Long-term follow-up of idiotype vaccination in human myeloma as a maintenance therapy after high-dose chemotherapy.

The aim of this work was to evaluate the long-term immunological and clinical impact of idiotype (Id) vaccination in multiple myeloma (MM) patients in first remission after high-dose chemotherapy. A total of 15 patients received a series of subcutaneous (s.c.) injections of autologous Id, conjugated to keyhole limpet hemocyanin (KLH) and in association with low doses of GM-CSF. The median duration of follow-up was 110 months from diagnosis. The vaccine induced immune responses that lasted almost 2 years after the end of treatment. Antibody responses included anti-KLH IgM and IgG (90% of patients), anti-KLH IgE (30%), anti-GM-CSF IgG (20%), anti-Id IgG (20%), and anti-Id IgE (30%). Id-specific delayed type hypersensitivity skin tests were positive in 85% of tested patients. Following vaccination, a progressive recovery of T-cell receptor (TCR) diversity was observed and the loss of oligoclonality was significantly correlated with the remission duration. Although Id/KLH conjugates did not eliminate the residual tumor burden, the median progression-free survival, and overall survival were 40 and 82 months, respectively. A retrospective case-matched analysis showed similar results in patients treated with IFN-alpha alone or in association with steroids. This vaccine formulation can overcome Id-specific immune tolerance by inducing clinical responses that are worthy of further investigation.

Idiotype vaccination in human myeloma: generation of tumor-specific immune responses after high-dose chemotherapy.

Igs contain unique portions, collectively termed idiotypes (Id), that can be recognized by the immune system. Id expressed by tumor cells in B-cell malignancies can be regarded as tumor-specific antigens and a target for vaccine immunotherapy. We have started a vaccination trial in multiple myeloma (MM) using Id-specific proteins conjugated to keyhole limpet hemocyanin (KLH) as immunogens and low doses of subcutaneous granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-2 (IL-2) as immunoadjuvants. Twelve patients who had previously been treated with high-dose chemotherapy followed by peripheral blood progenitor cell (PBPC) transplantation entered this study from August 1995 to January 1998. All patients were in first remission at the time of vaccination. They received subcutaneous injections of Id vaccines and immunoadjuvants in an outpatient setting. The generation of Id-specific T-cell proliferative responses was documented in 2 patients, whereas a positive Id-specific delayed-type hypersensitivity (DTH) reaction was observed in 8 of the 10 patients studied. DTH specificity was confirmed in 1 patient by investigating the reactivity to synthetic peptides derived from the VDJ sequence of the tumor-specific Ig heavy chain. None of the patients generated soluble immune responses to Id, whereas the generation of soluble and cellular immune responses to KLH was observed in 100% and 80%, respectively. Eleven patients completed the treatment, whereas 1 patient failed to finish owing to progression of disease. Freedom from disease progression (FFDP), measured from the date of first Id/KLH injection to the date of first treatment after vaccination or last follow-up, ranged from 9 to 36 months. These data indicate that the immune competence status of MM patients is still susceptible to specific immunization after high-dose chemotherapy and PBPC transplantation. It remains to be determined whether generation of Id-specific immune responses can reduce the relapse rate of patients with minimal residual disease.

[Mixed IgM kappa-IgM lambda cryoglobulinemia with a double monoclonal serum component. A case report]. / Crioglobulinemia mista IgM cappa-IgM lambda con doppia componente monoclonale sierica. Descrizione di un caso.

The paper describes a case of mixed cryoglobulinemia with a double serum monoclonal component. The serum immunofixation revealed that a monoclonal component was IgG lambda while the other was positive for antisera anti-IgM, anti-k and anti-lambda even if the EP proved monoclonal. Resuspended cryoglobulins had the same electrophoresis pattern and the same reactivity for the antisera as the IgM serum component.

[The problem of recurrence of rhino-sinusal polyposis: pilot trial with locally administered azelastine HCL in the prevention of relapses]. / Il problema delle recidive dei polipi naso-sinusali: studio pilota sul possibile impiego per via topica dell'azelastina HCL nella prevenzione delle recidive.

