N-nitroso-N-methylurea-induced mammary carcinogenesis: effect of prolactin on expression of Ia antigen by tumor cells.

Prolactin (PRL) increases of Ia antigen (Ia Ag) expression in female Sprague-Dawley rats with N-nitroso-N-methylurea [(NMU) CAS: 684-93-5]-induced mammary tumors were studied. The effectiveness of PRL was examined when cancers appeared about 2-3 months after the first NMU administration. Rats with NMU-induced mammary tumors were divided into 3 groups: Group 1 was treated with 30 micrograms ovine PRL (o-PRL) in daily sc injections for 5 days. Group 2 received 0.5 mg 2 alpha-bromoergocryptine (CB-154), a known inhibitor of pituitary gland secretion, daily in sc injections for 6 days. Group 3 was the control group. Ia Ags expressed by NMU-induced mammary tumor cells were then quantified successively by double labeling [protein membrane cells with iodine-131 and anti-Ia monoclonal antibody (MoAb) with iodine-125]; then isolation and quantification of the doubly labeled immune complex were performed by affinity chromatography and chromatofocusing successively. When the specific activity of glycoproteins is known, the amount of glycoproteins that bind specifically to the anti-Ia MoAb can be deduced. In NMU-induced rat mammary tumor controls, about 5% of the purified glycoproteins bound specifically to the MoAb, and the amount increased to 8% for NMU-induced rat mammary tumors treated with 30 micrograms o-PRL daily for 5 days and decreased to 2.5% in NMU-induced rat mammary tumors treated with 0.5 mg CB-154 daily for 6 days. Total PRL receptor levels were measured in all tumors tested. For control NMU-induced rat mammary tumors, total PRL receptor levels were 6.35 +/- 1.40 fmol/mg protein, 7.20 +/- 2.40 fmol/mg protein for NMU-induced rat mammary tumors treated with o-PRL, and 6.81 +/- 2.34 fmol/mg protein for NMU-induced rat mammary tumors treated with CB-154. Our results demonstrated that treatment of NMU-induced rat mammary tumors with PRL increased the amount of Ia Ag expression by tumor cells and should prove very useful to the understanding of the biology of PRL in the tumorogenesis of the mammary gland.

Clinical relevance of immunohistochemical detection of multidrug resistance P-glycoprotein in breast carcinoma.

In 20 women with breast carcinoma, 17 of whom had locally advanced cancer and 3 of whom had confirmed metastases, the expression of P-glycoprotein was evaluated before the start of a chemotherapy regimen that included multidrug resistance-related drugs. With the use of the C494 monoclonal antibody in an avidin-biotin-immunoperoxidase technique, P-glycoprotein was detected in 17 of 20 tumor samples. Results were expressed in a semiquantitative manner, taking into account the number of positive tumor cells (N index) and the specific staining intensity (I index). The 17 patients with nonmetastatic cancer were followed from the first cycle of chemotherapy to cancer recurrence; subsequent to six cycles of chemotherapy, all of these patients except one were rendered clinically disease-free through surgery and/or radiation. The end point was defined as either local/regional recurrence or metastasis. Strong P-glycoprotein-positive staining in a majority of tumor cells (the N+/I+ phenotype) was significantly correlated with no initial response to chemotherapy (P less than .02) and with a shorter progression-free survival (P less than .02). Thus, the pretreatment evaluation of P-glycoprotein expression may be of prognostic value in patients with locally advanced breast cancer.

Disposition of the carcinostatic agent 1-(2-chloroethyl)-3-[1'-(5'-p-nitrobenzoyl-2',3'-isopropylidene)-alpha,beta-d-ribofuranosyl]-1-nitrosourea in animals.

