Successful treatment with bortezomib and dexamethasone for proliferative glomerulonephritis with monoclonal IgG deposits in multiple myeloma: a case report

Background: Proliferative glomerulonephritis with monoclonal IgG deposits (PGNMID) is a form of renal involvement by monoclonal IgG deposits that was found in mesangial, subendothelial or subepithelial regions. The distribution of glomerular deposits was completely different from that in monoclonal immunoglobulin deposition disease. PGNMID is reported to be rarely associated with a hematological malignancy. Previously, only five cases of PGNMID with multiple myeloma have been reported. However, the pathogenic relationship between PGNMID and multiple myeloma was unclear because a detailed description was not provided. We report that a patient with PGNMID associated with multiple myeloma was treated with bortezomib and dexamethasone and underwent the second renal biopsy after treatment, showing that chemotherapy was effective for PGNMID clinically and pathologically. Case presentation: A 75-year-old man presented with progressive leg edema, had nephrotic range proteinuria, hypoalbuminemia, moderate renal failure, and occult blood in his urine. Electrophoresis results showed serum and urinary monoclonal spikes of IgGκ type immunoglobulin. A renal biopsy specimen showed lobular mesangial proliferation with mesangiolysis, glomerular micro-aneurysm, and endocapillary hypercellularity. Immunofluorescence results revealed strong granular capillary and mesangial staining for IgG1, C3 and κ light chain in glomeruli without tubular deposits of any immunoglobulin. Electron microscopy also showed dense granular deposits in subendothelial and mesangial areas. PGNMID associated with multiple myeloma (IgGκ type) was diagnosed on the basis of a subsequent bone marrow examination. Bortezomib and dexamethasone therapy significantly reduced proteinuria and elevated serum albumin level. Eight months later, the second renal biopsy showed no active lesions and that the IgG1 and κ light chain deposits had drastically disappeared. Conclusions: This is the first case of PGNMID with multiple myeloma successfully treated with bortezomib and dexamethasone in which comparative renal biopsies were performed before and after treatment. Our findings suggest the pathogenesis of PGNMID and therapeutic options for PGNMID

Platelets promote tumor growth and metastasis via direct interaction between Aggrus/podoplanin and CLEC-2.

The platelet aggregation-inducing factor Aggrus, also known as podoplanin, is frequently upregulated in several types of tumors and enhances hematogenous metastasis by interacting with and activating the platelet receptor CLEC-2. Thus, Aggrus-CLEC-2 binding could be a therapeutic molecular mechanism for cancer therapy. We generated a new anti-human Aggrus monoclonal antibody, MS-1, that suppressed Aggrus-CLEC-2 binding, Aggrus-induced platelet aggregation, and Aggrus-mediated tumor metastasis. Interestingly, the MS-1 monoclonal antibody attenuated the growth of Aggrus-positive tumors in vivo. Moreover, the humanized chimeric MS-1 antibody, ChMS-1, also exhibited strong antitumor activity against Aggrus-positive lung squamous cell carcinoma xenografted into NOD-SCID mice compromising antibody-dependent cellular cytotoxic and complement-dependent cytotoxic activities. Because Aggrus knockdown suppressed platelet-induced proliferation in vitro and tumor growth of the lung squamous cell carcinoma in vivo, Aggrus may be involved in not only tumor metastasis but also tumor growth by promoting platelet-tumor interaction, platelet activation, and secretion of platelet-derived factors in vivo. Our results indicate that molecular target drugs inhibiting specific platelet-tumor interactions can be developed as antitumor drugs that suppress both metastasis and proliferation of tumors such as lung squamous cell carcinoma

Humanized chimeric ChMS-1 antibody suppressed the <i>in vivo</i> growth of Aggrus-expressing lung squamous cell carcinoma PC-10 cells.

<p>A, interaction between the human Aggrus protein prepared from mammalian cells (R&D systems) and ChMS-1 antibody was estimated by SPR analysis. <i>K</i>_<i>D</i> of ChMS-1 antibody on human Aggrus is shown. B, antimetastatic activity of ChMS-1 antibody. BALB/c-<i>nu/nu</i> mice were intravenously injected with the indicated concentrations of antibodies. After 24 h, CHO/Aggrus cells were intravenously inoculated. After 20 days of tumor inoculation, lung surface metastatic foci were counted. The number of metastatic foci are shown. Bars, mean (n=8). **<i>P</i> < 0.01 by the Mann–Whitney <i>U</i> test. C, antitumor activity of ChMS-1 antibody in NOD-SCID mice. PC-10 (left) and A549 (right) cells were subcutaneously inoculated into the backs of eight-week-old female NOD.CB17-<i>Prkdc</i>^<i>scid</i>/J mice. After 10 days of tumor inoculation, antibodies were intravenously injected into the lateral tail vein and repeated 3 more times once a week (100 µg/mouse, arrow heads). Tumor volume was calculated as described in Materials and Methods. All data are shown as means ± SD. *<i>P</i> < 0.05 by the Mann–Whitney <i>U</i> test. D, PC-10 cells were cultured in the presence of control human IgG1 or ChMS-1 (10 µg/ml). Cell viability was measured by adding the CellTiter-Glo assay reagent. Briefly, a total of 1,500 cells were seeded in 96-well plates in triplet. On the following day, cells were treated with or without antibodies and incubated for another 24, 72, or 120 hours. Cell viability was determined by adding the CellTiter-Glo assay reagent (Promega) for 10 minutes and luminescence was measured using a Centro LB 960 luminometer (Berthold Technologies). E, representative image of infiltrated platelets (stained by anti-CD41 antibody, lower panels) and Aggrus expression in tumors (stained by D2-40, upper panels). PC-10 and A549 tumors were excised after 40 days of tumor inoculation. Bar, 0.1 µm. Magnification, x20.</p

