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.

Distinct chromosomal alterations associated with TP53 status of LoVo cells under PALA selective pressure: a parallel with cytogenetic pathways of colorectal cancers.

This study investigates the chromosomal alterations involved in the acquisition of PALA resistance of LoVo colorectal cancer cells homozygous for wild-type TP53 before and after transfection with a 143Ala-mutated TP53 gene. PALA resistance was always associated with an increased number of CAD gene copies, but gene amplification sensu stricto was rarely observed. Interestingly, distinct chromosome patterns were found in relation to the TP53 status of the cells. In parental LoVo cells, the CAD copy number was increased through gains of normal chromosome 2 whereas in transfectant clones, resistance mostly occurred through chromosome rearrangements. The relationship with the two different cytogenetic patterns described in colorectal tumors is discussed.

Frequently elevated content of immunochemically defined wild-type p53 protein in colorectal adenomas.

Mutations of the p53 tumour-suppressor gene are considered to be rare in human colorectal adenomas, a premalignant state of the digestive tract. We have analysed a series of 32 exophytic tumours of the colon and rectum for the presence of p53 protein. In 26 of the 28 pure adenomas, the presence of significant levels of p53 proteins was established by a sensitive two-point enzyme-linked immunosorbent assay. The detectability of p53 protein was frequently limited to PAb 1801, recognizing an N-terminal epitope. Immunoblotting of the fractions captured by the monoclonal antibodies revealed that PAb 421 reacted exclusively with a 53-kDa species, whereas an additional 48-kDa band was detected after incubation with PAb 1801. In the adenomas, the mutant conformation-specific PAb 240 was always negative and no mutations were detected on exons 5-8 in three large and highly dysplastic lesions, selected for their high p53 protein content. The remaining four of the 32 tumours presented foci of cancer. Three of these were shown to contain 'mutant' PAb 240-reactive p53, and gene mutations were identified in two by denaturing gradient gel electrophoresis and sequencing of the amplified products. Intense p53 nuclear immunohistochemical staining was associated with the malignant areas. We conclude that a novel mechanism affecting the regulation of p53 protein could occur in colorectal adenomas.

Different p53 mutations produce distinct effects on the ability of colon carcinoma cells to become blocked at the G1/S boundary after irradiation.

The LoVo colon carcinoma cell line that presents two wild type p53 alleles was used as the recipient for a series of transfections with p53 expression vectors coding for wild-type or three different mutants (143ala, 175his or 273his). The parental cell line as well as all clones that had rearranged the plasmid with consequent loss of p53 c-DNA were readily blocked at the G1/S boundary following 10 Gy of irradiation. For each mutation two clones with different levels of mutant protein expression were selected. Confirmation of the integration of the exogenous sequence was obtained by the expression of the mutant m-RNA, established by reverse transcription and DGGE or Southern blot. Flow cytometric measurements of 5-bromodeoxyuridine incorporation revealed a total G1/S block of the 143ala transfectants, similarly to the parental and control transfectant cells, but little or no cell cycle block for the 175his and 273his clones. Although it has been shown in vitro that all three mutations interfere with transcriptional activation by the wild-type protein, not only did we observe p53 protein induction and nuclear accumulation following irradiation, but WAF-1/CIP-1 m-RNA was increased in some of the clones for which the G1/S block was abolished. Our results show that mutant p53 proteins are to some extent submitted to the control of the cellular environment in cancer cells with wild type p53 alleles, but with an efficacy that depends on the mutation.

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.

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.

Breast tumour response to primary chemotherapy predicts local and distant control as well as survival.

The purpose of the present paper was to evaluate correlations between clinical response to chemotherapy and outcome in a subgroup analysis of premenopausal patients with tumours considered too large for breast conserving surgery, treated with primary chemotherapy (n = 200) from a previously published trial (Scholl S.M., Fourquet A., Asselain B, et al. Eur J Cancer 1994, 30A, 645-652). Objective response rates amounted to 65% following four courses. In a multivariate Cox regression analysis, comparing seven parameters, the following variables were associated with poor survival: clinically involved nodes [N1b:RR: 2.7 (95% CI 1.3-5.3)], the failure to respond to chemotherapy [D:RR: 2.62 (95% CI 1.3-5)] and a raised S phase fraction [SPF > 5%: RR: 2.4 (95% CI 1.2-5)]. Parameters associated with increased metastatic recurrence rates, by order of entry in the model, were: young age [< 35: RR: 2.46 (95% CI 1.2-5)], large clinical tumour size [T3: RR: 2.02 (95% CI 1.2-3.4)], poor histological grade (SBR III: RR: 1.93 (95% CI 1.1-3.3)] and the failure to respond to chemotherapy [D: RR: 1.91 (95% CI 1-3.4)]. The assessment of both tumour cell proliferation rates as well as possibly drug resistance markers (although not available in the present study) should be helpful in selecting patients likely to benefit from intensified chemotherapy regimens. The most accurate predictor of response in the present study appeared to be the response to chemotherapy treatment itself.

