Immunoregulatory T cell function in multiple myeloma.

Multiple myeloma is a malignancy characterized by uncontrolled monoclonal B cell differentiation and immunoglobulin production. In most instances, there is concomitant reduction in polyclonal differentiation and immunoglobulin synthesis both in vivo and in vitro. In in vitro pokeweed mitogen-induced B cell differentiation assays, proliferation and polyclonal immunoglobulin secretion optimally requires T cell help and can be inhibited both by monocytes and suppressor T cells. Helper function and monocyte-mediated suppression are relatively radio-resistant whereas T suppressor function is sensitive to 2,000 rad x-irradiation. We have examined myeloma T cell subset function in this assay using recombinations of isolated patient and normal B cells, T cells, and T cell subsets. Monocytes were removed by a carbonyl iron ingestion technique, normal and myeloma T cells were fractionated on the basis of Fc receptors for immunoglobulin (Ig) G (Tgamma) or IgM (Tmu or T non-gamma), and proliferation and IgG secretion after co-culture determined by [(3)H]thymidine incorporation and radio-immunoassay, respectively. Myeloma B cells demonstrate quantitatively and qualitatively normal blastogenic responses and are appropriately regulated by either autologous or allogeneic T helper and suppressor subsets. Despite normal proliferation, however, myeloma B cells remain deficient in subsequent differentiation and immunoglobulin secretion even when co-cultured in the absence of monocytes or suppressor T cells and the presence of normal helper cells. Myeloma T cell populations, in contrast, are entirely normal in helper capacity over a range of T:B ratios but are markedly deficient in radiosensitive and concanavalin A-induced suppressor activity. T suppressor cell dysfunction in multiple myeloma is apparently due to a deficit in the T non-gamma suppressor subset, whereas Tgamma cells, although proportionately reduced, are functionally normal. This unique T suppressor deficit reflects the heterogeneity of suppressor mechanisms in this disease and may represent a compensatory response to the monoclonal proliferation or the involvement of regulatory T cells in the pathogenesis of the malignancy.

Radioimmunoassay for myeloma idiotype.

Rabbit anti-idiotype antisera were prepared against four human myeloma proteins. These antisera demonstrated a capacity to bind the 125I-labeled autologous purified monoclonal IgG, but failed to demonstrate any binding to 125I-labeled normal IgG or to labeled myeloma IgG obtained from other myeloma patients. The anti-idiotypic antisera were used with 125I-labeled autologous myeloma IgG preparations and goat antirabbit IgG for specific radioimmunoassay with a sensitivity limit of 20 ng/ml. Little or no cross-reaction occurred between these anti-idiotypic antisera and normal IgG preparations or other myeloma IgG proteins.

Effects of systemic acidification of mice with Sarcoma 180.

The effects of dietary-induced acidosis on the growth and rates of complete regression of Sarcoma 180 in mice have been studied. The experiments here reported have demonstrated that mineral acidification of laboratory food produces a late decrease in tumor growth and significantly increases the rates of complete tumor regression. Blood acid-base studies also demonstrate the effects of these diets in altering the acid-base balance, and seemingly, this is independent of starvation and/or ketosis. The relationships of such in vivo acid-base metabolic changes to the control of tumor metabolism are briefly discussed. A therapeutic potential for this preliminary approach is considered.

High terminal deoxynucleotidyl transferase activity in acute myelogenous leukemia.

Although leukocytes from all 13 acute lymphoblastic leukemia patients examined had high terminal deoxynucleotidyl transferase (terminal transferase) activity (20 to 100 units/mg of cellular DNA, where 1 unit equals 1 nmole of nucleotide polymerized in 1 hr) and those from 21 acute myelocytic leukemia patients had low terminal transferase activity (0.2 to 2 units/mg of cellular DNA), the bone marrow and peripheral blood leukocytes from 2 patients with acute myelocytic leukemia, diagnosed on the basis of clinical features and the morphology, cytochemistry, and cytogenetics of the leukemic cells, had terminal transferase activity (39 to 52 units/mg of cellular DNA) equivalent to that found in leukemic lymphoblasts. These results bring under question the specificity of high terminal transferase activity outside of the thymus as a marker for leukemic lymphoblasts and, secondarily, the derivation of acute lymphoblastic leukemia cells in all cases from thymocytes. Perhaps malignant transformation in a pleuripotent stem cell with derepression of the genome for terminal transferase could account for high terminal transferase activity observed in certain leukemic cells.