MS-1 mAb suppressed spontaneous pulmonary metastasis and tumor growth <i>in vivo</i>.

<p>A to D, CHO/Aggrus cells were xenografted in BALB/c-<i>nu/nu</i> mice. Antibodies (30 µg/mouse) were intravenously injected on the following day of tumor inoculation and repeated two more times every fourth day (arrow heads). Tumor volume was calculated as described in Materials and Methods. All data are shown as means ± SDs (n=5). **<i>P</i> < 0.01 by the Mann–Whitney <i>U</i> test (A). Representative pictures of CHO/Aggrus tumor-bearing mice on day 18 are shown (B). After 29 days of tumor inoculation, spontaneously pulmonary metastatic foci were counted. The numbers of metastatic foci are shown. Bars, mean (n = 5). **<i>P</i> < 0.01 by the Mann–Whitney <i>U</i> test (C). Representative pictures of the lungs and lung surface metastatic foci are shown (D). E, a lung squamous cell line PC-10 (left) and a lung adenocarcinoma cell line A549 (right) cells were treated with control mouse IgG (gray area) or MS-1 mAb (bold lines). Aggrus expression was detected by flow cytometry. F and G, PC-10 cells were xenografted into BALB/c-<i>nu/nu</i> mice. Antibodies were intravenously injected on 1, 4, 7, and 11 days after tumor inoculation (arrow heads). Tumor volume was calculated as described in Materials and Methods. All data are shown as means ± SDs (n=8). **<i>P</i> < 0.01 by the Mann–Whitney <i>U</i> test (F). Representative pictures of the PC-10 tumor-bearing mice on day 18 are shown (G).</p

Characterization of the established anti-Aggrus neutralizing mAb MS-1.

<p>A, cells were treated with control mouse IgG (gray area) or MS-1 mAb (bold lines). B, cells were lysed and immunoblotted with the indicated antibodies. C, the GST-tagged recombinant human ∆N20-Aggrus protein and its point mutants were expressed in <i>E. Coli</i> and immunoblotted with the indicated antibodies. GST, anti-GST antibody. D, interaction between MS-1 mAb and the human Aggrus protein (red line) or the mouse Aggrus protein (blue line) was estimated by SPR analysis. Equilibrium dissociation constants (<i>K_D</i>) of MS-1 mAb on human Aggrus are shown. The <i>K</i>_<i>D</i> of MS-1 mAb on mouse Aggrus could not be calculated (N.C.).</p

Suppression of Aggrus-induced platelet aggregation and tumor metastasis by MS-1 mAb.

<p>A, recombinant CLEC-2 protein was immobilized on an ELISA plate and then incubated with the human IgG Fc-tagged recombinant human Aggrus protein in the presence of the indicated concentrations of control mouse IgG (Mouse IgG) or MS-1 mAb. The value of PBS-treated control was normalized to 100%. Data are means ± SDs of triplicate determinations. B, CHO/Aggrus cells were incubated with 10 µg/mL of antibodies, followed by incubation with mouse PRP. Light transmittance of samples was measured as the aggregation rate. C to F, BALB/c-<i>nu/nu</i> mice were intravenously injected with the indicated concentrations of antibodies (C and D) or F(ab’)₂ fragments (E and F). CHO/Aggrus cells were intravenously inoculated on the following day of antibody administration. After 20 days of tumor inoculation, lung surface metastatic foci were counted. Numbers of metastatic foci in each mouse were shown. Bars, mean (n=8). NS, not significant. **<i>P</i> < 0.01 by the Mann–Whitney <i>U</i> test (C and E). Representative pictures of the lungs and lung surface metastatic foci are shown (D and F).</p

Identification of recognition epitope of MS-1 mAb on human Aggrus.