Recurrent nasal polyposis is one of the most common unsolved problem in clinical rhinology. In the last few years a great number of histopathological, immunohistochemical and immunological studies on nasal polyps have been carried out by several Authors. Notwithstanding this, the aetiology of these formations still remains unknown. Many data suggest that the presence of polyps is the result of various inflammatory, allergic and pseudo-allergic processes which finally lead to the formation of the oedema constitutive of the polyp itself. In the present report a preliminary trial was carried out in order to evaluate the possibility of preventing recurrences by means of a locally administered anti-H1 receptors drug (Azelastine HCl). In the reported first phase of the study 10 allergic patients with bilateral polyposis were evaluated. Attention was given to the anti-edemigen activity of the drug, as well as to its influence on the local production of Secretory IgA, 7SIgA and albumines. Data are presented and discussed.

Retinoic acid inhibits the growth of human myeloma cells in vitro.

Retinoic acid has been shown to induce growth inhibition in a variety of cell types including human myeloma cell lines. Bone marrow plasma cells from 31 multiple myeloma (MM) patients were cultured to investigate the activity of 13-cis-retinoic acid (cRA), all-trans-retinoic acid (tRA), interferon-alpha (IFN-alpha), interferon-gamma (IFN-gamma), and dexamethasone (DEX), alone or in combination, on in vitro proliferation and immunoglobulin (Ig) secretion. Both cRA and tRA inhibited proliferation: the labelling index (LI) of treated cultures/controls, was 0.47 +/- 0.05 (mean +/- standard error mean, M +/- SEM) P < 0.0001, and 0.67 +/- 0.04 (M +/- SEM), P < 0.0001, respectively. The inhibitory effect of cRA was significantly superior to tRA (P = 0.0129) and IFN-alpha, similar to IFN-gamma and DEX. The combinations of cRA + IFN alpha, tRA + IFN-gamma, tRA + DEX did not show any synergistic effect on myeloma proliferation. In contrast, the combination cRA + DEX (0.29 +/- 0.04, M +/- SEM) markedly increased the effect of both cRA and DEX used as single agents. Ig synthesis was not significantly affected by CRA, tRA, IFN-gamma and the combination tRA + IFN-gamma. As expected, only IFN-alpha (P = 0.002) and DEX (P < 0.001) inhibited Ig production. The combinations cRA + IFN-alpha, cRA + DEX and tRA + DEX decreased Ig secretion to the same extent as IFN-alpha and DEX alone respectively. In conclusion, our data indicate that tRA and especially cRA strongly inhibited plasma cell proliferation but had no effect on Ig synthesis. The combination of cRA + DEX showed the highest degree of inhibitory activity of all cytokines, alone or in combination.

Recombinant interferon-gamma inhibits the in vitro proliferation of human myeloma cells.

Interferon-alpha (IFN-alpha), interferon-gamma (IFN-gamma) and dexamethasone (DEX) have shown anti-tumour effects in multiple myeloma (MM) cells. Bone marrow plasma cells from 39 MM patients were cultured to clarify the intensity and specific activity of each compound on bromo-deoxyuridine (BrdUrd) uptake and immunoglobulin (Ig) secretion. BrdUrd uptake was inhibited by recombinant human IFN-gamma (100 U/ml) and by DEX (10(-6) M). The stimulation index (StI), i.e. labelling index (LI) of treated samples/controls, was 0.49 +/- 0.09 (mean +/- standard error of the mean, M +/- SEM), P = 0.0003, and 0.52 +/- 0.07 (M +/- SEM), P < 0.0001, respectively. Ig secretion was reduced by IFN-alpha (100 U/ml) and DEX. The secretion index (SI), i.e. Ig quantitation of treated samples/controls, was 0.04 (M +/- SEM), P < 0.0001, and 0.52 +/- 0.04 (M +/- SEM), P < 0.0001, respectively. Finally, IFN-gamma inhibits BrdUrd uptake only and IFN-alpha secretion only. In 18 patients the simultaneous addition of IFN-alpha plus IFN-gamma mainly parallel the effect of IFN-gamma on BrdUrd uptake and IFN-alpha on secretion, but not result in any additive or synergistic effect, though both BrdUrd uptake and Ig secretion were decreased to about the same extent as with DEX. These data indicate that the combination of IFN-alpha plus IFN-gamma and DEX are the strongest inhibitors of both BrdUrd uptake and secretion. Since IFN-alpha and IFN-gamma appear to have a different mechanism of action, their combined use could be considered as a possible new treatment strategy.