The disposition of 1-(2-chloroethyl)-3-[1'(5'-p-nitrobenzoyl-2',3'-isopropylidene)-alpha,beta-D-ribofuranosyl]-1-nitrosourea (RFCNU) was investigated in animals following i.p. administration (35 micromol/kg) of the drug labeled with 14C in three different positions. The appearance and disappearance kinetics of the label in blood and plasma were strongly dependent upon the labeled species administered to animals. Protein binding of [carboxyl-14C]- and [carbonyl-14C]RFCNU greatly prolonged the blood and plasma half-life of these two labeled species. No intact RFCNU was found when rat plasma samples were analyzed using high-performance liquid chromatography, indicating the very short half-life of this compound. Most of the dose was recovered in the urine after administration of the carboxyl-14C and ethyl-14C species and in the expired air as 14CO2 after administration of the carbonyl-14C species. Except for the liver and kidney, no specific localization of the radioactivity was found when animals were given [carboxyl-14C]- or [carbonyl-14C]RFCNU. However, tissue distribution studies showed a marked specificity of the species carrying the ethyl-14C moiety for the liver, kidney, pancreas, and thymus, but not for the brain.

Disposition and metabolism in 1-(2-chloroethyl)-3-(2',3',4'-tri-o-acetyl, ribopyranosyl)-1-nitrosourea in rats.

The antineoplastic activity in animals of 1-(2-chloroethyl)-3-(2',3',4'-tri-O-acetyl, ribopyranosyl)-1-nitrosourea (RPCNU) has been widely demonstrated. The present study deals with the disposition and the metabolism of three 14C-labeled species of RPCNU. The chemical plasma half-life of the drug was less than 5 min. Within the first min after injections, most of the radioactivity derived from ethyl-14C groups were recovered as volatile products. Among these, 2-chloroethanol was identified as a main component. Analysis of labeled species in urine after administration of [ethyl-14C]RPCNU showed that thiodiacetic acid and its sulfoxide were major metabolites of RPCNU (62% of the urinary radioactivity). Traces of N-acetylcarboxymethyl- and N-acetylhydroxyethylcysteine) were also detected. The only labeled species concentrating in particular tissues was that carrying the chloroethyl moiety. Uptake to high levels of [ethyl-14C]RPCNU did occur in liver, kidney, pancreas, thymus, and Harder's gland.

Comparison of Class II HLA antigen expression in normal and carcinomatous human breast cells.

Class II HLA antigen expression in breast carcinoma and normal breast gland cells was compared using a method more accurate than immunofluorescence. This new method involves labeling membrane proteins with 131I and the anti-Class II HLA monoclonal antibody with 125I. The isolation and purification of the doubly labeled (125I-131I) immune complex was performed by affinity chromatography and chromatofocusing successively. When the specific activity of glycoproteins is known, the amount of glycoproteins which bind specifically to the anti-Class II HLA monoclonal antibody can be deduced. In breast carcinoma cells, 1.5 to 2% of the purified glycoproteins bind specifically to the monoclonal antibody, whereas less than 0.3% of normal breast gland cells binds. In contrast, leukemic cells, of which 80 to 90% possess Class II HLA antigens, 2 to 3% of Class II HLA glycoproteins bind specifically with the anti-Class II HLA monoclonal antibody.

Effects of cyclosporine A on Ia antigen expression in N-nitroso-N-methylurea-induced rat mammary tumors.

Cyclosporine A (an immunosuppressive agent) decreases Ia lymphoid differentiation marker in female Sprague-Dawley rats with N-nitroso-N-methylurea-induced mammary tumors. Presence of lymphoid differentiation antigens was determined on mammary tumor cells and lymphoid cells from bone marrow, spleen, and peripheral blood by flow cytometric analysis. Quantification of Ia antigen expression was also performed by affinity chromatography and chromatofocusing in mammary tumors. A significant decrease in Ia antigen expression by mammary tumors of animals treated with cyclosporine A was noted with the two different methods. Cyclosporine A acts as an antagonist to prolactin receptors in such hormone-dependent mammary cancer. Our results should prove very useful in understanding the mechanisms of prolactin regulation of Ia antigen in tumorigenesis of the mammary gland.

Variations in committed stem cells (CFU-GM and CFU-TL) in the peripheral blood of cancer patients treated by sequential combination chemotherapy for breast cancer.