The p53-positive phenotype of breast cancers.

One hundred and eighty-one breast cancer specimens were analyzed for nuclear p53 staining by immunochemical methods. There were 123 fine-needle cytological specimens and 58 frozen tissue sections of surgical biopsies. The microscopic evaluation of the staining fitted with a 4 group classification. Ninety-one samples (50.6%) were devoid of any staining (-), while 42 (23.3%) showed only few stained nuclei (+/-), typically around 1%. Thirty-two (17.8%) samples presented with strong nuclear staining (++) which in practically all cases concerned more than 50% of the nuclei, but a few cases showed staining heterogeneity. A further 17 cases (9.4%) presented with nuclear staining which concerned 10-20% of the cancer cells (+). This four class system was used to compare p53 expression with other prognostic parameters. A strong inverse correlation was observed with steroid hormone receptor content and p53 positivity was highly significantly associated with higher S-phase. All but one of the highly positive cases were aneuploid. Twenty-five percent (29/120) of the aneuploid tumors were strongly stained and a further 10% were considered positive (+). On the other hand, only 5 out of 59 DNA-diploid tumors were considered as + and one ++. The DNA index distribution according to p53 positivity showed peaks of positivity for hypodiploid, triploid and hypertetraploid values. Negative tumors were in all regards similar to those with only few stained nuclei, in particular mean S-phases of 2.8 and 3.3% respectively. Altogether, the typical strong p53 phenotype concerned a DNA-aneuploid tumor with above median S-phase fraction (mean of 7.1%), negative steroid hormone receptors and cytoprognostic index III. The p53 positive cases (+), were frequently steroid hormone receptor positive and had on the average intermediate S-phase fractions (4.3%). The proportion of immunochemical positivity (27% in our series), is compatible with the published frequency of p53 mutations detected in breast cancers, but the differences in the phenotype according to the level of positivity should be further investigated.

Immunocytochemical determination of estrogen and progesterone receptors on 50 fine-needle samples of breast cancer. A prospective study including biochemical correlation and DNA flow cytometric analysis.

In a prospective study of 50 consecutive fine-needle samples of breast cancer, determination of estrogen and progesterone receptors was performed by separate immunocytochemical and biochemical assays. Comparisons of immunocytochemical assays with biochemical assays were obtained in 42 cases (84%) for both steroid receptors and showed significant relationships for estrogen receptor (P less than 0.001, r = 0.85) and progesterone receptor (P less than 0.001, r = 0.89) when the percentage of labeled nuclei (immunocytochemical assay index) was considered. In addition, DNA flow cytometric analyses were performed on fine-needle samples in 34 cases, with measurable DNA ploidy and S-phase fraction in 33 (97%) and 31 cases (91%), respectively. These results show that (1) estrogen and progesterone receptor immunocytochemical assays are valuable tools for steroid receptor determination, and that (2) on the same fine-needle samples of breast cancer DNA flow cytometric analysis may be performed.

Prognostic value of the S-phase fraction of breast cancers treated by primary radiotherapy or neoadjuvant chemotherapy.