The effects of postinduction intensification treatment with cytarabine and daunorubicin in adult acute lymphocytic leukemia: a prospective randomized clinical trial by Cancer and Leukemia Group B.

Cancer and Leukemia Group B undertook a randomized trial of intensification treatment in adults aged 15 to 79 years with acute lymphocytic leukemia (ALL) in complete remission (CR). Daunorubicin (DNR), prednisone, vincristine (VCR), intrathecal (IT) methotrexate (MTX), and asparaginase produced 177 CRs in 277 patients. One hundred fifty-one patients were randomly assigned to receive treatment as follows: 74 received intensive cytarabine and DNR, and 77 received cycles of mercaptopurine (6-MP) and MTX, followed by 6MP, MTX, VCR, and prednisone for 3 years in all. One hundred twelve patients received CNS prophylaxis. Intensification produced major myelosuppression but did not improve remission duration (median, 21 months). Of the 151 patients with CRs who entered the intensification phase, 29% remain in continuous CR (43 to 117 months); in 19 patients, CRs have lasted for longer than 7 years. No relapses occurred after 60 months. Median survival from the time of randomization was 30 months. Those under 30 years of age responded more frequently, with longer CR and survival. While 53% of those aged 15 to 19 years remain in continuous CR, 92% of patients over 59 years have relapsed. The presence of a myeloid antigen on the leukemic cells was adversely prognostic for CR achievement and for survival. Pretreatment WBC and platelet levels independently affected CR duration and survival. Early M1 marrow development presaged longer remissions. CNS relapse occurred in 47 of 256 patients with normal CSF before treatment, in 29 before CNS prophylaxis. CNS disease occurred after CNS prophylaxis in 18 patients: 13 of 61 who had received standard premaintenance and five of 51 who received intensification. No advantage in CR duration or survival resulted from intensive treatment with DNR and cytarabine following induction of CR.

Cytogenetic studies in 77 patients with chronic lymphocytic leukemia: correlations with clinical, immunologic, and phenotypic data.

Cytogenetic analyses by G-banding and/or Q-banding techniques of polyclonal B cell mitogen-stimulated peripheral blood lymphocytes in 77 patients with chronic lymphocytic leukemia were carried out in the present study. Adequate metaphases were obtained in 65 patients (84%). Of 29 patients with abnormal karyotypes, ten (34%) had trisomy 12 as the sole abnormality, eight (28%) had trisomy 12 in combination with other karyotypic changes, and the remaining 11 had various karyotypic changes other than trisomy 12. There was a significant relationship between the abnormal karyotype and disease status, clinical stage, lymphocyte count, bone marrow infiltration pattern, monoclonal IgM gammopathy, and urinary monoclonal-free light chain status. Six of seven patients (87%) with trisomy 12 only had stage 0-11 disease, whereas all eight patients with trisomy 12 with other changes had stage III or IV disease (P less than .02). However, of nine patients with other karyotypic changes without trisomy 12, five had stage 0-II and four had stage III or IV disease. These observations suggest that trisomy 12 may be the primary or the earliest karyotypic change in a majority of aneuploid patients with chronic lymphocytic leukemia, and that other karyotypic changes in addition to trisomy 12 may develop as a result of clonal evolution, dedifferentiation, or therapy. Of nine patients in whom autopsy studies were carried out, four were found to have diffuse histiocytic lymphoma or Richter's syndrome (three with trisomy 12 in combination with other chromosome changes and one with normal karyotype). Our findings clearly demonstrate that cytogenetic study may be of value in the clinical and prognostic evaluation of patients with chronic lymphocytic leukemia.

