Panel sequencing for clinically oriented variant screening and copy number detection in 142 untreated multiple myeloma patients.

We employed a customized Multiple Myeloma (MM)-specific Mutation Panel (M(3)P) to screen a homogenous cohort of 142 untreated MM patients for relevant mutations in a selection of disease-specific genes. M(3)Pv2.0 includes 77 genes selected for being either actionable targets, potentially related to drug-response or part of known key pathways in MM biology. We identified mutations in potentially actionable genes in 49% of patients and provided prognostic evidence of STAT3 mutations. This panel may serve as a practical alternative to more comprehensive sequencing approaches, providing genomic information in a timely and cost-effective manner, thus allowing clinically oriented variant screening in MM.

Longitudinal analysis of 25 sequential sample-pairs using a custom multiple myeloma mutation sequencing panel (M(3)P).

Recent advances in genomic sequencing technologies now allow results from deep next-generation sequencing to be obtained within clinically meaningful timeframes, making this an attractive approach to better guide personalized treatment strategies. No multiple myeloma-specific gene panel has been established so far; we therefore designed a 47-gene-targeting gene panel, containing 39 genes known to be mutated in ≥3 % of multiple myeloma cases and eight genes in pathways therapeutically targeted in multiple myeloma (MM). We performed targeted sequencing on tumor/germline DNA of 25 MM patients in which we also had a sequential sample post treatment. Mutation analysis revealed KRAS as the most commonly mutated gene (36 % in each time point), followed by NRAS (20 and 16 %), TP53 (16 and 16 %), DIS3 (16 and 16 %), FAM46C (12 and 16 %), and SP140 (12 and 12 %). We successfully tracked clonal evolution and identified mutation acquisition and/or loss in FAM46C, FAT1, KRAS, NRAS, SPEN, PRDM1, NEB, and TP53 as well as two mutations in XBP1, a gene associated with bortezomib resistance. Thus, we present the first longitudinal analysis of a MM-specific targeted sequencing gene panel that can be used for individual tumor characterization and for tracking clonal evolution over time.

BCL-2 family proteins as 5-Azacytidine-sensitizing targets and determinants of response in myeloid malignancies.

Synergistic molecular vulnerabilities enhancing hypomethylating agents in myeloid malignancies have remained elusive. RNA-interference drug modifier screens identified antiapoptotic BCL-2 family members as potent 5-Azacytidine-sensitizing targets. In further dissecting BCL-XL, BCL-2 and MCL-1 contribution to 5-Azacytidine activity, siRNA silencing of BCL-XL and MCL-1, but not BCL-2, exhibited variable synergy with 5-Azacytidine in vitro. The BCL-XL, BCL-2 and BCL-w inhibitor ABT-737 sensitized most cell lines more potently compared with the selective BCL-2 inhibitor ABT-199, which synergized with 5-Azacytidine mostly at higher doses. Ex vivo, ABT-737 enhanced 5-Azacytidine activity across primary AML, MDS and MPN specimens. Protein levels of BCL-XL, BCL-2 and MCL-1 in 577 AML patient samples showed overlapping expression across AML FAB subtypes and heterogeneous expression within subtypes, further supporting a concept of dual/multiple BCL-2 family member targeting consistent with RNAi and pharmacologic results. Consequently, silencing of MCL-1 and BCL-XL increased the activity of ABT-199. Functional interrogation of BCL-2 family proteins by BH3 profiling performed on patient samples significantly discriminated clinical response versus resistance to 5-Azacytidine-based therapies. On the basis of these results, we propose a clinical trial of navitoclax (clinical-grade ABT-737) combined with 5-Azacytidine in myeloid malignancies, as well as to prospectively validate BH3 profiling in predicting 5-Azacytidine response.

Prognostic value of chromosome 1q21 gain by fluorescent in situ hybridization and increase CKS1B expression in myeloma.