Multiple independent immunoglobulin class-switch recombinations occurring within the same clone in myeloma.

Multiple myeloma (MM) is characterized by the expansion of terminally differentiated plasma cells. It is still uncertain whether the clonogenic fraction is confined to the plasma cell or pre-plasma cell compartment. We examined the immunoglobulin (Ig) rearrangement of myeloma heavily infiltrated bone marrow cells with a probe from the heavy chain J region (JH) and the BamHI, EcoRI and HindIII restriction enzymes which are appropriate for the detection of clonal VDJ recombination. In 23/39 MMs, clonal Ig gene rearrangement was detected with BamHI, EcoRI and HindIII enzymes. Unexpectedly, in 14/39 patients both BamHI and EcoRI failed to detect Ig rearrangement, whereas HindIII consistently demonstrated VDJ recombination. The 5' sites of BamHI, EcoRI and HindIII restriction fragments are precisely defined by the VDJ rearrangement. Since the 3' ends of BamHI and EcoRI restriction fragments are downstream from the switch mu region and change in size during switch recombination, the absence of rearranged bands is determined by several autonomous recombinations affecting the switch region. By contrast, the 3' ends of HindIII restriction fragments are upstream, their size does not vary during isotype switch allowing the constant detection of clonality. Accordingly, in 35% of patients the clonogenic fraction seems to originate from a pre-switch B cell. This B cell will differentiate to a mature plasma cell developing multiple independent switch recombinations, as the variable mechanism of switch recombination suggests.

Early disappearance of murine plasmocytoma stem cells in long-term bone marrow culture.

Long-term bone marrow culture (LTBMC) was evaluated as a purging procedure in the murine plasmocytoma MOPC-315s system. MOPC-315s cells injected in Balb-c mice rapidly proliferate both in marrow and spleen, where macroscopic tumor colonies develop. A linear relationship between the number of injected cells and spleen colonies was observed, consistent with the presence of 1 clonogenic myeloma stem cell out of 1800 cells. In vitro, MOPC-315s cells are easily identifiable as rosette-forming cells (RFC+) with trinitrophenil acid (TNP) coated sheep red blood cells. When bone marrow (BM) cells containing 20-40% RFC+ were seeded in LTBMC, RFC+ rapidly decreased and were no longer detectable by day 14 of culture. Clonal Ig gene rearrangement was evident at time 0, but it was no more detectable later on. In addition, cells taken at days 14 and 21 of culture were no more tumorigenic when injected in vivo. The results suggest the efficacy of the LTBMC for the in vitro elimination of myeloma cells, including the neoplastic stem cells.

[Local immunity following treatment with S-carboxymethylcysteine-lysine in tracheotomy patients]. / Immunità locale in seguito a trattamento con S-carbossimetilcisteina sale di lisina nei soggetti tracheotomizzati.

Pathological modification in secretory IgA values as well as in circadian rhythms were found in tracheotomized patients in both nasal and tracheo-bronchial secretions. The protective role of the mucosal immune system in addition to the frequency of severe infectious respiratory diseases in laryngectomized patients justifies the efforts of clinicians to prevent and treat such modifications. Mucoregulatory drugs have a peculiar role in these therapeutical attempts. Forty tracheotomized subjects with neoplastic disease were studied. Twenty received SCMC-Lys as a mucoregulatory drug. IgA 7S, 11S and albumin values in nasal and tracheo-bronchial secretions, were evaluated at surgery, 15 and then 40 days later. In accordance with clinical data, an improvement in local antibody production, damaged by tracheostomy, was found in the treated group. The influence of SCMS-Lys on SIgA production, whose evaluation was made by means of an original, highly selective and sensitive Immuno-IsoElectroFocusing procedure, seems to encourage the use of mucoregulatory drugs in laryngectomized patients.

Immunologic characteristics of fibrillary glomerulonephritis.