Splenectomy in advanced chronic lymphocytic leukemia.

Between February 1972 and February 1989, splenectomies were performed in 30 patients with chronic lymphocytic leukemia (CLL) and three with prolymphocytic leukemia (PLL) at our institution. Indications for splenectomy included anemia and/or thrombocytopenia (hypersplenism) in 31 patients and symptomatic splenomegaly in two patients. Median time from the diagnosis of CLL to splenectomy was 25 months. Twenty (87%) of the 23 patients splenectomized for thrombocytopenia with or without anemia had platelet increments of greater than or equal to 50 x 10(9)/liter. Hemoglobin increments of greater than or equal to 3 gm/dl were noted in 12 (71%) of 17 patients splenectomized for anemia with or without thrombocytopenia. The median duration of platelet response was 18 months for 19 evaluable patients, and the median duration of hemoglobin response was 62 months for 10 evaluable patients. Median survival time from splenectomy was 36 months. Median survival from diagnosis was 103 months for 10 patients with stage III or IV disease at diagnosis and 79 months for 10 patients with stage II. A prospective study of the effect of splenectomy in a larger number of patients with advanced CLL should be considered.

Phase III trial of brief intensive treatment of adult acute lymphocytic leukemia comparing daunorubicin and mitoxantrone: a CALGB Study.

This paper reports a study of the Cancer and Leukemia Group B (CALGB) comparing daunorubicin (DNR) or mitoxantrone (DHAD) in induction followed by multidrug intensification over 8 months in adult patients with acute lymphocytic leukemia (ALL). A total of 164 newly diagnosed patients were randomly assigned to either DNR or DHAD plus vincristine, prednisone and methotrexate given intravenously (i.v.) and interthecally (i.t.). Patients received four more intensification courses of chemotherapy and then all therapy was stopped. Central nervous system (CNS) prophylaxis consisted of nine infusions of intermediate dose methotrexate (MTX) and intrathecal MTX. DHAD and DNR were equally effective in producing complete remissions (63 and 65%, respectively). The estimated median remission duration is 10.2 and 12.3 months for the DHAD and DNR arms, respectively (p = 0.56). This study was stopped earlier than planned when it became apparent that remission duration for both arms was shorter than seen in our prior study in which all patients received more than 1 year of maintenance therapy. The estimated median survival is 18.3 and 20.6 months for the DHAD and DNR arms, respectively (p = 0.90). Younger patients and patients with a pre-treatment white blood count of less than 30,000/microliters had a significantly longer remission duration and survival. Eleven per cent of patients who achieved a complete remission have had a CNS relapse to date, which is not different from the rate in our prior study using cranial irradiation and i.t. MTX, implying that intermediate dose MTX with i.t. MTX may be as effective as cranial irradiation and i.t. MTX. This study suggests that some form of maintenance chemotherapy is required for the eradication of residual leukemia cells.

Staphylococcal bacteremia in a "Germ-free" unit.

The rate of carriage and infections due to strains of Staphylococcus aureus were evaluated in adults with acute leukemia in isolators characterized by laminar air flow and barrier isolation. Patients were randomly given antimicrobial prophylaxis with oral nonabsorbed antibiotics and a nasal antibiotic ointment. In four years S aureus was isolated from the nostrils or other sites in 36 patients. Persistent isolation was noted in 24 patients. Suppression of gut flora was associated with a higher carriage rate of S aureus. Five episodes of bacteremia due to S aureus occurred at the nadir of leukopenia induced by chemotherapy. Death occurred within five days in the three patients whose peripheral white blood cell count did not rise. Patient isolation and suppression of gut flora helped reduce infections due to Pseudomonas sp and fungi, but S aureus emerged as a life-threatening pathogen.