A specific role for increased level of expression of CKS1B, as a consequence of chromosome 1q21 copy number gain, has been postulated as both pathogenic, as well as a powerful clinical prognostic factor in multiple myeloma (MM). The purpose of this study is to determine the clinical associations and prognostic impact of copy number gain at chromosome 1q21 (with a bacteria artificial chromosome clone containing CKS1B) and CKS1B gene level of expression in MM. We studied the chromosome region 1q21 for copy number change in a cohort of myeloma patients treated by high-dose therapy with stem-cell rescue (HDT) (n = 159). A separate cohort of patients, treated by HDT was studied for CKS1B messenger RNA expression by gene expression profiling (n = 67). 1q21 gain was then correlated with clinical parameters and survival. Gain of 1q21 copy number was detected in about a third of MM and was associated with more proliferative disease and poor-risk cytogenetic categories such as t(4;14), and chromosome 13 deletion. Both 1q21 gain and increase gene expression level were significantly associated with reduced survival. However, neither is an independent prognostic marker in MM on multivariate Cox proportional hazard analysis.

Prognostic factors for hyperdiploid-myeloma: effects of chromosome 13 deletions and IgH translocations.

Chromosomal hyperdiploidy is the defining genetic signature in 40-50% of myeloma (MM) patients. We characterize hyperdiploid-MM (H-MM) in terms of its clinical and prognostic features in a cohort of 220 H-MM patients entered into clinical trials. Hyperdiploid-myeloma is associated with male sex, kappa immunoglobulin subtype, symptomatic bone disease and better survival compared to nonhyperdiploid-MM (median overall survival 48 vs 35 months, log-rank P = 0.023), despite similar response to treatment. Among 108 H-MM cases with FISH studies for common genetic abnormalities, survival is negatively affected by the existence of immunoglobulin heavy chain (IgH) translocations, especially those involving unknown partners, while the presence of chromosome 13 deletion by FISH did not significantly affect survival (median overall survival 50 vs 47 months, log-rank P = 0.47). Hyperdiploid-myeloma is therefore a unique genetic subtype of MM associated with improved outcome with distinct clinical features. The existence of IgH translocations but not chromosome 13 deletion by FISH negatively impacts survival and may allow further risk stratification of this population of MM patients.

Ploidy status rarely changes in myeloma patients at disease progression.

Hyperdiploid and non-hyperdiploid multiple myeloma represents distinct biological entities characterized by different patterns of genetic changes. We sought to determine whether ploidy category (non-hyperdiploid versus hyperdiploid) remains stable over time from diagnosis to progression. Of the 43 patients studied (39 by flow cytometry DNA index and 4 by a FISH-based index), only five (12%) altered their ploidy status at progression. In three of these patients, the change may possibly be attributable to technical artifacts because of the low absolute change in DNA index. For those who retain their ploidy subtypes, the DNA index change minimally (3.75+/-4.87%). It would appear that the initiating genetic events underlying hyperdiploid and non-hyperdiploid MM that marks them out as distinct entities continue to dominate and persist during disease evolution and progression.

Systemic therapy of myeloma xenografts by an attenuated measles virus.

Conditionally replicating viruses are promising agents for the treatment of malignancy. Here it is shown that the live attenuated Edmonston-B vaccine strain of measles virus (MV-Edm) replicates selectively in human myeloma cells and has potent antitumor activity. In vitro, replication of MV-Edm was restricted in phytohemagglutinin (PHA)-stimulated peripheral blood lymphocytes (PBLs) but proceeded efficiently in a panel of 6 myeloma cell lines-ARH-77, RPMI 8226, JJN-3, MM1, KAS-6/1, and KMS-11-and in primary myeloma cells isolated by CD138 sorting from the bone marrow aspirates of 6 patients. MV-Edm infection induced potent cytopathic effects in these myeloma cells, resulting in the formation of multinucleated syncytia that eventually became nonviable. In contrast, syncytial formation in PHA-stimulated PBLs was minimal after MV-Edm infection. In vivo, MV-Edm was antitumorigenic and inhibited the establishment of myeloma cells as xenografts in immunocompromised mice. When injected directly into ARH-77 myeloma xenografts in the mice, MV-Edm caused complete regression of these xenografts. MV-Edm administered intravenously into the tail veins of mice also showed significant antineoplastic activity against established RPMI 8226 and ARH-77 xenografts. In particular, the ARH-77 myeloma xenografts were exquisitely sensitive to MV-Edm therapy, and tumors in all mice regressed completely. In light of its selectivity for myeloma cells and its potent antineoplastic activity against myeloma xenografts in vivo, MV-Edm merits further development for the treatment of multiple myeloma.

