Immune response induced in small-cell lung cancer by maintenance therapy with interferon gamma.

BACKGROUND: Chemotherapy, with or without radiotherapy, results in a 30%-40% complete response rate in small-cell lung cancer (SCLC), but approximately 90% of patients who have complete remission die within 2 years after relapse with chemoresistant disease. Randomized clinical studies of maintenance chemotherapy after complete response have failed to demonstrate survival advantage. However, studies have shown that the human cytokine interferon gamma (IFN-gamma) induces immune response in humans, including T-cell activation and expression of class II major histocompatibility complex (HLA-DR) and receptor for the Fc portion of immunoglobulin on monocytes. It has also been demonstrated that recombinant IFN-gamma (rIFN-gamma) induces immunomodulation and has antiproliferative activity. PURPOSE: In vivo effects of rIFN-gamma treatment were characterized by flow cytometric analysis of peripheral blood mononuclear cells in patients with SCLC who received rIFN-gamma as maintenance treatment. METHODS: After induction chemotherapy and radiotherapy, 100 patients who achieved a complete remission were randomly assigned to receive rIFN-gamma at a dose of 0.2 mg (4 x 10(6) units) once a day, subcutaneously, for 6 months, or observation only. In 31 patients, peripheral mononuclear cells were obtained prior to the study and at weeks 4, 8, and 12 for serial monitoring of immune response. By flow cytometric analysis, we identified the lymphocyte and monocyte populations using characteristic differences in electronic volume and right-angle scatter. In these populations, we determined the mean fluorescence channel after staining for CD14 (antigen expressed on monocytes), CD3 (antigen expressed on T lymphocytes), and HLA-DR (HLA class II expressed by monocytes and activated lymphocytes). To determine the number of Fc receptors per cell, an Fc receptor assay was performed using the monocyte cell line U937 as a standard. RESULTS: At weeks 4, 8, and 12, expression of HLA-DR and Fc receptors on monocytes in patients who received rIFN-gamma was significantly higher than that in untreated patients, and the difference was statistically significant. The number of Fc receptors per monocyte consistently increased during the rIFN-gamma treatment and reached a fivefold elevation at week 12. There was no statistically significant difference in lymphocyte surface antigen expression between the treated and untreated groups. CONCLUSION: The dose of rIFN-gamma used in this study resulted in immune stimulation in patients with SCLC who had complete remission after induction therapy. The in vivo immunomodulatory activity of rIFN-gamma in such patients is characterized by a strong monocyte activation but no significant alteration in T-cell activation.

Control by positive and negative regulatory cells of the generation of antigen-specific suppressor T cells that inhibit cytotoxic T cell responses to alloantigens.

The induction of a cytotoxic T cell response requires interaction with T helper (TH) cells and can be suppressed by T suppressor (TS) cells. However the control mechanisms that regulate the regulatory cells themselves have not been clearly elucidated. We have designed a three-step sequential culture system to analyze the regulatory controls operating on antigen-specific Ts cells active in the inhibition of CTL responses to alloantigen. The generation of T suppressor cells requires a positive collaborative interaction with a Ts-directed helper cell and is negatively regulated by interaction with a TS-directed inhibitory T cell. In the system described, both Lyt 1+2- and Lyt 1-2+ radioresistant effector TS are observed. The suppressive effect of these is subject to negative control by a T cell inhibitor. The Lyt 1-2+ Ts have been directly shown to be strongly inhibited by Lyt 1+2+ TS-directed inhibitors. Analysis of the regulatory effects mediated by populations of cells treated with anti-Thy 1.2 or anti-Lyt antibodies indicates a very complex network of TH and T inhibitors acting to control antigen-specific TS generation. An antigen-specific immune network is proposed to explain the control of CTL responses and the regulators of CTL responses and is discussed in light of our experimental observations on the positive and negative control of the generation of TS.

Phyllodes tumor: clinicopathologic review of 60 patients and flow cytometric analysis in 30 patients.