Neoadjuvant chemotherapy for large operable breast cancers is being increasingly used for the purpose of "downstaging," so that the lesions become accessible to conservative treatment. Since tumor proliferative activity has been shown to be a major prognostic factor for breast cancers, we have studied the value of S-phase fractions established by flow cytometry on cytological samples at diagnosis, in 184 stage II or IIIa patients entered in a randomized trial comparing neoadjuvant to adjuvant chemotherapy. All patients were pre- or perimenopausal, and the median follow-up was for 43 months (24-64). Using the median value (5%) as cutoff, a high SPF was found to be associated with relapse (p < 0.0008), locoregional recurrence (p < 0.02), or metastasis (p < 0.003). However, when the patients were analyzed according to the type of treatment, significance was maintained for the patients in the primary radiotherapy arm (p < 0.003) but not for those in the neoadjuvant chemotherapy arm (p < 0.06). The overall rate of response to neoadjuvant chemotherapy was significantly lower for tumors with low SPF (56.5%), compared to tumors with high SPF (85.6%). Thus, SPF was no longer predictive of outcome when the tumors regressed by more than 50% after chemotherapy (p = 0.66), whereas it was highly predictive in the nonresponding patients (p < 0.0001). Our study has revealed that patients with low-SPF tumors, irrespective of response or treatment schedule, had similar prognosis (around 70% free of disease at 45 months), while the high-SPF nonresponders had a dismal prognosis, with less than 25% free of disease at 24 months. If our results are confirmed with a longer follow-up, proliferative activity of breast cancers should prove to be instrumental for the initial therapeutic decision of stage II or IIIa patients.

Colorectal carcinogenesis: from chromosomal evolution pathways to molecular pathogenesis.

Since the mid-1980s, research in the field of colorectal carcinogenesis has seen a series of breakthroughs such, as the process of loss of heterozygosity for large chromosomal segments and the consequent characterization of a series of suppressor genes considered to be the targets of the allelic deletions. More recently, a new perspective has been opened, with the discovery of germinal mutations of genes involved in mismatch repair in certain inherited forms of the disease. Through the retrospective analysis of our data on colorectal adenomas and cancers, we have tried to critically reassess a number of theoretical considerations relating to the instability of the genome viewed at the chromosome level and its consequence on tumor progression.

Cytogenetic and molecular approaches of polyploidization in colorectal adenocarcinomas.

We present the cytogenetic analysis of 23 cases of polyploid colorectal adenocarcinomas. We took advantage of the high intratumoral heterogeneity of the karyotypes to identify clones, subclones, and cell-to-cell variations. This allowed us to reconstruct the chromosomal evolution of each tumor and to propose a schema of the chromosomal changes in relation to the endoreduplication process. All but one case were characterized by a relative deficiency of chromosomes 17p and 18. Other deficiencies affecting the late-replicating X, and to a lesser degree, 1p, 5q, 14, 15, 8p, 10, 21, and 4, and excesses affecting the early-replicating X, 8q, 13, 16, 17q, and 11 were frequently associated. This pattern of imbalances is very similar to that of the monosomic type previously described in near-diploid tumors. The pattern of the 23rd tumor corresponded to those of the trisomic type tumors. These data largely confirm the existence of two distinct processes of chromosomal evolution in colorectal adenocarcinomas, with a strong tendency to undergo endoreduplication for the monosomic type near-diploid tumors. To correlate cytogenetic and molecular data, allelic losses analyses were investigated for probes of chromosomes 17p and 18. In all 12 informative tumors, a loss of heterozygosity for probes of the short arm of chromosome 17 indicated the occurrence of a rearrangement of chromosome 17 before the endoreduplication. The same was true for allelic losses for probes of chromosome 18 found in 11 of 12 informative tumors. The correlation between cytogenetic and molecular data is thus excellent and indicates that losses of 17p and 18 are early events in the tumor process.

Clinical and biological characteristics of breast cancers in post-menopausal women receiving hormone replacement therapy for menopause.

The paradox of an excess breast cancer incidence among current users of hormone replacement therapy (HRT) without excess in breast cancer mortality raises the question of possible differences in the clinical and biological characteristics of cancers in current HRT users compared to non-users. A consecutive series of 129 post-menopausal patients under HRT for at least 6 months, in whom an operable breast cancer was diagnosed from January 1992 to December 1996, were identified retrospectively. In most cases women had received combination HRT (estrogen and progestative) and the mean duration was 60.4 (range: 6-360) months. Breast cancers diagnosed in post-menopausal patients during 1992-1993 at the Institut Curie constituted the reference series. Cancers in patients receiving HRT were smaller: 78% versus 32% T1 and 12 mm in larger diameter versus 29.5 mm. They were also more often diagnosed radiologically (49 versus 33%). A second group of 420 post-menopausal breast cancer patients whose samples had been referred for steroid hormone receptor and flow cytometric analysis in 1992-1996 were used for comparing biological and pathological information. Cancers of patients receiving HRT tended to be more often grade I and rarely grade III in the Scarf Bloom Richardson classification. Percentage of cells in S-phase as measured by flow cytometry was considerably lower in HRT users compared to control (mean 2.4 versus 3.7, median 2.2 versus 2. 6). Lymph node invasion, ploidy, and steroid hormone receptor expression did not differ significantly between the 2 groups. This apparently more favourable phenotype of breast cancers diagnosed in post-menopausal patients receiving HRT compared to unselected non-HRT users was not confirmed when analysis was restricted to breast cancers of less than 25 mm in diameter. If, as expected, the phenotypic information bears out in terms of prognosis, this may contribute to overcome the reticence in prescribing HRT due to the increased risk of breast cancer. However, it is still not clear whether the biologically less aggressive phenotype is related to the hormone treatment or is simply due to early detection.