Primary chemotherapy for localized non-Hodgkin's lymphomas with diffuse histologic characteristics. Preliminary report of a prospective study.

Thirty-three patients with diffuse non-Hodgkin's lymphoma (stages I and II) received intermediate doses of oral methotrexate followed by leucovorin calcium every four weeks, on days 1 and 8, followed on day 15 by intravenous cyclophosphamide and vincristine sulfate. Prednisone was given for four weeks on alternate courses of treatment. A total of six such four-week courses was planned. Involved-field radiation (3000 or 3600 rad [30 or 36 Gy]) was given between three courses of chemotherapy to 18 patients who presented with tumors exceeding 7 cm in greatest diameter and who had responded to the initial chemotherapy. On completion of treatment, 27 patients (82%) were in complete remission; all the failures were in patients with large intra-abdominal masses. The presence of high lactate dehydrogenase levels, large tumor size, and age over 60 years had a suggestive negative correlation with the achievement of complete remission. The median follow-up was 26 months (range, ten to 59 months). At 48 months, the actuarial disease-free survival, remission duration, and overall survival were 53%, 72%, and 68% respectively. No deaths from toxic effects and no septic episodes were observed during treatment. The complete remission rate achieved with this program is comparable with those of other intensive programs of treatment reported previously.

Prognostic significance of karyotypic abnormalities in B cell chronic lymphocytic leukemia: an update.

Cytogenetic analyses by G-banding and/or Q-banding techniques of leukemic B cells were performed in 102 patients with chronic lymphocytic leukemia (CLL), including six with prolymphocytic leukemia (PLL), one with hairy cell leukemia (HCL), and one with Waldenstrom's macroglobulinemia (WM) from 1979 through 1983. Follow-up after cytogenetic study ranged from 24 to 70 months. Seventeen patients had stage 0, 10 had stage I, 31 had stage II, and 44 had stage III or IV. Adequate metaphases were obtained for karyotypic analysis in 86 (84%) of 102 patients. Of these 86 patients with adequate metaphases, 43 had normal karyotypes (50%) and 43 had abnormal karyotypes (50%), of which trisomy 12 was the most frequent. Ten patients had trisomy 12 as the sole abnormality, 14 had trisomy 12 in combination with other abnormalities, and the remaining 19 had other abnormalities without trisomy 12. Abnormal karyotypes were more frequently associated with patients with advanced stages than those with early stages of the disease. Response rate to chemotherapy was significantly higher in patients with normal karyotypes than in those with abnormal karyotypes. Of eight patients who subsequently developed Richter's syndrome, seven initially had complex karyotypic changes with or without trisomy 12. These observations suggest that the chances of development of Richter's syndrome in CLL patients with multiple chromosome changes may be much higher than in those with either simple trisomy 12 or a normal karyotype. Mean frequency of abnormal metaphases was significantly higher in patients with complex trisomy 12 in combination with other changes than in those with trisomy 12 as the sole abnormality.(ABSTRACT TRUNCATED AT 250 WORDS)

Staging laparotomy and splenectomy in early Hodgkin's disease. No therapeutic benefit.