We reviewed 66 phyllodes tumors of the breast from 60 patients. Our patients included 59 women and one man ranging in age from 16 to 72 years. Fifty patients presented for primary treatment of newly diagnosed breast masses, nine presented with recurrent tumors, and one presented with soft tissue metastases 9 years after bilateral subcutaneous mastectomies and multiple chest wall recurrences of phyllodes tumor. After 0.3 to 53.2 years (mean, 15.5 years) of follow-up, 26 (43.3%) patients are free of disease without recurrence, 26 (43.3%) patients are dead of other (17 patients) or unknown (nine patients) causes, four (6.7%) patients had locally recurrent tumor 0.7 to 2.9 years after lumpectomy and are free of disease 3 months to 12 years after re-excision or simple mastectomy, two (3.3%) patients are lost to follow-up, and two (3.3%) patients died with metastatic disease 1.8 and 7 years after diagnosis. Histologic features and flow cytometric analysis showed no correlation with outcome. Fifty-six breast tumors were biphasic and nine were purely stromal tumors. Twenty-six (47%) biphasic tumors showed stromal overgrowth. Tumor margins were pushing in 20 (39%) and infiltrative in 29 (61%) of 49 evaluable cases. Twenty-one tumors were highly cellular and 17 showed cytologic atypia. Necrosis was identified in 16 tumors. Mitotic rates ranged from 0/10 high-power fields to 48/10 high-power fields. Twenty-four diploid, six aneuploid, three tetraploid, and one polyploid tumor were identified by flow cytometry. S-phase fractions tended to be higher in nondiploid tumors. Neither DNA content nor S-phase fraction correlated with outcome. Our results indicate that most mammary phyllodes tumors, including purely stromal tumors, behave as low-grade, nonmetastasizing neoplasms. Neither histologic evaluation nor DNA content provides reliable clues concerning the natural history of an individual tumor.

Comparison of fluorescence in situ hybridization with flow cytometry and static image analysis in ploidy analysis of paraffin-embedded prostate adenocarcinoma.

Nuclear DNA ploidy has been shown to have an important prognostic association for patients with adenocarcinoma of the prostate. Flow cytometry and static image analysis are ploidy methods that have been used in prostate carcinoma. Fluorescence in situ hybridization (FISH) using chromosome-specific probes can be used to evaluate the ploidy of interphase nuclei. In this study FISH was compared with flow cytometry and static image analysis in determining ploidy in paraffin-embedded tissue from 34 prostatic adenocarcinomas. Ploidy status using FISH was determined by enumerating centromeres of two chromosomes (8 and 12) by use of directly-labeled alpha-satellite DNA probes in isolated whole nuclei obtained by the Hedley technique. All three methods identified 11 of 34 cases as diploid and 17 of 34 cases as nondiploid (82% concordance). Six cases were discordant; two cases had discrepant results by each method. Ploidy classification as determined by FISH had an 88% concordance with ploidy classification by either flow cytometry or static image analysis. In conclusion, FISH was found to be a sensitive method of ploidy analysis in isolated paraffin-embedded nuclei from prostate adenocarcinomas. When the chromosomes commonly involved in aneuploidy have been identified in prostate adenocarcinoma, FISH has the potential to provide greater sensitivity for aneuploidy detection compared with currently available methods.

Symptomatic subependymoma: a clinicopathological and flow cytometric study.

Twenty-one intracranial subependymomas were reviewed with regard to presentation, diagnosis, operative findings, and long-term follow-up data. The histopathological features were critically reviewed, and deoxyribunucleic acid analysis was performed by flow cytometry. The patients' mean age was 48.5 years (range 32 to 72 years). In 14 cases the tumor was located in the fourth ventricle, in six within a lateral ventricle, and in one in the third ventricle with extension into the lateral ventricle. Radiographic characteristics included isodensity with minimal enhancement on computerized tomography, frequent dystrophic calcification, and isointensity on T1-weighted or slight hyperintensity on T2-weighted magnetic resonance images. The predominant histological features in all cases were those of classic subependymoma. Nonetheless, pathological examination showed a minor (less than 20%) ependymoma component in five cases, significant cytological atypia in seven, mitoses in 11, endothelial prominence in four, and focal hemorrhage-associated necrosis in two. Flow cytometry revealed a diploid pattern in 12 patients, tetraploidy in two, and aneuploidy in one. Two patients died in the perioperative period. Of the remaining 19, 12 underwent gross total resection (two of whom received postoperative irradiation) and seven underwent subtotal resection (five of whom received irradiation). None of the 12 non-irradiated patients developed tumor progression or died of direct tumor-related causes. Of the seven irradiated patients, follow-up imaging studies demonstrated their tumors to be radioresponsive, particularly with doses of 5000 cGy or greater. Despite the presence of cytological atypia and mitotic activity in the majority of cases, the prognostic effects of such factors as tumor location and the extent of surgical resection outweighed those of the standard histopathological parameters. Routine postoperative irradiation is not recommended, but should be reserved for cases with a symptomatic residual or recurrent subependymomas following surgery.