Neo adjuvant Tamoxifen in post menopausal patients with operable breast cancer.

AIMS: Tamoxifen is widely used as adjuvant therapy in receptor positive post menopausal breast cancer. Little is known about its efficacy as neo adjuvant therapy in terms of breast conservation and improved survival. METHODS: We analyzed the tumour response to 20-30 mg Tamoxifen for 6 months in post menopausal patients with oestrogen receptor positive tumours. Treatment included Tamoxifen for 6 months, surgical resection, and irradiation for post menopausal patients refusing initial mastectomy; aged > or =70 years; or with other factors delaying surgery. RESULTS: Between April 1994 and June 1998, 102 patients, age 73+/-87 (54-90) were studied. There were 24 T1, 56 T2, 14 T3, and 8 T4 tumours. Clinical response to Tamoxifen was observed in all patients, with a median size reduction from 31+/-15 (9-70) to 16+/-9 mm (0-50), 15 clinical and 6 complete responses. 88/102 patients were treated conservatively. Radiotherapy was given to 80 and a flash technique to 8 patients. All patients but one are still alive. CONCLUSION: Neo adjuvant Tamoxifen in operable post menopausal ER positive breast cancer is associated with a good clinical response rate and facilitates conservative surgery. Tamoxifen has a valuable role as neo-adjuvant treatment in terms of breast conservation and survival.

Studies on the influence of the presence of an activated ras oncogene on the in vitro radiosensitivity of human mammary epithelial cells.

The clonogenic cell survival to gamma-rays was determined in a human mammary epithelial cell line transfected by an activated ras oncogene in comparison to either the host untransfected cell line or the same cell line transfected by the neo gene. In contrast to previous observation of fibroblasts derived from a murine cellular system (NIH 3T3), the analysis of survival curves did not demonstrate an acquired radioresistance in the human ras transfected cells as opposed to the two other cell lines. In other words, the appearance of resistance to ionizing radiation does not seem to be a feature which can be generalized to all situations associated to activation of a specific oncogene (ras).

[Two colons--two cancers? Proximal or distal adenocarcinoma: arguments for a different carcinogenesis]. / Deux côlons--deux cancers? Adénocarcinomes coliques proximal ou distal: arguments en faveur d'une cancérogenèse distincte.

The purpose of this review is to study the literature concerning normal colon and colorectal cancer for evidencing that the location of the primary proximal or distal tumor may determine two categories of carcinogenesis. The proximal or distal normal colon can be considered as two different organs. Their embryologic origins are different, the splenic flexure starts the distal segment. Antigenic pattern as well as the use of metabolic pathways, such as that of glucose, butyrate and polyamines differ. Epidemiologic, macroscopic and histological features and the inherited and acquired genetic abnormalities allow in some cases to distinguish between two types of cancer according to their proximal or distal location. These two forms are not entirely different and these features can overlap. However the recognition of these two different forms of colon cancer, must be taken in account for clinical or basic research and evaluation of data.

[Immunochemical evidence of a mutated p53 protein expressed in human colorectal adenocarcinoma]. / Mise en évidence immunochimique de l'expression d'une protéine p53 mutée dans les adénocarcinomes colorectaux humains.

