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Monoclonal antibodies against a 24,000 dalton intracellular estrogen-regulated protein in human breast cancer cells were used to study storage conditions and the effects of monoclonal antibody concentrations on immunohistochemical antigen localization. Both hybridoma supernatants and ascites fluid obtained from mice injected with hybridoma cells were used as sources of monoclonal antibodies; the monoclonal antibodies in the ascites fluid were concentrated and purified. Both antibody preparations were stored at 4, -20, or -70 degrees C and periodically tested for activity at these storage conditions. There was no difference in activity for the antibodies between storage at -20 and -70 degrees C. However, when highly diluted antibody was stored at 4 degrees C, the activity was lost within 2 weeks if carrier proteins were not added. These monoclonal antibodies were applied to immunohistochemical staining of different mouse and human tissues processed for routine paraffin sections, using the avidin-biotin-peroxidase procedure. A monoclonal antibody of unrelated specificity was used as control. When these antibodies were used at high concentrations, all the different tissues examined were immunostained. With reduction of the antibody concentration, an immunohistochemical dissection of the tissues was seen until specific immunostaining was reached. When even more highly diluted monoclonal antibody was used, heterogeneity in the staining pattern became very high. On the basis of these results, certain immunohistochemical criteria are proposed for the selection of the optimum concentration of monoclonal antibodies for specific antigen detection.

From January 1983 through August 1988, 318 consecutive patients with refractory germ cell neoplasms were treated with ifosfamide-containing combination chemotherapy. The patients received ifosfamide at 1.2 gm/m2 day with cis-platin 20 mg/m2 day for 5 days and etoposide 75 mg/m2 day for 5 days or vinblastine 0.11 mg/kg on days 1 and 2 for each cycle. Of 277 evaluable patients, NAC was used as an uroprotector in the initial 86 patients while the latter 191 consecutive patients received mesna to reduce urothelial toxicity. Dosages of NAC was 2.0 gm po q 6 hr and for mesna 120 mg/m2 IV push prior to ifosfamide and then 1200 mg/m2/day as continuous infusion of 5 consecutive days. All patients received 3.0 liters of normal saline per day. The number of courses of chemotherapy given in the two groups were similar. Twenty-four of the 86 patients (27.9%) receiving NAC developed hematuria (13 patients — grade 1, 4 patients — grade 2, and 7 patients — grade 3 toxicity). While 8 out of 191 (4.2%) mesna patients developed hematuria (6 — grade 1 and 2 — grade 3) (p<0.0001). The incidence of severity of renal toxicity was similar in the two groups. Ifosfamide dosage was reduced solely for urothelial toxicity in 11 patients receiving NAC compared with none of the patients receiving mesna (p<0.0001). Chemotherapy response was similar in the two groups. In conclusion, mesna provides better urothelial protection from ifosfamide-induced toxicity than NAC and allows better maintenance of the drug dosage.

Seventy-one patients with poor-prognosis breast cancer were enrolled after informed consent in a multicentre randomized study to evaluate the use of selected peripheral blood CD34+ cells to support haematopoietic recovery following high-dose chemotherapy. Patients who responded to conventional chemotherapy were mobilized with chemotherapy (mainly high-dose cyclophosphamide) and/or recombinant human granulocyte colony-stimulating factor (rhG-CSF). Patients who reached the threshold of 20 CD34+ cells per microl of peripheral blood underwent apheresis and were randomized at that time to receive either unmanipulated mobilized blood cells or selected CD34+ cells. For patients in the study arm, CD34+ cells were selected from aphereses using the Isolex300 device. Fifteen patients failed to mobilize peripheral blood progenitors and nine other patients were excluded for various reasons. Forty-seven eligible patients were randomized into two comparable groups. CD34+ cells were selected from aphereses in the study group. Haematopoietic recovery occurred at similar times in both groups. No side-effect related to the infusion of selected cells was observed. The frequency of epithelial tumour cells in aphereses was low (8 out of 42 evaluated patients), as determined by immunocytochemistry. We conclude that selected CD34+ cells safely support haematopoietic recovery following high-dose chemotherapy in patients with poor-prognosis breast cancer.