<p>A, cells were lysed and immunoblotted with the indicated antibodies. B, CHO cells that had been transfected with WT-, G45A- or D49A-Aggrus expression plasmids (CHO/Aggrus, CHO/Aggrus-G45A or CHO/Aggrus-D49A, respectively) were treated with control mouse IgG (0.1 µg/ml, gray area) or MS-1 mAb (0.1 µg/ml, bold lines in upper panels). In some experiments, cells were treated with an anti-human Aggrus mAb D2-40 antibody (0.1 µg/ml, bold lines in lower panels). After incubation with the Alexa Fluor 488-conjugated secondly antibody, Aggrus expression was detected by flow cytometry. C, CHO/mock, CHO/Aggrus, CHO/Aggrus-G45A, and CHO/Aggrus-D49A cells were treated with control mouse IgG (0.1 µg/ml, gray area) or mouse IgG Fc-conjugated recombinant human CLEC-2 protein (4 µg/ml, bold lines). After incubation with the Alexa Fluor 488-conjugated secondly antibody, CLEC-2 binding to Aggrus-expressing cells was confirmed by flow cytometry. D and E, BALB/c-<i>nu/nu</i> mice were intravenously injected with control mouse IgG (Mouse IgG) or MS-1 mAb (3 µg/mouse). After 24 h, cells (2.5 × 10⁵ cells/mouse) were intravenously inoculated into mice. After 20 days of tumor inoculation, lung surface metastatic foci were counted. The number of metastatic foci are shown (D). Bars, mean (n = 7 or 8). NS, not significant. **<i>P</i> < 0.01 by the Mann–Whitney <i>U</i> test. Representative pictures of the lungs and lung surface metastatic foci are shown (E).</p

Involvement of Aggrus-platelet interaction in <i>in vivo</i> tumor growth.

<p>A, establishment of Aggrus-knockdowned PC-10 cells. Aggrus expression of PC-10 transfectants was confirmed by immunoblotting with the indicated antibody. B, <i>in vitro</i> growth of PC-10 transfectants that have been stably transfected with ZsGreen-expressing plasmid. Cells were cultured in medium containing 10% FBS for the indicated times. Relative cell viability was calculated as measuring the fluorescence of ZsGreen. C, <i>in vivo</i> growth of PC-10 transfectants. PC-10 cells were subcutaneously inoculated into the backs of eight-week-old female BALB/c-<i>nu/nu</i> mice (n=3). Tumor volume was calculated as described in Materials and Methods. D, the effects of platelets on PC-10 cell growth <i>in vitro</i>. PC-10 transfectants that have been stably transfected with ZsGreen-expressing plasmid were co-cultured with washed platelets in medium containing 0.5% FBS. Relative cell viability was calculated as measuring the fluorescence of ZsGreen.</p

Prediction of ALK mutations mediating ALK-TKIs resistance and drug re-purposing to overcome the resistanceResearch in context

Background: Alectinib has shown a greater efficacy to ALK-rearranged non-small-cell lung cancers in first-line setting; however, most patients relapse due to acquired resistance, such as secondary mutations in ALK including I1171N and G1202R. Although ceritinib or lorlatinib was shown to be effective to these resistant mutants, further resistance often emerges due to ALK-compound mutations in relapse patients following the use of ceritinib or lorlatinib. However, the drug for overcoming resistance has not been established yet. Methods: We established lorlatinib-resistant cells harboring ALK-I1171N or -G1202R compound mutations by performing ENU mutagenesis screening or using an in vivo mouse model. We performed drug screening to overcome the lorlatinib-resistant ALK-compound mutations. To evaluate these resistances in silico, we developed a modified computational molecular dynamic simulation (MP-CAFEE). Findings: We identified 14 lorlatinib-resistant ALK-compound mutants, including several mutants that were recently discovered in lorlatinib-resistant patients. Some of these compound mutants were found to be sensitive to early generation ALK-TKIs and several BCR-ABL inhibitors. Using our original computational simulation, we succeeded in demonstrating a clear linear correlation between binding free energy and in vitro experimental IC50 value of several ALK-TKIs to single- or compound-mutated EML4-ALK expressing Ba/F3 cells and in recapitulating the tendency of the binding affinity reduction by double mutations found in this study. Computational simulation revealed that ALK-L1256F single mutant conferred resistance to lorlatinib but increased the sensitivity to alectinib. Interpretation: We discovered lorlatinib-resistant multiple ALK-compound mutations and an L1256F single mutation as well as the potential therapeutic strategies for these ALK mutations. Our original computational simulation to calculate the binding affinity may be applicable for predicting resistant mutations and for overcoming drug resistance in silico. Fund: This work was mainly supported by MEXT/JSPS KAKENHI Grants and AMED Grants. Keywords: ALK-rearranged lung cancer, Resistant mutation, Computational simulation, MP-CAFEE, Compound mutation, Quantum chemistr