Evidence is accumulating that the p53 anti-oncogene is a key gene in the genesis of carcinoma in human colon and rectum. Although mutations of the p53 gene have been shown to be frequent, the protein was present in only approximately 50 percent of specimens examined. However only one monoclonal antibody recognizing an epitope present on wild-type p53 had been used. We studied the p53 expression in a series of 16 colorectal carcinoma specimens using 3 different monoclonal antibodies (pAb 421, 1801, 240). Specific immunofluorescent staining was quantified by dual parameter (DNA/p53) flow cytometry. Two different types of preparations were compared in order to verify the conservation of the antigen. Nuclear suspensions prepared from frozen tumor fragments were shown to produce results equivalent to those of whole cell preparations originating from fresh surgical specimens. The p53 protein was detected in 9 of the 16 cancers with pAb 421 and 240 monoclonal antibodies (8 of which were also positive for pAb 1801 antibody). Four additional tumors were considered positive for pAb 240 antibody alone. Overall, 13/16 cancer specimens were shown to present immunoreactivity for pAb 240 antibody. Topography of staining was investigated by immunohistochemistry with peroxidase methods. Eight cases were informative, 6 of which presented nuclear staining compatible with the cytometry results. There was one discordant case i.e. pAb 240 antibody being positive on cytometry and entirely negative on immunohistochemistry. This small series allowed us to show that 81 percent of tumor samples stained with monoclonal antibody pAb 240, considered to be specific to mutated protein, and that some tumors express a p53 protein which is not detected with terminal sequence-specific antibodies.

[Genetic heterogeneity of certain sporadic colorectal cancers]. / Hétérogénéité génétique de certains cancers colorectaux sporadiques.

Intratumoral heterogeneity provides information concerning the timing of genetic events which occur during colorectal carcinogenesis. In a series of 14 colorectal adenocarcinomas, the following genetic alterations were characterized: loss of heterozygosity on chromosomes 17 p, 1 p, 18 q, 5 q and 22 q, point mutations on TP53 and K-RAS genes, change in DNA index. Six of the 14 initial samples were investigated for putative genetic heterogeneity. In two cases, variations in genetic alterations on chromosome 17 p and gene TP53 were demonstrated. These events seem to occur at a late stage in colorectal carcinogenesis.

[A joint study of mutation of the Ki-ras oncogene and overexpression of the Tp53 oncogene in colorectal cancer]. / Etude conjointe des mutations de l'oncogène Ki-ras et de la surexpression de l'oncogène Tp53 dans des cancers colorectaux.

Twenty-five colorectal tumors (rectum 6, left colon 13, right colon 6) were studied with respect to the overexpression of p53 and the activation by point mutation of the Ki-ras oncogene. Single point mutations on codon 12 and codon 13 were analyzed after PCR amplivication, dotblotting and sequential hybridization with 12 different oligonucleotides. The intranuclear concentration of p53 protein was measured by flow cytometry after immunofluorescence staining with monoclonal antibody Pab 421. Twelve tumors were found to significantly overexpress p53 and 6 of them had an activated Ki-ras (5 on codon 12, 1 on codon 13). Of 13 tumors which failed to demonstrate over expression of p53, 8 had an activated Ki-ras (5 on codon 12, 3 on codon 13). In our series, p53 overexpression and ki-ras activation appeared to be independent.

[Sentinel node and operable breast cancer: utilization of blue dye injection. Pilot study]. / Ganglion sentinelle et cancer opérable du sein: utilisation du bleu patent. Etude pilote.

STUDY AIM: Sentinel node detection in breast cancer can be realized with colorimetric and isotopic procedures often associated. The aim of this study was to report results obtained with blue dye injection only. PATIENTS AND METHOD: From September 1998 to July 1999, blue dye injection was performed in 73 consecutive patients (mean age: 51 years, range: 36-71 years); 51/70 70% were post-menopausal and half of them were under substitute hormonal treatment; 70% of cancers were discovered through routine mammography. There were 12 bilateral cancers, six of them synchronous, and 84% of cancers were located in the external quadrants. Individualization of sentinel node was performed through blue dye injection into the tumor in case of preoperative diagnosis or in the tumoral site in case of discovery of the cancer through extemporaneous histological examination. RESULTS: 71 out of 73 cancers were classified pT1 and 70% measured 10 mm and over. Individualization of sentinel node failed in two obese patients. Sentinel node invasion concerned one node (n = 7), two nodes (n = 1) and three nodes (n = 1). Conservative treatment was performed in 72 patients out of 73; in case of sentinel node invasion, axillary irradiation was performed without reoperation. CONCLUSION: Blue dye injection for sentinel node individualization is an accurate technique in selected patients in case of small tumors. Reoperation can be avoided and replaced by axillary irradiation in case of N+ tumors. Duration of hospitalization was 48 hours or under in 70/73 patients. Nevertheless isotopic procedure must be recommended as a routine technique in learning centers and for most surgical teams.