In a prospective randomized study of treatment for early-stage Hodgkin's disease presenting above the diaphragm, 76 patients had staging by laparotomy (Group I) and 28 had staging by closed techniques (Group II). Treatment consisted of involved-field radiotherapy alone (44 patients), involved-field radiotherapy followed by chemotherapy (38 patients), total nodal radiotherapy alone (15 patients), or total nodal radiotherapy followed by chemotherapy (seven patients). On presentation, both groups had similar clinical features and similar treatment distribution. With similar follow-up (87 months), no significant differences in remission or survival were observed between Groups I and II: remission 59 versus 68 percent; survival 74 versus 92 percent; p value 0.27 and 0.09, respectively. Multiple areas of relapse were more frequently observed in Group I (11 of 32 had relapse) as compared with Group II (none of nine had relapse, p less than 0.082). In Group I, relapse in the abdomen was observed as an isolated event or as part of disseminated relapse in 12 percent of patients compared with 3 percent (one patient) in Group II with abdominal relapse alone. Seven patients in Group I and two patients in Group II died with Hodgkin's disease. Six other patients in Group I died with complete remission of non-Hodgkin's lymphoma (one patient), leukoencephalopathy (one patient), sepsis during chemotherapy (two patients), myocardial infarction (one patient), and cerebrovascular accident (one patient). Three other patients in this group had other secondary malignancies successfully controlled (histiocytic lymphoma, squamous cell carcinoma of the cervix, and malignant schwannoma). No second primary lesions or death with complete remission were observed in Group II. Staging laparotomy with splenectomy in early-stage Hodgkin's disease did not improve the duration of remission or survival or decrease the number of abdominal relapses compared with closed staging.

In vitro and in vivo cytotoxic activity of anti-human leukemia monoclonal antibodies SN5c and SN6 daunorubicin conjugates.

Murine monoclonal antibodies SN5c specific for the common acute lymphoblastic leukemia antigen (CALLA) and SN6 specific for a novel GP160 tumor associated antigen expressed on non-T ALL and myelomonocytic leukemia cells were conjugated to daunorubicin via an intermediate dextran carrier. The resulting monoclonal antibody-daunorubicin conjugates retained the immunoreactivity of the unlabeled antibody to antigen positive leukemia target cells. In addition, these conjugates demonstrated selective cytotoxic activity when tested against a panel of human leukemia cell lines and/or human leukemia patient samples of peripheral blood or bone marrow origin. The SN5c and SN6-daunorubicin immunoconjugates were superior to a non-specific isotype matched MOPC-daunorubicin conjugate in in vitro cytotoxicity assays. Free daunorubicin, however, was more cytotoxic than either immunoconjugate but lacked selectivity. SN5c-daunorubicin and SN6-daunorubicin combined were as effective as free daunorubicin when used for in vivo therapy and led to complete ablation of established NALM-6 tumors in an athymic nude mouse model. The SN5c-daunorubicin conjugate was also shown to be significantly less toxic than free daunorubicin in non-tumor bearing Balb/c mice. These studies indicate that mAb-daunorubicin conjugates can be constructed which retain specific binding and exhibit selective cytotoxicity against human leukemia cells and suggest that they may have therapeutic applications.

Glycol methacrylate embedding for light microscopy. I. enzyme histochemistry on semithin sections of undecalcified marrow cores.

A simple, routine procedure for water miscible glycol methacrylate (GMA) embedding of undecalcified bone marrow cores, which preserves the activity of enzymes useful in diagnosing various haematopoietic disorders, is described. The GMA used in this study has a low acid content that eliminates background staining, and the modified May-Grünwald-Giemsa stain provides good definition and excellent colour differentiation of various haematopoietic cells in the bone marrow, thereby providing optimal conditions for the study of the morphology and enzyme activity of bone marrow cells in the same preparation. The method is simple, reproducible, requires no expensive equipment, and is suitable for routine processing of small bone marrow cores in any histopathology or haematology laboratory.

Plastic embedded core biopsy: a complementary approach to bone marrow aspiration for diagnosing acute myeloid leukaemia.

Bone marrow aspirates and biopsy specimens were taken at diagnosis from 51 patients with acute myeloid leukaemia (AML). The diagnosis was based on morphological and cytochemical analyses, and the leukaemias were classified by FAB criteria. A considerable difference was observed between the results of bone marrow aspirates and the findings of plastic-embedded bone marrow biopsy specimens, particularly in marrow cellularity, extent of blast cell infiltration, and cell type involved in the leukaemic process. The myelomonocytic cell type seemed to predominate in the sections. In four cases there was considerable marrow infiltration with maturing, but dysplastic, granulocytic cells in the sections, but not in the aspirate smears. Features of potential prognostic importance, such as bone marrow infiltration with inflammatory cells, were easily recognised and quantified in the sections. These results indicate that plastic embedded bone marrow biopsy sections complement the findings of bone marrow aspiration in the diagnosis of AML and may also provide information of independent prognostic importance that cannot be obtained by other means.