The present work compared the blood variations in some committed stem cells (CSC), and corresponding differentiated WBC during the first three courses of a 6-day sequential chemotherapy given to 16 breast cancer patients for 28 days each. The granulomonocytic and lymphocytic colony-forming unit levels in blood were significantly lowered in cancer patients before treatment. These CSC appeared to have cyclic variations following each course of chemotherapy. In granulomonocytic colony-forming units, a nadir was observed by Day 15, followed by a sharp rebound above the initial values by Days 22 to 24, which was not affected in magnitude by the continuation of treatment. In lymphocytic colony-forming units, the Day 1 level increased with the continuation of chemotherapy. The initial decrease was less marked, but by Day 15 a minimal level was also observed, followed by a progressive increase to reach a maximum by Day 28. Leukocytes and granulocytes reached a nadir by Days 16 to 17 and recovered by Day 25. The cyclic evolution of monocytes was less apparent as was that of lymphocytes; however, there was less restoration of monocytes and lymphocytes than of granulocytes at the end of the resting period. This study showed: (a) an apparent relationship between the level of CSC in blood, and subsequent variations in the corresponding mature cells of the same lineage; and (b) a weak interval of time between the nadir and peaks of CSC and the corresponding mature cells, which was more evident in the granulocytic lineage. These observations seemed to be of physiological importance, but the possible prediction value of peripheral granulomonocytic colony-forming unit peak magnitude following treatment remains to be established.

High-dose chemotherapy with hematopoietic rescue in patients with stage III to IV ovarian cancer: long-term results.

PURPOSE: A series of 53 patients with poor-prognosis epithelial ovarian cancer treated with high-dose chemotherapy (HDC) followed by hematopoietic rescue was retrospectively studied from the day of diagnosis for toxicity and long-term survival analysis. PATIENTS AND METHODS: Patients were treated with surgery followed by cisplatin combination chemotherapy. After second-look operation (SLO), HDC was administered: 23 patients received melphalan (140 mg/m2 on day 1) and 30 patients received a combination of carboplatin (400 mg/m2 on days 1 to 4) and cyclophosphamide (1.6 g/m2 on days 1 to 4). After HDC, autologous stem-cell transplantation was performed for hematologic support. RESULTS: One patient died of cardiac failure after HDC, but the acute toxicity was acceptable for the other patients. With a median follow-up of 81.5 months, the 5-year overall survival rate for the 53 patients was 59.9% and the disease-free survival (DFS) rate at 5 years was 23.6%. Twenty-four patients (45.3%) were alive, 12 with no evidence of disease and 12 with recurrent disease. The best results were achieved in 19 patients with pathologic complete response at SLO (74.2% 5-year overall survival; 32.8% 5-year DFS). CONCLUSION: HDC followed by autologous stem-cell support is a well-tolerated therapeutic approach for patients with poor-prognosis ovarian carcinoma. In this report, the 59.9% survival of 53 patients at 5 years must be compared to the 20% to 30% 5-year survival observed after conventional therapy. These results should be confirmed by an ongoing prospective randomized trial.

Adjuvant treatment of node-positive breast cancer with cyclophosphamide, doxorubicin, fluorouracil, and vincristine versus cyclophosphamide, methotrexate, and fluorouracil: final report after a 16-year median follow-up duration.

PURPOSE: To determine the long-term impact on disease-free survival (DFS) and overall survival (OS) of adjuvant anthracycline-based chemotherapy, when prospectively compared by random allocation with standard cyclophosphamide, methotrexate, and fluorouracil (CMF) in node-positive (N+) breast cancer patients. PATIENTS AND METHODS: Two hundred forty-nine patients with N+ breast cancer, recruited from eight French cancer centers, were randomized to receive 12 monthly cycles of adjuvant chemotherapy, either CMF (n = 112) or doxorubicin, vincristine, cyclophosphamide, and fluorouracil (AVCF) (n = 136). All had a negative metastatic work-up before inclusion, which was stratified by accrual center, tumor stage (International Union Against Cancer [UICC]), and menopausal status. RESULTS: No severe adverse effect related to grade 4 (World Health Organization [WHO]) toxicity was observed. There was no difference in second primary tumor incidence between the two arms. The treatment given was 88% of planned for AVCF and 75% for CMF in both premenopausal and menopausal patients. With a median follow-up time of 16 years (range, 13 to 17), the OS and DFS rates are significantly longer in the AVCF arm (56% v 41% [P = .01] for OS, and 53% v 36% [P = .006] for DFS). These differences are significant, irrespective of tumor stage (T1 to T2 v T3 to T4), and remain positive in patients with or without postoperative locoregional radiotherapy (55% of cohort). When analyzed according to menopausal status, the differences remain significant only for premenopausal patients. CONCLUSION: This set of mature controlled data confirms the added value of anthracycline-based combination adjuvant therapy for N+ breast cancer patients when compared with CMF, with both regimens given for 1 year.