IgG and IgA immune complexes, mononuclear phagocytic system function, interleukin-2 (IL-2) production by peripheral blood lymphocytes (PBL), serum-soluble IL-2 receptors, tumor necrosis factor, beta 2-microglobulin and IL-1 beta, HLA-DNA polymorphisms, immuno-isoelectrofocusing, phenotype of PBL, lymphocyte cytotoxicity, activation of lymphokine-activated killer cells and natural killer cell activity were evaluated in 8 patients with tubular/fibrillary glomerulonephritis (GN). No common serologic, immunologic or immunogenetic features suggestive of plasma cell dyscrasias were found. No elements to state whether these GNs represent a new entity or just atypical forms of known GN were found.

Increased serum neopterin concentration as indicator of disease severity and poor survival in multiple myeloma.

In this study we investigated serum neopterin levels in 73 multiple myeloma (MM) patients (63 determinations at diagnosis, 58 in remission, and 35 at relapse), in 56 monoclonal gammopathies of undetermined significance (MGUS), and in 70 normal controls. Median neopterin level was 5.3 nmol/l in normal controls, 6.8 nmol/l in MGUS, and 10.7 nmol/l in MM patients. In comparison to healthy subjects, significantly higher levels were observed in MM patients (p less than 0.0001). A statistical difference was observed between MGUS and MM patients at diagnosis (p less than 0.007). Compared to diagnosis, a further increase was noticed during relapse, suggesting a correlation between neopterin and disease activity. The prognostic significance of raised neopterin levels was confirmed by a survival analysis. Median survival for patients with high values was 20 months, whereas it was 63.9 months for those with low values (log-rank test p less than 0.003). Serum neopterin concentrations also correlated to beta 2 microglobulin levels and the percentage of CD38+ circulating lymphocytes, indicating a link between neopterin and other myeloma prognostic factors.

Dual rearrangement of immunoglobulin and T-cell receptor gene in a case of T-cell hairy-cell leukemia.

We report a case of T-cell hairy-cell leukemia with a dual rearrangement of Ig- and T-cell receptor genes. The cytochemical, transmission electron microscopy, and surface antigens data (CD3+, CD8+, CD11+, HLA-DR+, CD19-, CD20-) were consistent with a T-cell hairy-cell leukemia. Molecular analysis according to Southern revealed a dual rearrangement of immunoglobulin heavy-chain (JH) and T-cell receptor beta (TcR beta) chain genes. Our findings suggest that the coexistence of JH and TcR gene rearrangements, frequently detected in acute leukemia, may also be observed in hematologic malignancies derived from more differentiated cells.

[Determination of 11S IgA in nasal secretions in the monitoring of local immunomodulating therapy]. / Il dosaggio delle IgA 11S nel secreto nasale nel monitoraggio delle terapie immunomodulanti locali.

Twenty pediatric patients with recurrent infectious diseases of the upper respiratory tract (tonsillitis, adenotonsillitis with or without involvement of the ear and/or lower respiratory tract) were studied. An immunological assay of the nasal secretum was performed at time of diagnosis and following treatment with a local immunomodulator drug, administered by spray. The 7S, 11S IgA and albumin rates were evaluated. The authors emphasize the importance of SIgA in mucose defense mechanisms as well as the need for a selective method for determining the 11S IgA level. An original method for immuno-isoelectrofocusing (IIEF) determination was employed in the present study. After treatment a significant increase in 11S IgA level was observed, especially in those patients with a significant basal SIGA deficit. The authors describe details of the technique for determination and discuss the results.

Analysis of the breakpoint cluster region in essential thrombocythemia.

Essential thrombocythemia (ET) is a myeloproliferative disorder characterized by a platelet count higher than 1000 x 10(9)/l. Bone marrow karyotype aberrations are occasionally observed. The presence of cytogenetic and molecular markers of chronic myeloid leukemia (CML) was assessed in 25 patients with the clinical features of ET. One displayed a complex translocation (9; 15; 22) (q34.1 or q34.3; q26.1; q11), and another a Philadelphia chromosome with standard translocation (9; 22) (q34; q11). Southern blot analysis revealed a rearranged breakpoint cluster region (bcr) in each case. Both patients experienced a stormy disease course without a leukemic transformation. These data indicate that the Philadelphia chromosome rarely occurs in ET and strongly influences patient outcome.