Glycol methacrylate (GMA) embedding for light microscopy. II. Immunohistochemical analysis of semithin sections of undecalcified marrow cores.

A routine method allows bone marrow biopsy specimens to be embedded in glycol methacrylate (GMA), a water miscible plastic, and to benefit from the advantages of good morphology with immunoperoxidase detection of a wide range of cellular antigens useful in diagnosing and classifying various haematopoietic disorders. Marrow cores were fixed in cold Bouin's solution, rinsed in cold phosphate buffer, dehydrated in cold methanol, infiltrated and embedded in cold GMA, then polymerised at 4 degrees C. Sections were cut at 2 micron thickness with a Tungsten carbide knife in a Jung's high performance microtome (Autocut). Antigenecity was preserved when drying slides at room temperature but pronase digestion was necessary to re-expose the antigens in bone marrow biopsy sections embedded in GMA. Histostik, a new adhesive, was used to coat the glass slides to prevent section loss during enzyme digestion and immunostaining procedures. This method of adapting plastic embedding to undecalcified marrow cores preserves marrow architecture and cellular details and it can serve as a useful adjunct to analyse the bone marrow from patients with myeloproliferative and lymphoproliferative disorders. This technique may also be applicable in non-haematological malignant conditions which affect the marrow.

Clonal chromosome abnormalities in prison-acquired lymphoproliferative syndrome.

Lymph nodes from five male patients with prison-acquired lymphoproliferative syndrome (PALS), which seems to be a prodrome of acquired immune deficiency syndrome (AIDS), were examined cytogenetically. Two had clonal chromosome abnormalities, i.e., 11q- and -11, and another had multiple nonclonal chromosome changes, including t(2p-;3q+),6q-,+12,14q+. These chromosome changes are also common in malignant lymphoma and suggest that the patients with PALS may be predisposed to develop malignant lymphoma.

Cytogenetic studies in hairy cell leukemia.

Cytogenetic analyses were performed on cells from 17 patients with hairy cell leukemia stimulated with polyclonal B-cell activators (in 155 different cultures). No mitosis was obtained in samples from four cases (23.5%). Of 14 bone marrows, four (28.6%) showed mitoses, two with clonal abnormalities. All four samples from the spleen had mitoses with four clonal changes; eight of 13 (37.5%) blood samples had mitoses with three clonal changes. Of the polyclonal B-cell activators (PBA), lipopolysaccharide and protein A seemed to be effective for the detection of clonal abnormalities in hairy cell leukemia. Among the clonal aberrations, chromosomes #3, #10, and #17 were affected in two cases each; frequent numerical changes were monosomies of #10 and #17 and structural changes were deletions at band 3p21 (two cases), 6q-, and der(9)t(9;?)(p22;?). The chromosomal bands involved in structural changes were close to accepted constitutive fragile sites.

Cytogenetic studies of a diffuse mixed cell lymphoma of T cell origin.

The chromosome finding obtained from a lymph node of a patient with T cell lymphoma is described. Two different abnormal clones were found; one of them had a t(14;18)(q32;q21), along with other structural and numerical abnormalities, including 1p+q-,1p-,2p+q+, der(11),t(11;?)(q13;?),+20,+21,22p+. The other clone contained der(13),t(13;?)(q22;?).

Primary prostatic involvement in non-Hodgkin lymphoma.

We report 3 cases of primary extranodal lymphoma of the prostate, an unusual extranodal presentation rarely diagnosed antemortem. Symptoms of prostatism associated with an enlarged hard prostate with pyuria and hematuria in younger patients should suggest the diagnosis. Urine cytologic examination should aid in the diagnosis of this condition.