Detection of Epstein-Barr virus sequences in primary brain lymphoma without immunodeficiency.

We searched for Epstein-Barr virus (EBV) sequences by enzymatic DNA amplification in nine primary brain lymphomas from patients without immunodeficiency. We used seven nonlymphoma brain tumors as negative controls, and the Raji cell line as a positive control. We detected EBV DNA, using ethidium bromide-stained-agarose minigel electrophoresis and dot blot hybridization, in the positive control and in only one brain lymphoma tumor; we did not detect EBV DNA in the other tumors. The EBV-positive patient had a second B-cell monoclonal population in the peripheral blood without detectable EBV DNA, suggesting a direct role for EBV in the development of the brain lymphoma.

Dihydropyrimidine dehydrogenase activity in cancer patients.

Dihydropyrimidine dehydrogenase (DPD) is the major catabolic enzyme of pyrimidines and fluoropyrimidines. The clinical course of 2 patients with suspected DPD deficiency is described. Both patients had significantly delayed clearance of fluorouracil (5-FU), elevated plasma uracil concentrations, and subsequent lethal toxicity. The prevalence of DPD deficiency in the general population is unknown, but given the large number of cancer patients treated with 5-FU, it may be of great clinical significance. Lymphocytes have been previously shown to be a useful marker of systemic DPD activity. Because DPD activity has not been previously reported in a large population of cancer patients using 5-FU as the substrate, we determined DPD activity in lymphocytes from 66 patients with cancer. DPD activity was determined by a sensitive high performance liquid chromatography method. The mean DPD activity (S.D.) in 66 patients with head and neck cancer was 0.189 (0.071) nomol/min/mg protein with wide interpatient variability (range 0.058-0.357). DPD activity was not correlated to age (r = -0.164, P = 0.188). The mean DPD activity in men [0.192 (0.074)] was not significantly different from that in women [0.172 (0.057); t-test P = 0.418]. Likewise, there was no statistical difference in DPD activity in patients who had not received prior chemotherapy [0.195 (0.066)] to patients receiving one or more cycles of chemotherapy [0.186 (0.074); t-test P = 0.638].

Electrophoretic mobility of human lymphocytes purified by nylon wool columns and spontaneous sheep red blood cells rosetting techniques correlation with immunofluorescence.

Cell electrophoresis allows separation of normal human lymphocytes into two principal groups which are a function of their relative rates of migration. In 42 healthy adults, 19.9% of the lymphocytes have a slower migration rate, and 80.1%, a faster migration rate than the reference speed (1 mum - sec-1 - cm). Two methods are used for the selection of the lymphocytic populations: spontaneous rosetting with sheeps red blood cells, a property of T-lymphocytes, and adherence to nylon wool columns, which preferentially selects B-lymphocytes. The cells which do not form spontaneous rosettes, but adhere to nylon wool columns show mainly slow migration. Cells which do not adhere to nylon columns show a faster migration rate. These findings affirm the T-nature of the rapidly migrating lymphocytes, and the B-nature of the slow-migrating lymphocytes. Results by the immunofluorescence technique confirm this.

New cysteamine (2-chloroethyl)nitrosoureas. Synthesis and preliminary antitumor results.