Use of fluorescent in situ hybridization for deoxyribonucleic acid ploidy analysis of prostatic adenocarcinoma.

Fluorescent in situ hybridization using 2 chromosome specific centromere probes was evaluated as a method of ploidy analysis in touch preparations from 50 radical prostatectomy specimens. Tumors were classified as aneuploid by fluorescent in situ hybridization when nuclei had an abnormal copy number (aneusomic) for either chromosome centromere 8 or 12. Tetraploid tumors were defined as those with 4 copies (tetrasomic) of chromosome centromeres 8 and 12. The fluorescent in situ hybridization ploidy patterns were compared to the deoxyribonucleic acid (DNA) ploidy patterns subsequently obtained by flow cytometry on the same tissue following paraffin embedding. Concordant fluorescent in situ hybridization and flow cytometry ploidy classification was obtained in 82% of the cases (p < or = 0.0001). Of 7 aneuploid tumors 3 were identified by both methods. Trisomy 8 was detected by fluorescent in situ hybridization in 3 cases that were classified as DNA diploid (2 tumors) and DNA tetraploid (1 tumor). Conversely, flow cytometry detected aneuploidy (hypotetraploidy) in 1 tumor when the fluorescent in situ hybridization results were consistent with tetraploidy. Overall, fluorescent in situ hybridization was more sensitive in aneuploidy detection (6 of 7 cases) than flow cytometry (4 of 7). Of 19 tetraploid cases 5 had discordant fluorescent in situ hybridization and flow cytometry results. However, all 5 cases contained low levels of tetraploidy and the discrepant results were most likely due to the limits of precision of 1 or both methods. In conclusion, we demonstrated that fluorescent in situ hybridization ploidy analysis can be rapidly performed on fresh touch preparations of prostate tissue. This preliminary study demonstrates that the ploidy result determined by fluorescent in situ hybridization correlates well with that obtained by flow cytometry. More complete fluorescent in situ hybridization studies of prostate carcinoma will require additional probes for other chromosomes.

Specificity repertoire of lymphocytes from multiple myeloma patients. I. High frequency of B cells specific for idiotypic and F(ab')2-region determinants on immunoglobulin.

The specificity repertoire of B lymphocytes from 14 multiple myeloma patients has been studied using the technique of Epstein-Barr virus (EBV) transformation of peripheral blood lymphocytes (PBL) coupled with clonal analysis by limiting dilution. We find that up to 100% of the B cells from myeloma patients undergoing EBV transformation secrete IgM specific for determinants on the F(ab')2 region of autologous and/or heterologous monoclonal immunoglobulin. In normal individuals 0.02-0.73% of the transformed B cells secrete IgM specific for F(ab')2 determinants. Two patients with monoclonal gammopathy of undetermined significance had only a weak reactivity to F(ab')2 fragments. The number of anti-F(ab')2 B cells was up to 145-fold greater in patients than in normal donors. The majority of antibodies from patient clones recognized determinants shared among 3-12 different F(ab')2 fragments, whereas those originating from normal donor B cells saw determinants expressed on only one or two of the panel of test F(ab')2 fragments. There was a preference for autologous M components and a high proportion of antiidiotypic reactivity in five of eight patients so analyzed. We speculate that these findings indicate the existence of an anti-F(ab')2 immunoregulatory network mediating patient immunodeficiency network mediating patient immunodeficiency, thereby creating an abnormality that may enable the progression of multiple myeloma.