monica giordano

In many cell systems, resistance to cytotoxic drugs is acquired by the amplification and/or overexpression of the multidrug resistance (mdr) gene, which codes for the glycoprotein, p170 (P-glycoprotein). Moreover, in a variety of malignant tumours there is increasing evidence of the relationship between the DNA ploidy pattern of patients and their prognosis. In this study we aimed to evaluate these two potential indicators of constitutive drug resistance in human colorectal tumours. We employed a method to quantify simultaneously, on a per cell basis, mdr gene expression (using the C219 monoclonal antibody for P-glycoprotein) and nuclear DNA content with high-resolution bivariate flow cytometry. The study was performed on a human colon-carcinoma-derived cell line (LoVo) and its doxorubicin-resistant variant (LoVo/Dx) and on tumour samples and adjacent normal mucosa from 35 untreated patients with colon cancer. The P-glycoprotein was found in both LoVo and LoVo/Dx cells with levels slightly lower in the parental than in the resistant subline (P, NS). A multi-drug-resistant specific probe for mRNA expression and Western blot assay confirmed the specificity of p170 expression. All of the colon cancer with unimodal diploid DNA distribution and all the normal colonic mucosa samples showed P-glycoprotein expression, without a statistically significant difference in median values between tumours and normal samples. Tumours with bimodal DNA distribution showed median values of P-glycoprotein expression of their hyperdiploid cell clones significantly higher than those of their diploid clones and of the tumours with unimodal DNA distribution (P less than 0.005). Our results show the feasibility of bivariate flow-cytometric analysis of P-glycoprotein expression and DNA content on clinical material and support the hypothesis that the MDR phenotype and DNA ploidy together may influence the biological behaviour of colon cancer in vivo.

A Phase II multicenter trial testing an accelerated regimen of radiotherapy in locally advanced and inoperable cancers of the head and neck, in patients selected on the basis of 5-bromo-2-deoxyuridine/DNA flow cytometry-derived tumor potential doubling time (Tpot). From September 1992 to September 1993, 23 patients consecutively diagnosed to have locally advanced, inoperable carcinomas of the oral cavity and the oropharynx, with Tpot of < or = 5 days, received an accelerated radiotherapy regimen (AF) based on a modification of the concomitant boost technique: 2 Gy/fraction once a day, delivered 5 days a week up to 26 Gy, followed by 2 Gy/fraction twice a day, with a 6-h interval, one of the two fractions being delivered as a concomitant boost to reduced fields, up to 66 Gy total dose (off-cord reduction at 46 Gy), shortening the overall treatment time to 4.5 weeks. A contemporary control group of 46 patients with Tpot of >5 days or unknown was treated with conventional fractionation (CF): 2 Gy/fraction once a day, 5 days a week, up to 66 Gy in 6.5 weeks, with fields shrinkage after 46 Gy. All patients completed the accelerated regimen according to protocol and in the prescribed overall treatment time. Immediate tolerance was fairly good: 65% of the patients in the AF group experienced Grade 3 mucositis vs. 45% in the CF group (p = n.s.). Symptoms related to mucosal reactions seemed to persist longer in AF than in CF patients. The crude proportion of mild (Grades 1 and 2) late effects on skin (p < 0.01) and salivary glands (p < 0.05) was higher in AF than in CF patients, although these reactions did not exceed the limits of tolerance. Three patients in the AF and 1 in the CF arm experienced a late Grade 4 bone complication. Actuarial estimates of severe (Grades 3 and 4) late complications showed a 2-year hazard of 33.3% in the AF arm and 49.7% in CF (p = NS). The actuarial 2-year local control rate of the AF patients was 49.4%, while actuarial 2-year overall survival for the same patients was 43.5%. The results suggested that this accelerated regimen is worth testing in a controlled randomized trial to compare different accelerated schedules. Our findings also confirmed the 5-bromo-2-deoxyuridine/DNA flow cytometry technique as a suitable method of evaluating tumor cell kinetics in multicenter clinical studies, on condition that all measurements are carried out by one most experienced laboratory.

The influence on survival of 21 basic clinical and hematologic parameters was evaluated in 72 patients with previously untreated myelodysplastic syndromes (MDS). Only five parameters were significant by both survival curves and multiple regression analyses: hemoglobin level, bone marrow (BM) cellularity (estimated from trephine BM biopsies), BM blast percentage, age and BM erythro/myeloid (E/M) ratio. Using these parameters, multiple regression analysis enabled us to predict 34% of the survival of all MDS patients (p less than 0.002), 38% of that of patients who had stable disease (p less than 0.04) and over 80% of that of patients who developed acute leukemia (p less than 0.02). High BM cellularity was the most predictive factor for the development of leukemia. No factor was predictive for patients who died of cytopenic or other complications.

The proliferative characteristics of acute nonlymphoblastic leukemia (ANLL) were studied in vivo, and data were correlated with response to chemotherapy and survival. Sixty-five patients with untreated ANLL and 15 patients with solid tumors and normal bone marrow (BM) received 250 mg/m2 of bromodeoxyuridine (BUdR); bivariate flow cytometric (FCM) analysis then was used to measure cell BUdR incorporation and DNA content to obtain a complete set of kinetic parameters (i.e., BM BUdR-labeling index, DNA-synthesis time, potential doubling time [Tpot], and cell production rate). The percentage of blasts with positive results for proliferating cell nuclear antigen (PCNA) also was obtained by FCM analysis on the same BM samples, and these kinetic parameters were derived specifically for the ANLL proliferating compartment (growth fraction). Induction therapy, consisting of vincristine, arabinosylcytosine, and daunomycin, was administered subsequently to the patients with ANLL. Overall ANLL proliferative activity was lower than normal myelopoiesis, and a short Tpot was found to be a favorable factor for achieving complete remission (CR), the duration of CR, and survival. When the growth fraction was considered, however, ANLL proliferative activity was higher and more like that of normal BM. The kinetic differences detected in the PCNA-positive cells of patients with CR and no response and those with CR and survival durations above and below the median values for the entire series were highly significant in univariate analysis and retained a strong independent prognostic value when multivariate analysis was performed. These data show the clinical feasibility of a detailed study of cell kinetics by means of new FCM-based techniques and reinforce the clinical value of pretreatment proliferative activity in ANLL.

PC10 is a monoclonal antibody to proliferating cell nuclear antigen, a nuclear protein associated with the cell cycle. We have evaluated the effects of tissue fixation on PC10 immunoreactivity in sections of paraffin embedded rat tissues. Immunoreactivity was well preserved in tissues after fixation with alcohol-based solutions for 3-24 hr. Fewer PC10-positive cells were detectable in samples fixed with formaldehyde-containing solutions compared with samples fixed with alcohol for the same time. Loss of PC10 immunoreactivity in formaldehyde fixed tissues was progressive, and quantifiable as early as after 3 hr fixation. Consequently, alcohol-based fixatives are strongly recommended for any immunocytochemical prospective study using PC10 antibody. In contrast, loss of PC10-immunoreactivity is always predictable, but difficult to quantitate, using formaldehyde fixed specimens. This aspect should be considered when using PC10 antibody in retrospective studies with routinely-processed archival material.