Three chemical pathways were used for the synthesis of four new N'-(2-chloroethyl)-N-[2-(methylsulfinyl)ethyl]- and N'-(2-chloroethyl)-N-[2-(methylsulfonyl)ethyl]-N- or N'-nitrosoureas. These compounds are plasma metabolites of CNCC, a promising antineoplastic (2-chloroethyl)nitrosourea. Preliminary antitumor evaluation was performed against L1210 leukemia implanted intraperitoneally in mice. Among these compounds, two of them exhibited a greater antitumor activity compared to that of the parent mixture.

Correlation between active E-rosettes and antigens defined on peripheral lymphocytes by OKT 4 and OKT 8 monoclonal antibodies.

A relationship has been sought between the classical marker, active E-rosette and the surface antigens of T-lymphocytes, defined by OKT 4 and 8 monoclonal antigens, in the peripheral blood of normal human people. It was found that no significant correlation existed in the case of OKT 4 antigen; conversely the OKT 8-positive cells were significantly enriched after E-rosette-forming cell isolation (about 2.5 times in comparison with the negatively selected cells). In this latter case, some partial correlation could exist between SRBC-high affinity of T-lymphocytes and the subsets defined by OKT 8 antigen.

The correlation of adenosine deaminase and purine nucleoside phosphorylase activities in human lymphocytes subpopulations and in various lymphoid malignancies.

Adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) were measured in normal human and in malignant lymphoid cells. Thymocytes had high ADA activity (21.2 +/- 6.8 10(3) nM/h/mg) and low PNP activity (1.2 +/- 0.6), whereas T peripheral blood lymphocytes (PBL) had low ADA activity (1.20 +/- 0.22) and high PNP activity (2.8 +/- 1.3). Moreover cortico-thymocytes had higher ADA and lower PNP levels than medullary thymocytes. A linear correlation was observed between ADA and PNP activities in both thymocytes and T-PBL. Cells from 13 patients with T acute lymphoblastic leukemia (ALL) and 10 patients with T lymphoblastic lymphoma (LL) had very high levels of ADA (respectively 13.0 +/- 5.4 and 22.8 +/- 14) and low levels of PNP (respectively 1.9 +/- 0.8 and 2.5 +/- 1.4). However no clear relationship appeared between subgroups of these T-cell malignancies defined by their patterns of surface antigens, revealed by reactivity with monoclonal antibodies, and ADA and PNP levels, and there was no correlation between the two enzymes. In contrast, cells from 31 patients with HLA-DR+ common ALL had significantly low values of ADA as compared to cells from six patients with HLA-DR- common ALL and a linear correlation was observed between ADA and PNP in cells from children with non-T, non-B ALL. These results show that specific stages of T-cell development may be characterized by the relationships and the correlation between the two enzymes and suggest that T-ALL and T-LL appear to be the group of lymphoid malignancies with a high degree of incoordination between ADA and PNP activities.

Presence of the mouse mammary-tumor virus (mmtv) pol gene in breast-cancer.

We have used the polymerase chain reaction (PCR) to detect sequences related to the mouse mammary tumor virus (MMTV) reverse transcriptase gene in DNA from breast cancer cell lines and an extensive series of breast tumors. Similar MMTV-related sequences were observed in DNA from normal tissues. The segments amplified by PCR showed over 90% homology to the nucleotide sequence of the MMTV pol gene and no significant differences were noted between the DNA from normal or tumor tissue. When the amplified DNA was used to probe Southern blots, a unique restriction fragment indicative of a single copy locus was detected in all DNAs tested, but the high background of hybridization suggested that many closely related sequences may occur in the human genome.

Potential allogeneic graft-versus-tumor effect in a patient with ovarian cancer.

A 33-year-old woman developed progressive ovarian cancer resistant to classical chemotherapy agents. We performed a bone marrow allograft after a myeloablative regimen. During hematological recovery, she developed acute graft-versus-host disease (GVHD). From this time her tumor diminished progressively. One year post transplant she has limited chronic liver GVHD and is still free of disease. The complete remission of advanced ovarian cancer was probably related to the GVHD which might therefore provide a new treatment option for this disease.