Translocations involving the immunoglobulin heavy-chain locus are possible early genetic events in patients with primary systemic amyloidosis.

Primary systemic amyloidosis (AL) is a plasma cell (PC) dyscrasia with clinical similarities to multiple myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS), but its molecular basis is poorly understood. Translocations at the immunoglobulin heavy-chain (IgH) locus, 14q32, are likely early genetic events in both MM and MGUS and involve several nonrandom, recurrent, partner chromosomes such as 11q13, 16q23, and 4p16.3. Given the similarities between MM, MGUS, and AL, bone marrow clonal PCs were evaluated in 29 patients with AL using interphase fluorescence in situ hybridization (FISH) combined with immunofluorescence detection of the cytoplasmic light-chain (cIg-FISH) for the presence of 14q32 translocations and the t(11;14)(q13;q32). Of 29 patients studied, 21 (72.4%) showed results compatible with the presence of a 14q32 translocation, and 16 (76.2%) of those had translocation (11;14)(q13;q32) for an overall prevalence of the abnormality of 55%. IgH translocations are common in AL, especially the t(11;14)(q13;q32).

Fish demonstrates treatment-related chromosome damage in myeloid but not plasma cells in primary systemic amyloidosis.

Conventional cytogenetic analysis is limited in the evaluation of plasma cell disorders because, relative to normal hematopoietic elements, plasma cells divide slowly. Moreover, it is difficult to know whether abnormal metaphases originate from malignant plasma cells or myeloid cells harboring other abnormalities. We studied a patient with primary systemic amyloidosis who had previously been treated with an alkylating agent. Bone marrow cells were analyzed by cytoplasmic-immunoglobulin fluorescent staining combined with fluorescent in situ hybridization (cIg-FISH). Both chromosome enumeration probes for chromosome 1 and 7 and loci-specific probes for the short and long arm of chromosome 7 were used. Cytogenetic analysis disclosed the following abnormality: +der(1;7)(q10;p10). On cIg-FISH, the myeloid cells had fusion signals between chromosome enumeration probes for chromosomes 1 and 7, whereas plasma cells had the normal appearance of two pairs of signals. There was a second clone of abnormal myeloid cells with monosomy of chromosome 7. The bone marrow did not show any evidence of myelodysplasia. Interphase cIg-FISH is a useful technique for assigning the lineage of chromosomal abnormalities in plasma cell disorders.

Clinical significance of the translocation (11;14)(q13;q32) in multiple myeloma.

The most common chromosomal translocation in multiple myeloma (MM) is t(11;14)(q13;q32). Here, we describe the clinical characteristics of patients with MM who have this translocation. We have identified 24 patients at our institution who had t(11;14)(q13;q32) as determined by standard cytogenetic analysis (CC). Seven patients had the translocation detected at the time of original diagnosis and 17 at the time of relapse. Median survival in all patients after original diagnosis was 43 months; median survival after the translocation was detected was 11.9 months. Four patients had a clinical diagnosis of plasma cell leukemia. Most patients had an elevated beta2-microglobulin (13/20 had >4 microg/ml). The bone marrow (BM) labeling index (LI) of patients, at the time of translocation detection, was elevated in most (median 1.4%, 17/23 patients had BMLI > or = 1%). Of the 24 patients, 19 (79%) died of disease progression and 5 (21%) were alive with disease at last follow-up. Lytic lesions, bone pain, or compression fractures eventually developed in all patients. Patients with MM who have t(11;14)(q13;q32) detected by standard cytogenetics seem to have an aggressive clinical course.

Comparison of interleukin-1 beta expression by in situ hybridization in monoclonal gammopathy of undetermined significance and multiple myeloma.