Human homologue of Moloney leukemia virus integration-4 locus (MLVI-4), located 20 kilobases 3' of the myc gene, is rearranged in multiple myelomas.

The structure of the c-myc locus and the flanking chromosomal region was investigated by Southern blot analysis of DNA from bone marrow aspirates from 42 patients with multiple myeloma. The main abnormality detected was the rearrangement of the MLVI-4 locus, 20 kilobases 3' of c-myc, which was observed in seven cases (16%). Two of these rearrangements were detected at the time of the initial diagnosis, four during treatment, and one at relapse, and their presence correlated with unresponsiveness to therapy. The MLVI-4 locus represents the human homologue of the Moloney leukemia virus integration-4 locus (Mlvi-4), a common region for provirus integration in Moloney murine leukemia virus-induced T-cell lymphomas in rodents. Provirus integration in this locus activates c-myc, and two additional genes, Mlvi-4 and Mlvi-1. The c-myc gene was rearranged in one patient; mutations involving the first exon of c-myc, frequently detected by altered restriction enzyme recognition sites in Burkitt's lymphomas, were not observed in these myelomas.

Multiple myeloma: increased circulating lymphocytes carrying plasma cell-associated antigens as an indicator of poor survival.

In multiple myeloma (MM) an increase in circulating lymphocytes expressing plasma cell-associated antigens (PCAA) has been described. Its prognostic significance was evaluated in this study. The immunologic phenotype of peripheral blood lymphocytes was analyzed with a panel of monoclonal antibodies specific for B, T, natural killer lymphocytes, and PCAA (CD38, PCA1) in 52 MM patients at diagnosis, remission, and during relapse, 18 monoclonal gammopathy of undetermined significance (MGUS), and 25 normal controls. No significant phenotypic alteration was observed in MGUS. In MM, the number of B lymphocytes was in the normal range at diagnosis and during the subsequent phases. A CD4/CD8 ratio decrease, during relapse, was due to both a CD4+ reduction and to an expansion of a subset of CD8+ activated suppressor lymphocytes. CD38+ and PCA1+ lymphocytes at diagnosis were significantly higher than in MGUS, and a further increase was observed during relapse, suggesting a correlation between PCAA expression and disease activity. The prognostic significance of increased PCAA was confirmed by a survival analysis of 32 patients evaluated at diagnosis using a CD38 cutoff of 0.45 x 10(9)/L positive lymphocytes. Median survival for patients with high values was only 14 months, whereas it was not reached at 32 months by those with low values (P less than .0007).

Multiple myeloma: altered CD4/CD8 ratio in bone marrow.

The expression of CD3, CD4 and CD8 antigens was simultaneously evaluated in peripheral blood and bone marrow lymphocytes from 22 multiple myeloma (MM) patients. The coexpression of CD11b and HLA-DR antigens was also analyzed within the CD4+ and CD8+ subpopulations in 4 MM patients. In peripheral blood the percentage of CD3+ and CD8+ cells was in the normal range, and the percentage of CD4+ was slightly reduced, leading to an altered CD4/CD8 ratio (less than 1) in only 7 patients. On the contrary, in bone marrow the percentage of CD4+ was profoundly reduced, leading to an altered CD4/CD8 ratio in all MM patients. As we previously reported in peripheral blood, an expansion of the CD11b+ and HLA-DR+ lymphocytes was observed within the CD4+ and CD8+ bone marrow T-cell subpopulations. A T-lymphocyte subset imbalance is more evident in bone marrow than in peripheral blood, and it is not due to a different lymphocyte distribution within the hematological compartments.

Paraproteinaemias and platelet aggregation: role of whole blood aggregometry.

Whole blood and optical platelet aggregation were measured in normals and in patients with paraproteinaemias; extent of aggregation was correlated with paraprotein concentrations in patients and in normals after addition of different doses of paraproteins; threshold aggregating concentrations of several agonists were also determined in whole blood and in PRP from both groups of subjects. The results indicate that patients with macromolecular monoclonal component bear a "hyperaggregable" state which can be probably ascribed also to plasma hyperviscosity and which is better detected with the impedance aggregometer.