Concurrent irinotecan and 5-fluorouracil plus levo-folinic acid given every other week in the first-line management of advanced colorectal carcinoma: a phase I study of the Southern Italy Cooperative Oncology Group.

OBJECTIVES: To determine the maximum tolerable doses (MTDs) of irinotecan (CPT-11) and 5-fluorouracil (5-FU) plus levofolinic acid (LFA) administered together every two weeks, to define the toxicity profile of this regimen, and to have a preliminary evidence of its activity in the first-line management of advanced colorectal cancer patients. PATIENTS AND METHODS: Patients with histologically proven colorectal carcinoma, no prior chemotherapy for their advanced disease, and with at least one measurable or evaluable indicator lesion, were admitted to this study. The starting dose of CPT-11 was 150 mg/m2 given i.v. (90 min infusion) on day 1, followed on day 2 by a fixed dose of LFA (250 mg/m2) as a two-hour i.v. infusion plus a starting dose of 5-FU 600 mg/m2 as i.v. bolus. No intra-patient dose escalation was allowed. If no dose limiting toxicity (DLT) was observed among three patients of each cohort, CPT-11 and 5-FU were alternately escalated in the subsequent cohort. Otherwise, three more patients were enrolled at the same dose level. DLT was defined as: WHO grade 3 non-haematological toxicity (except for vomiting or alopecia), grade 3 febrile neutropenia, grade 4 neutro- or thombocytopenia, or a > 2-week delay in recycling. The MTDs were defined as the doses at which two of three, or four of six, patients showed the same DLT. RESULTS: Thirty-one patients (five pretreated in adjuvant setting) were enrolled in this study, and a total number of 293 cycles (median 6/patient) were administered. Dose escalation safely proceeded to 210/950/250 mg/m2 of CPT-11/5-FU/LFA. These dosages were considered as MTDs, since four of six patients showed grade 4 neutropenia, in one case associated with grade 3 stomatitis. A mild decrease of both the CPT-11 and 5-FU doses to 200 and 850 mg/m2, respectively, caused different DLTs (neutropenia and diarrhoea) in two out of seven patients. At these dosages, transient grades 3 or 4 neutropenia affected two patients each during their treatment, while only one patient suffered from a severe delayed diarrhoea. Other non-haematological toxicities were mild and manageable. Therefore, we recommend this latter dose level for further study. Major responses (3 complete and 11 partial) were reported in 14 patients, for an overall response rate of 45% (95% CI: 27%-64%) according to an intent-to-treat analysis. Responses were observed from first dose level, and in four of five previously treated patients. Median failure-free and overall survivals, after a median follow-up of 39 weeks, were 42 and 55 weeks, respectively. CONCLUSIONS: The concurrent administration of CPT-11 and modulated 5-FU every two weeks is feasible at the recommended dosages. This regimen demonstrated interesting activity in the management of advanced colorectal cancer patients, and it probably better exploits the synergism between CPT-11 and 5-FU than recently tested alternating schedules. A phase II study is ongoing to more precisely define its activity and toxicity.

Image and flow cytometric analyses of DNA content in human solid tumors. A comparative study.

OBJECTIVE: To compare the sensitivity and potentials of flow and image cytometry in assessing DNA content. STUDY DESIGN: The study was performed on 152 tumors (oral cavity, uterine cervix, bladder, colorectum, breast). Flow cytometry was carried out on cell suspensions from frozen samples, and the results were expressed as the DNA index. Image cytometry was performed on Feulgen-stained sections, and the results were expressed as the rate of cells exceeding 2.5c or 5c. For colorectal and breast cancers, DNA content by image cytometry was also measured on imprints and was expressed as the DNA index or rate of cells exceeding 2.5c and 5c. RESULTS: Among flow cytometric diploid tumors, image cytometric analysis performed on histologic sections showed about 80% diploid tumors from the uterine cervix and breast cancers. The frequency decreased to 36% for oral cavity cancers. Generally satisfactory concordance was observed when flow cytometric aneuploid tumors were analyzed. A highly significant correlation was observed between DNA indices observed by flow and image cytometry on imprints. CONCLUSION: Image cytometry appears more sensitive than flow cytometry in detecting small, aneuploid clones, but its main limitation is the low power in resolving near-diploid cell populations. The results on imprints indicate that image cytometry is a potential alternative approach for small tumor samples.