We investigated whether interleukin-1beta (IL-1beta) is differentially expressed in plasma cells from monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM) patients because IL-1beta appears to play a major role in the development of lytic bone lesions, the major clinical feature distinguishing MGUS from myeloma. In situ hybridization (ISH) for IL-1beta was performed using bone marrow aspirates from 51 MM, 7 smoldering MM, 21 MGUS, and 5 normal control samples. Using the ISH technique IL-1beta mRNA was detectable in the plasma cells from 49 of 51 patients with active myeloma and 7 of 7 patients with smoldering myeloma. In contrast, 5 of 21 patients with MGUS and 0 of 5 normal controls had detectable IL-1beta message. Bone lesions were present in 40 of the 51 MM patients analyzed, and all 40 patients had IL-1beta mRNA by ISH. These results show that greater than 95% of MM patients but less than 25% of MGUS patients are positive for IL-1beta production. In the future, continued follow-up of IL-1beta positive and negative MGUS patients should determine whether aberrant expression of plasma cell IL-1beta is predictive of those MGUS patients that will eventually progress to active myeloma.

Chromosomal abnormalities in systemic amyloidosis.

Primary systemic amyloidosis (AL) is a plasma cell disorder characterized by deposition of monoclonal light chains in different organ systems. Although multiple and complex numerical chromosomal abnormalities have been described in patients with multiple myeloma, it is currently unknown whether such changes occur in systemic amyloidosis. Bone marrow samples from 21 patients with AL were studied by standard cytogenetics and interphase fluorescence in situ hybridization (FISH) for the presence of numerical chromosomal abnormalities. We tested for six chromosomes (7, 11, 9, 15, 18 and X) using centromere-specific probes. The monoclonal plasma cells were identified by simultaneous fluorescent staining of the monotypic cytoplasmic immunoglobulin. We compared these results with those obtained from 19 patients with monoclonal gammopathy of undetermined significance (MGUS) and normal controls. Multiple numerical chromosomal abnormalities were detected in AL by interphase FISH, including trisomy of chromosomes 7 (42%), 9 (52%), 11 (47%), 15 (39%), 18 (33%) and X (13% in women and 54% in men). Monosomy of chromosome 18 was seen in 72% of cases. Previous exposure to alkylator therapy did not appear to correlate with these abnormalities. No significant difference was observed in the prevalence of these abnormalities between AL and MGUS. Multiple chromosomal numerical abnormalities were detected by interphase FISH analysis in patients with AL, especially monosomy of chromosome 18. Aneuploidy in the monotypic plasma supports a neoplastic nature for the disorder.

Is multidrug resistance (P-glycoprotein) an intrinsic characteristic of plasma cells in patients with monoclonal gammopathy of undetermined significance, plasmacytoma, multiple myeloma and amyloidosis?

Multiple myeloma is not a curable disease, and most patients relapse after plateau phase. Drug resistance is a major problem in effective chemotherapy in this kind of disease. Current approaches are aimed at reversing or preventing drug resistance late in the disease. We studied a drug resistance marker, P-glycoprotein (P-gp), in a total of 43 patients with monoclonal gammopathy. This group included eight with monoclonal gammopathy of undetermined significance (MGUS), five with plasmacytoma (PCM), nineteen with multiple myeloma (MM; six newly diagnosed, seven plateau, five refractory, one relapse) and eleven amyloidosis (seven newly diagnosed, four after treatment). Using 3-color flow cytometry, a plasma cell gate was selected on the basis of CD38+/45-(dim) staining and the population was examined for the expression of P-gp using two monoclonal antibodies (MRK16 and UIC2). P-gp expression was positive on marrow plasma cells in 42/43 patients. The resistance index (RI) in these cases (range 2.0-7.07) is comparable to that in the positive cell line KG-1A (3.05-3.08). In 2 of 5 patients with refractory MM, the RI for P-gp (5.4, 6.36) was higher than in plateau phase. These data suggest that relative resistance due to the P-gp mechanism is likely to be an intrinsic property of plasma cells in monoclonal gammopathies and may provide a partial explanation as to why these diseases always relapse. The results of our study support strategies for MDR reversal earlier in the course.

Contrast in cytokine expression between patients with monoclonal gammopathy of undetermined significance or multiple myeloma.