Breast cancers often progress from a hormone-dependent, nonmetastatic, antiestrogen-sensitive phenotype to a hormone-independent, antiestrogen- and chemotherapy-resistant phenotype with highly invasive and metastatic growth properties. This progression is usually accompanied by altered function of the estrogen receptor (ER) or outgrowth of ER-negative cancer cells. To understand the molecular mechanisms responsible for metastatic growth of ER-negative breast cancers, the activities of the transcription factor NF-kappaB (which modulates the expression of genes involved in cell proliferation, differentiation, apoptosis, and metastasis) were compared in ER-positive (MCF-7 and T47-D) and ER-negative (MDA-MB-231 and MDA-MB-435) human breast cancer cell lines. NF-kappaB, which is usually maintained in an inactive state by protein-protein interaction with inhibitor IkappaBs, was found to be constitutively active in ER-negative breast cancer cell lines. Constitutive DNA binding of NF-kappaB was also observed with extracts from ER-negative, poorly differentiated primary breast tumors. Progression of the rat mammary carcinoma cell line RM22-F5 from an ER-positive, nonmalignant phenotype (E phenotype) to an ER-negative, malignant phenotype (F phenotype) was also accompanied by constitutive activation of NF-kappaB. Analysis of individual subunits of NF-kappaB revealed that all ER-negative cell lines, including RM22-F5 cells of F phenotype, contain a unique 37-kDa protein which is antigenically related to the RelA subunit. Cell-type-specific differences in IkappaB alpha, -beta, and -gamma were also observed. In transient-transfection experiments, constitutive activity of an NF-kappaB-dependent promoter was observed in MDA-MB-231 and RM22-F5 cells of F phenotype, and this activity was efficiently repressed by cotransfected ER. Since ER inhibits the constitutive as well as inducible activation function of NF-kappaB in a dose-dependent manner, we propose that breast cancers that lack functional ER overexpress NF-kappaB-regulated genes. Furthermore, since recent data indicate that NF-kappaB protects cells from tumor necrosis factor alpha-, ionizing radiation-, and chemotherapeutic agent daunorubicin-mediated apoptosis, our results provide an explanation for chemotherapeutic resistance in ER-negative breast cancers.

Nerve-sparing retroperitoneal lymph-node dissection (RPLND) maintains the patient's ability to ejaculate postoperatively. However, since testicular cancer patients sometimes have diminished spermatogenesis, questions have been raised as to the advisability of nerve preservation relative to ultimate fertility. Fertility status was assessed in clinical stage A patients by two methods. These included standard semen analysis and a post-RPLND survey. The results show that approximately 75% of nonseminomatous testicular cancer patients who present in clinical stage A have fertility potential as based on semen analysis. Additionally, of those patients responding to the post-RPLND survey who had attempted pregnancy following RPLND, 76% reported attainment of pregnancy. Nerve-sparing RPLND maintains fertility potential in clinical stage I patients; furthermore, this fertility potential appears to be worth preserving as many patients will be capable of impregnating their partners.

Tumor samples are available from over 19,600 Stage I-III breast cancer patients treated according to evolving British Columbia guidelines from 1978 to 1990. A tissue mico-array (TMA) was constructed from 930 of these patients, all of whom participated in randomized or phase II studies. Outcome was defined as 20-year Breast Cancer specific Survival (BrCaSS), with events defined as Breast Ca death. Follow up was median 17.8 years (ranges 11–28). Multiple tumor markers were tested, and results correlated with 20-year BrCaSS for markers expressed versus non-expressed. No difference in BrCaSS was found for aromatase, integrin-linked kinase (ILK), IGF-1 and Topo-isomerase-2. The negative predictive value of IHC versus FISH and ACIS-IHC versus FISH was 96 and 97%, respectively. The positive predictive value of IHC versus FISH and ACIS-IHC versus FISH was 84 and 84%, respectively. All tests, with the exception of HER-2 FISH were done by IHC. Results of other markers (VEGF, ER/PgR, hypoxia markers, etc.), and an interactive multivariate analysis adjusting for conventional prognostic factors and for all above markers, are in progress. Conclusions 1. The TMA is a technique which provides opportunity for rapid screening of multiple genetic markers. 2. Expression of Her-2/Neu, uPA, Cox-2 and Cytokeratin 17/5,6 (but not of Aromatase, ILK, TOPO-II and IGF-1) is associated with inferior BrCaSS. 3. HER-2 determination by ACIS-IHC provides comparable results to IHC done manually (with a potential for more uniform reporting), and both provide comparable results to Her-2 assessment by FISH. **ACIS-IHC:IHC red by Automated Cell Image System (M.L.)