Cell proliferation-related markers in colorectal liver metastases: correlation with patient prognosis.

PURPOSE: We analyzed the relation between phenotypic (DNA ploidy) and functional markers (S-phase cell fraction, p53, and bcl-2 protein expression) and defined their relevance on clinical outcome on a retrospective series of radically resected liver metastases from colorectal cancer. PATIENTS AND METHODS: Among 104 patients with resectable liver metastases from colorectal cancer, DNA ploidy was determined by flow cytometry, 3H-thymidine labeling index (TLI) by autoradiography, and expression of p53 and bcl-2 by immunohistochemistry. RESULTS: TLI was a significant indicator for relapse at 4 years from radical surgery, DNA ploidy was a suggestive indicator of clinical outcome, and p53 and bcl-2 expression provided no clinical information. By multivariate analysis, cell proliferation rate and Dukes' stage remained independent prognostic parameters. In the most representative subgroup of patients with H1 liver lesions (86 cases), TLI was always associated with relapse, and DNA ploidy and p53 expression provided discriminant information within slowly proliferating liver lesions. CONCLUSION: Tumor-cell proliferation of liver lesions should be used with stage of the primary colorectal cancer for a more accurate prognosis in patients submitted to curative hepatic resection.

Biological indicators of survival in patients treated by surgery for squamous cell carcinoma of the oral cavity and oropharynx.

Squamous cell carcinomas of the head and neck are a heterogeneous group of tumours with regard to anatomical site, natural history and response to various treatments. Assessment of the role of biomarkers as indicators of prognosis or response to treatment is thus complex. In the last decade, different biomarkers have been investigated in the search for objective and reproducible indicators of prognosis. In 69 squamous cell carcinomas of the oral cavity or oropharynx from patients treated with radical surgery alone, we determined cell kinetics, evaluated as in vitro 3H-thymidine labelling index (TLI), p53, bcl-2 and glutathione S-transferase pi (GST pi) expression, by using immunohistochemical methods. The biological variables were unrelated to one another or to established clinical and pathological prognostic factors. Univariate analysis showed that a low proliferative activity was associated to a significantly higher risk of death than that observed in patients with a high TLI, whereas p53, bcl-2 and GST pi expression did not provide prognostic information. Multivariate analysis showed that cell proliferation, gender and nodal status retained their clinical relevance. In the subset of node-negative patients, TLI and p53 expression were indicators of survival. Moreover, the combined analysis of TLI and p53 expression identified a subgroup of node-negative patients with slowly proliferating and highly p53-expressing tumours who died within 1 year of radical surgery. These results indicate that in patients with operable oral cavity and oropharyngeal cancer, biomarkers can provide important information on clinical outcome.

Biological predictors of response to radiotherapy in cervical carcinomas.

On 21 patients with T1b-T3b tumours subjected to external radiotherapy and brachytherapy, the expression of P53 and glutathione S-transferase pi [GST-pi], immunohistochemically detected, the S-phase cells fraction (H-3-thymidine labeling index, TLI) and DNA content evaluated by image analysis were determined on biopsies before and after the first 10 Gy. P53 accumulation was reduced in 60% of P53-overexpressing tumours and not induced in P53-negative tumours, GST-pi was induced in about 40% of pretreatment GST-pi-negative cases, TLI was reduced in 70% of the cases regardless of pretreatment values, and DNA profiles remained unchanged in two-thirds of the cases. P53 accumulation was a predictor of 3-year relapse-free survival after radiotherapy, followed by GST-pi expression, whereas TLI did not influence prognosis.