Hematological recovery and peripheral blood progenitor cell mobilization after induction chemotherapy and GM-CSF plus G-CSF in breast cancer.

In order to determine the effect of GM-CSF plus G-CSF in combination in breast cancer patients receiving an effective induction regimen, we compared hematological recovery and peripheral blood progenitor cell (PBPC) mobilization according to colony-stimulating factor (CSF) support. Forty-three breast cancer patients were treated by TNCF (THP-doxorubicin, vinorelbine, cyclophosphamide, fluorouracil, D1 to D4) with CSF support: 11 patients received GM-CSF (D5 to D14); 16 patients G-CSF (D5 to D14) and 16 patients GM-CSF (D5-D14) plus G-CSF (D10-D14). Between two subsequent cycles, progenitor cells were assessed daily, from D13 to D17. The WBC count was similar for patients receiving G-CSF alone or GM-CSF plus G-CSF, but significantly greater than that of patients receiving GM-CSF alone (P<0.001). The GM-CSF plus G-CSF combination led to better PBPC mobilization, with significantly different kinetics (P<0.001) and optimal mean values of CFU-GM, CD34+ cells and cells in cycle, at D15 compared to those obtained with G-CSF or GM-CSF alone. The significantly greater PBPC mobilization obtained with a CSF combination by D15 could be of value for PBPC collection and therapeutic reinjection after high-dose chemotherapies.

Mobilization of peripheral blood progenitor cells after induction chemotherapy (THP-doxorubicin-vinorelbine-cyclophosphamide-fluorouracil) and granulocyte colony-stimulating factor in breast cancer.

In order to evaluate the mobilization of peripheral blood progenitor cells (PBPC) after an effective induction regimen in breast cancer, we performed a study on 15 breast cancer patients. Between January 1995 and June 1996, these patients received TNCF (THP-doxorubicin. vinorelbine, cyclophosphamide, fluorouracil for four days, every 21 days) with G-CSF support (5 microg/kg for 10 days after chemotherapy) to reduce aplasia. This regimen is known to result in a complete pathological response in 30% of patients. Between two cycles of TNCF treatment, hematological recovery was observed. Progenitor cells (CFU-GM and CD34+ cells) and mononuclear cells in DNA synthesis (MCDS) counts were performed daily, between the 12th and 17th post-chemotherapy days (81 samples). The results showed a similarity for hematological recovery and PBPC mobilization kinetics depending on the number of treatment cycles. The three methods used for PBPC evaluation were well correlated (P < 0.01) with an optimal mean PBPC recruitment by the last day of G-CSF administration: respectively, 11 520 (1729-26539) CFU-GM/ml of blood, 249 (14-1160) CD34+ cells/microl of blood and 211 (21-554) MCDS/microl of blood. These results suggested that a daily injection of G-CSF after one or two TNCF cycles will produce an effective PBPC mobilization in comparison with currently used regimens.

High dose melphalan and autologous marrow rescue in advanced epithelial ovarian carcinomas: a retrospective analysis of 35 patients treated in France.

We retrospectively evaluated the feasibility and antitumour efficacy of high dose melphalan (HDM) followed by autologous marrow rescue in 35 patients with common epithelial ovarian cancers. All patients initially had advanced disease (FIGO III-IV) and received HDM after extensive surgery and a median of 6 cycles of cis-DDP containing regimens CAP or CHAP. All, except three patients who showed evidence of progression, had a second surgical exploration before high dose chemotherapy. Melphalan was given at a dosage greater than or equal to 140 mg/m2 followed 24 h later by autologous marrow rescue. Severe but reversible aplasia and mucositis were the most common toxicities: three patients died from the procedure, two from infection and one from secondary leukaemia. HDM was effective in 75% of evaluable patients; this was evidence of activity in patients who failed to respond to first line chemotherapy. The duration of response was short, particularly for patients treated with progressive disease at the time of high dose chemotherapy rather than in partial or complete remission. With a median follow-up of 23 months (range 8-54) after high dose chemotherapy, 19 patients are alive (15 with non-progressive disease) with a projected survival of 47% between 2 and 5 years.