Silvano Battaglio

Early responder myeloma: kinetic studies identify a patient subgroup characterized by very poor prognosis.

In order to assess the prognostic value of rapid tumor mass reduction in responding multiple myeloma (MM) patients, 100 consecutive patients were analyzed, and bone marrow plasma cell kinetic characteristics were evaluated at diagnosis. Forty-two patients obtained a tumor mass reduction greater than or equal to 50% with three cycles of chemotherapy and within 3 months (early responder myeloma [ERM]), and 23 in greater than 3 months (slow responder myeloma [SRM]). Survival rates in these two groups were not statistically different (P = .07). The labeling index (LI) of bone marrow plasma cells was significantly higher in ERM patients than in SRM patients (1.8 +/- 2.0 v 0.8 +/- 0.7, P = .006). The LI was used to separate the ERM patients into two well-defined subgroups. ERM patients with a LI greater than or equal to 2% showed a median survival of 16.4 months, whereas ERM patients with a LI less than 2% did not reach the median survival at 46.9 months (P less than .0044). Remission duration was also significantly different: 12.2 months in the high LI subgroup and 26.3 months in the low LI subgroup (P less than .0025). Early response itself does not correspond to shorter remission duration and shorter survival, but it is a poor prognostic factor if associated with a high plasma cell proliferative activity.

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-α 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.

Human myeloma: Several subsets of circulating lymphocytes express plasma cell-associated antigens

Peripheral blood lymphocytes from 9 monoclonal gammopathies of undetermined significance (MGUS) and 27 multiple myelomas (MM) were studied with a panel of monoclonal antibodies (MoAb) that recognize B and T lymphocytes and plasma cells. No difference in the percentage of B lymphocytes, identified by B1 and B4 MoAb, was observed in MGUS and MM patients versus normal controls. However, high percentages of circulating lymphocytes expressing plasma cell‐associated antigens were detected in MM (HAN‐PC1 + = 29.4 ± 20.4%; TEC‐T10+ = 27.8 ± 19.2%) whereas they were in the normal range in MGUS (HAN‐PC1 + = 8.8 ± 5.8% p = 0.006; TEC‐T10+ = 5.7 ± 4.7% p<0.001). Almost identical results were obtained using PCA‐1 MoAb in 17 of these patients. TEC‐T10+ and PCA‐1 + lymphocytes were sorted and re‐analyzed with phycoerythrin conjugated MoAb in 3 healthy subjects, 2 MGUS, and 4 MM patients. In normal subjects and in MGUS the majority of PCA‐1 + cells belonged to the B lineage (Leu 2‐, Leu3‐, Leu 15‐, HLA‐Dr +), whereas the majority of TEC‐T10+ cells are represented by activated T cells and NK cells (Leu 15 +). In MM an abnormal expansion of T lymphocytes was chiefly responsible for the high values of lymphocytes expressing plasma cell‐associated antigens. Moreover, in MM a clinical evaluation showed a correlation between the presence of these lymphocytes and an aggressive disease. Indeed, they can be considered a useful prognostic marker.

Recombinant interferon‐γ inhibits the in vitro proliferation of human myeloma cells

Summary. Interferon‐α (IFN‐α), interferon‐γ (IFN‐γ) 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‐γ (100 U/ml) and by DEX (10−6m). The stimulation index (StI), i.e. labelling index (LI) of treated samples/controls, was 0·49 0·009 (mean standard error of the mean, MSEM), P=0·0003, and 0·52·0·07 (MSEM), P<0·0001, respectively. Ig secretion was reduced by IFN‐α (100 U/ml) and DEX. The secretion index (SI), i.e. Ig quantitation of treated samples/controls, was 0·04 (MSEM), P<0·0001, and 0·52·0·04 (MSEM), P<0·0001, respectively. Finally, IFN‐γ inhibits BrdUrd uptake only and IFN‐α secretion only. In 18 patients the simultaneous addition of IFN‐α plus IFN‐γ mainly paralleled the effect of IFN‐γ on BrdUrd uptake and IFN‐α on secretion, but did 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‐α plus IFN‐γ and DEX are the strongest inhibitors of both BrdUrd uptake and secretion. Since IFN‐α and IFN‐γ appear to have a different mechanism of action, their combined use could be considered as a possible new treatment strategy.

Polyclonal immunoglobulin E levels are correlated with hemoglobin values and overall survival in patients with multiple myeloma.

Purpose: Polyclonal IgG, IgA, and IgM immunoglobulins are often decreased in sera of patients with multiple myeloma (MM), whereas very few data are available on polyclonal IgE levels. We have determined IgE levels in a large series of MM patients at diagnosis and subjects with monoclonal gammopathy of undetermined significance (MGUS) and correlated IgE levels with survival and prognostic factors in MM. Experimental Design: IgE were determined with a commercially available ELISA kit in 201 MM patients at diagnosis, 144 subjects with MGUS, and 77 age-matched controls. Results: IgE levels progressively decreased from controls to MGUS and from MGUS to MM (P = 0.001). MM patients with IgE levels of >11.5 IU/mL (median) had a better survival than patients with IgE of <11.5 IU/mL (P = 0.048). The difference was even more significant when MM patients were divided according to clinical cutoff values. Patients with elevated IgE levels (>100 IU/mL) had from 2 to 3 years longer survival than those with low (<10 IU/mL) or intermediate values (10-100 IU/mL; P < 0.01). IgE levels were positively and negatively correlated with hemoglobin (P = 0.006) and β2-microglobulin levels (P = 0.007), respectively. Univariate and multivariate analyses confirmed that high IgE levels are positive predictors of overall survival (P = 0.03 and 0.08, respectively) and strongly correlated with hemoglobin values. Conclusions: Because IgE levels are dependent on Th2 responses, these data open new perspectives in the interpretation of antitumor immune responses and pathogenesis of anemia in MM.

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 BarnHI, 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 μ 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.

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‐α (IFN‐α), interferon‐γ (IFN‐γ), 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‐α, similar to IFN‐γ and DEX. The combinations of cRA + IFNα, tRA + IFN‐γ, 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.

Multiple Myeloma: Beta-2-Microglobulin is not a Useful Follow-Up Parameter

Serum beta-2-microglobulin (beta 2M) has been suggested as the most powerful prognostic factor in multiple myeloma (MM). This paper investigates its ability to detect remission and relapse in individual patients. A correlation analysis was carried out between beta 2M and M component determinations, at diagnosis and monthly during follow-up in 21 consecutive MM patients with normal renal function. A statistically significant correlation was observed in 52.4% only. The lack of correlation in the remaining cases was due to low beta 2M production at diagnosis, or independent fluctuation of these 2 parameters. Serum beta 2M proved a much less reliable parameter for the detection of tumour variations than simpler M-component determination.

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 (TcRβ) 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.

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.