Validation of p53 accumulation as a predictor of distant metastasis at 10 years of follow-up in 1400 node-negative breast cancers.

Most studies are in favor of a prognostic relevance of p53 accumulation determined by immunohistochemistry in breast cancer, but negative results are not lacking. On a series of 1400 patients with lymph node-negative cancers treated with local-regional therapy alone until relapse and with a median follow-up of 10 years, we validated the prognostic relevance for overall relapse and death of p53 accumulation observed in a pilot study and analyzed its predictivity on different adverse events. p53 protein accumulation was immunocytochemically detected using PAb1801. The case series had also been previously characterized for hormone receptor content [estrogen receptors (ERs) and progesterone receptors (PgRs)] and for cell proliferation [[3H]thymidine labeling index ([3H]dT LI)]. p53 expression, considered as a dichotomous variable with a cutoff value of 5% positive cells, significantly predicted the occurrence of overall relapse, distant metastasis, and death with an interaction with cell proliferation. p53 accumulation, cell proliferation, and their interaction term, along with tumor size and patient age, retained a predictive role for overall relapse, and together with tumor size and PgR, also for overall survival. When considered as continuous variables, we observed that the hazard of metastasis increased linearly with the increase of [3H]dT LI and decreased linearly with the increase of ER and PgR. Conversely, the hazard increased with the increase of p53-positive cells only for tumors with a [3H]dT LI lower than 7.5%. In multivariate analysis, the same prognostic factors for distant metastasis were identified when the biomarkers were considered as continuous or dichotomous variables.

Biological prospectives to define prognosis and treatment strategies in liver metastases from colorectal cancer.

Liver metastases arise in about a third of patients with colorectal cancer. Although important clinical results have been obtained by surgical treatment in patients with limited liver involvement, other intra-arterial or systemic therapies do not provide important long term clinical benefits in patients with unresectable liver metastases. Better knowledge of the biology of liver metastases could imply a more appropriate use of the available therapeutic approaches and a retrospective definition the biologic subgroups of patients who benefit from them. Phenotypic and molecular aspects of tumor cells have been investigated and have proven to be important determinants of clinical outcome in patients with different human tumor types. Liver metastases from colorectal cancer have been scarcely studied, but cell proliferation has been shown to be a discriminant of freedom from progression and even more of long-term clinical outcome in subsets in patients treated with radical surgery. Moreover, in patients with resectable liver metastases, DNA and entity of DNA abnormalities are significantly associated with patient survival. A few recent reports have indicated a potential prognostic relevance of abnormal activation or expression of the p53 tumor suppressor gene, bel-2 protein and ras oncogene. In conclusion, prognostic biologic factors are acquiring an important role as indicators of clinical outcome in patients with liver metastases. However, all information is derived from retrospective analyses heterogeneous for patient population and biomarkers analyzed. Therefore, the comparison among results from different studies is difficult, and prospective studies are needed to develop a prognostic classification which integrates biologic and pathologic factors.

Image analysis for the evaluation of p53 expression in human cancers.

We compared the results of a subjective microscopic evaluation and an interactive image analysis of p53 expression in different human tumours. Expression of p53 was immunocytochemically detected by using the monoclonal antibody PAb 1801, and the evaluation was made blindly by two observers. Image analysis was performed on the same sections using DISCOVERY. The image was segmented into objects and background by interactive thresholding. Several morphological and densitometric features were selected in order to remove artefacts, lymphocytes, stromal cells and overlapping nuclei from the counting. The study was performed on series of 30 cases for different tumour types: breast, colon, oral cavity, ovary and lung cancer. Spearman's correlation coefficient ranged from 0.72 to 0.95 (P = 0.0001). Image analysis can therefore be considered as a quick and alternative approach to microscopic evaluation of nuclear immuno-determinations.

P53 expression, DNA ploidy and S-phase cell fraction in operable locally advanced non-small-cell lung cancer.

