modified cell cycle compartment (64%). The measurement of the pretreatment tumour proliferative activity as well as the early kinetic modifications, as indicators of response, may prove interesting parameters for the future management of neoadjuvant chemotherapy.

N-MYC amplification, loss of heterozygosity on the short arm of chromosome 1 and DNA ploidy in retinoblastoma.

Recurrent genetic alterations different from the alteration of the RB1 gene on chromosome 13q14 have been described in retinoblastoma, including structural alterations on the short arm of chromosome 1 and amplification of the N-MYC oncogene. These two genetic alterations are major prognostic factors in neuroblastoma, another embryonic neuro-ectodermal tumour. In order to assess the frequency of these alterations and their possible association with clinical parameters in retinoblastoma, we studied a series of 46 retinoblastoma tumour samples. Ploidy was assessed by flow cytometry, N-MYC copy number was evaluated by a spot-blot procedure using the pNb-1 probe and loss of heterozygosity was investigated by PCR analysis at mini- and microsatellites located on the short arm of chromosome 1. Most tumours were in the diploid or near diploid range; only one case exhibited tetraploidy. N-MYC amplification was observed in only one of the 45 tumours. Loss of heterozygosity on the short arm of chromosome 1 was observed in 9/43 tumours (21%); in particular, its incidence was higher in metastatic than in localised disease (P < 0.05). We suggest that alterations of one or several genes on chromosome 1p might play a role in the oncogenesis or progression of retinoblastoma. Analysis of the long term follow-up of these and additional patients should determine the prognostic value of this parameter.