The identification of biomarkers to complement pathological stage for a more accurate prognosis and help clinicians decide on treatment is still an open problem for patients with lung cancer. Expression of P53 protein was detected by an immunohistochemical approach using the monoclonal antibody PAb1801 on paraffin-embedded sections of tumours obtained surgically from 102 stage II - IIIa patients with non-small-cell lung cancer (52 squamous cell carcinomas, 50 adenocarcinomas). [3H]Thymidine labelling index, an indicator of the S-phase cell fraction, was evaluated on histological sections of [3H]thymidine-labelled tumour samples. DNA ploidy was defined by flow cytometric analysis on frozen tumour tissue. The biomarkers, histology and pathological stage were analysed in relation to relapse-free survival in univariate and multivariate analyses. Stage and interaction between [3H]thymidine labelling index and histology provided significant prognostic information for the overall series. [3H]thymidine labelling index was an independent prognostic indicator of 3 year relapse-free survival in patients with adenocarcinoma. The results indicate the importance of cell proliferation to complement prognostic information provided by pathological stage in patients with stage II-IIIa adenocarcinomas.

P53, Bcl-2 expression and cell proliferation in benign breast lesions.

The role of proliferation-related markers on breast cancer pathogenesis has been only occasionally investigated. The immunocytochemical expression of P53 and Bcl-2 (using PAb1801 and anti-bcl-2 monoclonal antibodies) and cell proliferation (evaluated as the H-3-thymidine labeling index [H-3-dT LI]) were determined on 62 benign breast lesions at different risk. Accumulation of the P53 protein was never observed; Bcl-2 was detected in 50% of cases and it was unrelated to biologic and clinicopathologic features. Median H-3-dT LI was about three times lower than that observed on large series of invasive breast cancer. It was only slightly higher in lesions from patients younger than 35 years or with a positive family history than in those under 35 or with a negative family history and appeared unrelated to histology or risk classification. Such findings indicate that the investigated biomarkers fail to identify women at increased risk for breast cancer.

p53 gene point mutations in relation to p53 nuclear protein accumulation in colorectal cancers.

It is known that structural alterations of the p53 tumour suppressor gene cause malignant transformation and tumour progression in colorectal mucosa. In this study, 38 colorectal cancers were analysed for mutations detected in the p53 gene by single-strand conformational polymorphism and DNA sequence analysis, and the results were compared with p53 protein expression detected by immunohistochemistry. A very strict association (P < 0.0001) was found between genetic alterations and protein accumulation, as detected by the PAb 1801 monoclonal antibody. p53 expression and gene mutations were more frequent in rectal than in colonic cancers. No relation was observed with Dukes' stage, even though most of the mutations were at exon 7 in Dukes' A-B cancers and almost all mutations at exon 8 were observed in Dukes' C-D cancers. DNA ploidy was not generally associated with p53 protein expression or gene mutations. However, 83 per cent of cases with exon 5 and 6 mutations were diploid or near-diploid and 71 per cent of cases with mutations at exons 7 and 8 were aneuploid. Tumours with p53 gene mutations at exon 5 had a higher median [3H]thymidine labelling index (17 per cent) than those with mutations at exons 6, 7, and 8 (11.8 per cent).

Relationship between the levels of purine salvage pathway enzymes and clinical/biological aggressiveness of human colon carcinoma.

A large series of samples obtained after surgical resection of intestinal mucosa of patients affected by intestinal carcinoma was examined in order to define possible relationships between levels of enzymes involved in the purine salvage pathway and clinical/biological parameters of aggressiveness and invasiveness. The results confirm our previous observation on a different pattern of purine salvage enzymes in tumor as compared to normal colon tissues (Camici et al., 1990). In fact, we observed in human colon tumor tissues a significant enhancement of the three enzymes involved in the synthesis of IMP, hypoxanthine guanine phosphoribosyltransferase (HGPRT), adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP). On the other hand, no variation was observed in the 5'-nucleotidase and alkaline phosphatase activities. While we could not find a significant correlation between HGPRT, ADA and PNP activities and histologic grading or biological parameters of tumor aggressiveness, the significant correlation with the extent of disease, as expressed by the Dukes' stage, would demonstrate at least for human colon tumors, a relationship between enzyme activity and tumor invasiveness.

Cell kinetics of colorectal liver metastases.

PURPOSE: Liver metastases are the main cause of death in patients with colorectal cancer. We defined the proliferative activity of a metastatic liver cell population to investigate its potential role as an indicator of clinical outcome in patients with metastatic disease of the colorectum. METHODS: The S-phase cell fraction, evaluated as in vitro [3H]thymidine-labeling index, was determined on liver metastases of primary colorectal cancers from 75 patients. RESULTS: Cell proliferation was not related to the degree of liver involvement, the site of the primary cancer, or the time of presentation of liver metastases. Survival at 2 years was statistically different for patients with slowly proliferating (78 percent) or rapidly proliferating liver metastases (47 percent) (P = 0.024). The risk of death for patients with high [3H]thymidine-labeling index lesions was consistently threefold that of patients with low [3H]thymidine-labeling index lesions throughout the observation period. Bivariate analysis showed that cell proliferation was a further prognostic discriminant within the subsets characterized by a different degree of liver involvement. CONCLUSIONS: These results indicate that cell proliferation is a good prognostic marker even in patients with liver metastases from colorectal cancer. The clinical implications of the marker could be further potentiated by considering it in association with some clinical aspects of known prognostic relevance.

Characterization of radiation-induced apoptosis in the small intestine and its biological implications.

The small intestine with its high cell proliferation, well-accepted hierarchy, high radiation susceptibility and low cancer incidence is a useful model for studying the controls of cell replacement. Apoptosis, which represents part of the overall homeostatic process, occurs spontaneously at the stem cell position in the crypts, and very small doses of radiation elevate the levels of apoptosis rapidly in this region. Other cytotoxic agents also target cells in this region including several mutagenic chemicals. Yet other drugs target cells at higher positions in the crypt indicating that all crypt cells possess the programme for apoptosis, but this is normally suppressed in many of the cells. In contrast, high doses of radiation are required to reproductively sterilize the crypts and, using clonal regeneration techniques, the number of clonogenic cells is dependent on the levels of damage induced (dose), i.e. the more injury that is induced the greater number of cells that are recruited into the clonogenic compartment. All doses of radiation trigger rapid changes in proliferation in the stem cell region which suggests that the detection of the induced cell death (even small levels, such as one apoptotic cell per crypt) is efficient and has rapid consequences. p53 may be involved in this damage recognition and apoptosis initiation. The studies to date suggest that apoptosis plays an important role in this tissue in terms of its homeostasis and its protection against carcinogenesis by removal of potentially carcinogenic damaged cells.

Flow cytometric analysis of ploidy in colorectal cancer: a multicentric experience.

Ploidy and cell proliferation determined by flow cytometry were assessed on colorectal cancers from patients admitted to two Italian cancer research centres. A total of 181 patients were followed prospectively for 4 years at the Istituto Regina Elena (IRE) of Rome and at the Istituto Nazionale Tumori (INT) of Milan. Fresh (at the IRE) or frozen (at the INT) tumour material and similar procedures were used for subsequent sample preparation. Similar frequencies of aneuploid tumours (63% vs 66%) and superimposable median DNA indices (1.6) were observed for the two case series. In both series, DNA ploidy was generally unrelated to clinico-pathological factors, except for a higher frequency of aneuploid tumours in Dukes' D (88%) than in Dukes' A stage (33%) in the IRE experience. DNA ploidy was a weak prognostic indicator at 3 years but not at 4 years in the IRE case series, and it never exhibited a clinical relevance in the INT experience. Conversely, multiploidy was an indicator of worse relapse-free and overall survival at 4 years in the IRE and INT case series.

Biological markers in hepatocellular carcinoma: potential clinical implications.

The assessment of biological markers as potential indicators of disease aggressiveness is still an open problem in hepatocellular carcinoma. Cell proliferation and extracellular matrix (ECM)-associated antigens and tumor-cell products can be associated with clinical aggressiveness in this tumor type, as has already been demonstrated for others. Cell proliferation, expressed as the in vitro [3H]thymidine labeling index, and ECM-associated antigens, such as type IV collagenase and laminin receptors, were assessed on the same paraffin-embedded samples. A strong association (P = 0.0001) was observed between the expression of collagenase and laminin receptors, with a correlation coefficient (rs) of 0.68. No relationship was found between cell proliferation and ECM-associated antigens. Moreover, the biological markers were generally independent of clinicopathologic features, except for a higher number of collagenase- and laminin receptor-positive cells in large (> or = 5 cm) compared with small (< 5 cm) tumors. In the present series of hepatocellular carcinoma patients, the 3-year clinical outcome was significantly affected by cell proliferation and ECM-associated antigens.

Autoradiographic and flow-cytometric assessment of cell proliferation in primary colorectal cancer: relationship to DNA ploidy and clinico-pathological features.

Two cell-kinetic parameters, the [3H]thymidine-labeling index [3H]dT LI and the flow-cytometric S-phase cell fraction (FCM-S), and DNA ploidy were determined for a prospective series of 110 primary colorectal cancers. Aneuploidy was observed in 66% of tumors and more than one aneuploid peak was present in 12%. The frequency of aneuploid tumors was higher in rectal (80%) and left-colon (70%) cancers than in right-colon cancers (51%), and multiple aneuploid clones were detected more frequently in men than in women (p = 0.03) and more frequently in advanced Dukes' D-stage patients (p = 0.08). The median [3H]dT LI value (17.4%) was similar to the FCM-S value determined by a planimetric model (16.2%) and somewhat higher than the FMC-S value obtained by an optimization procedure (11.2%). However, there was no significant relationship between the [3H]dT LI value and either FCM-S value for individual tumors. Moreover, FCM-S values were higher in aneuploid than in diploid tumors, whereas [3H]dT LI values were independent of DNA-ploidy status; [3H]dT LI and FCM-S were also related differently to some clinical and pathological features such as tumor site and histology. These findings suggest different biological meanings for these 2 cell-kinetic parameters, which should not be used interchangeably.

Reliability of 3H-thymidine labeling index: cell proliferation of colorectal carcinoma in three different centers.

Although the 3H-thymidine labeling index (TLI) has been used extensively as a biologic marker of increased susceptibility to neoplasms or as a prognostic indicator of the clinical outcome in cancer patients, there is still some concern regarding its accuracy and reliability as a marker of cell replication. A study on the prognostic value of TLI in colorectal cancer gave us the ability to evaluate how such a measurement may vary in different laboratories. A total of 150 malignant tumors of the large bowel were studied in the period from 1988 to 1989. Microautoradiography was carried out in tumor fragments taken at surgery. There were only slight differences among the three centers involved in the investigation, mainly as regards the culture medium, exposure time, and the addition of O2 + CO2 during the incubation. No significant difference was observed among TLI values recorded in the three centers (18.3 +/- 0.9; 17.4 +/- 0.9; 15.7 +/- 1.0 mean +/- SE), and the ranges of values were almost identical. Similarly, the numbers of total and of labeled cells counted for each patient were comparable among the three centers. The frequency distribution of TLI showed a peak value between 10 and 19.9% in all three centers. Moreover, more than 80% of the observed values were within the range of 10 to 29.9%. In addition, the neoplastic areas with the highest proliferative activity ("high" TLI) showed a frequency distribution once again rather similar among the three centers.(ABSTRACT TRUNCATED AT 250 WORDS)