We investigated whether differences in IL-6 and IL-1beta expression could be detected in monoclonal plasma cells from patients with MGUS or MM. Expression of IL-6 and IL-1beta in bone marrow cells was determined using cell sorting to enrich for plasma cells followed by reverse transcriptase/polymerase chain reaction (RT/PCR). Nineteen patients (six MGUS, two primary amyloid (AL), 11 MM) were studied. IL-6 mRNA expression was detectable in the sorted CD38+/CD45- plasma cell populations from 0/6 MGUS, 0/2 AL and 5/11 MM patients. All five MM patients with autocrine IL-6 expression demonstrated an elevated plasma cell labeling index. IL-1beta mRNA was detectable in the sorted CD38+/CD45- plasma cell populations from 1/6 MGUS, 0/2 AL and 10/11 MM patients. In situ hybridization (ISH) confirmed that the IL-1beta producing cells were plasma cells. In conclusion, autocrine production of IL-6 parallels a high labeling index and aberrant expression of IL-1beta correlates with the diagnosis of MM. Follow-up of IL-1beta-positive MGUS patients will determine whether aberrant expression of IL-1beta will predict those MGUS patients that will eventually progress to MM.

A novel three-color, clone-specific fluorescence in situ hybridization procedure for monoclonal gammopathies.

We have developed a three-color cytoplasmic immunoglobulin (cIg) and fluorescence in situ hybridization (FISH) technique to detect plasma cell chromosomal aneuploidy in patients with multiple myeloma (MM), monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), and amyloidosis (AL). Immunofluorescent-labeled antibodies to detect light chain expression and six directly labeled alpha-satellite chromosome specific enumeration probes (CEP) were used simultaneously to detect aneuploidy of the plasma cells. The six probes were specific for chromosomes 7, 9, 11, 15, 18, and X. The technique was tested in 12 consecutive patient samples (5 MM, 2 MGUS, 3 SMM, and 2 AL). Based on the alpha-satellite signals, we found trisomic clones for CEP 7 (4 of 12), CEP 11 (4 of 12), CEP X (1 of 12), CEP 9 (8 of 12), CEP 15 (7 of 12), and CEP 18 (5 of 12). Trisomic clones of at least one of the six chromosomes were present in 9 of 12 patients. We believe that this technique efficiently identifies monotypic plasma cells and permits simultaneous analysis of numeric chromosome anomalies by FISH in emerging neoplastic cells. We are in the process of applying this technique to a series of about 100 newly diagnosed monoclonal gammopathy patients.

Detection of myeloma cells in the peripheral blood by flow cytometry.

Bone marrow plasma cells (PC) from patients with multiple myeloma (MM) express monoclonal cytoplasmic immunoglobulin (clg) light chain, strongly express CD38, and usually lack or dimly express CD45. The detection of malignant plasma cells in the peripheral blood (PB) by immunofluorescence microscopy (IM) distinguishes patients with active MM from those with stable disease. The aim of this study was to learn whether two-color (CD38 and CD45) flow cytometry (FC) on whole blood specimens (WBFC) and three-color FC (CD38, CD45, and anti-kappa or lambda clg) on mononuclear cells could identify circulating PC as well as the standard, more labor intensive IM technique. Split-samples of PB from 73 patients with plasma cell proliferative disorders were examined by both techniques. WBFC detected CD38+ CD45- cells in 94% (33/35) of patients with circulating monoclonal PC detected by IM and three-color FC detected monoclonal CD38+ CD45- cells in 77% (27/35) of these cases. The absolute number of monoclonal PC detected by IM was compared to the FC methods and the Spearman rank correlations were 0.77 with WBFC and 0.80 with three-color FC. This study indicates that WBFC, using antibodies to CD38 and CD45, offers a practical and reliable method to detect and quantify circulating malignant PC in patients with MM.

Establishment and characterization of three myeloma cell lines that demonstrate variable cytokine responses and abilities to produce autocrine interleukin-6.

A consensus regarding myeloma cell growth factor responsiveness and ability to produce autocrine interleukin (IL)-6 has not yet been obtained. In this study, we have established three new human myeloma cell lines (DP-6, KAS-6/1 and KP-6) from patients with aggressive disease. Extensive characterization of these cell lines revealed considerable heterogeneity at several levels. Growth factor responsiveness was initially addressed. Although the potent myeloma cell growth factor, IL-6, induced the proliferation and allowed for the expansion of all three cell lines, a panel of other cytokines elicited heterogeneous responses in each cell line. IL-3, IL-10, IL-11, insulin-like growth factor-I and tumor necrosis factor-alpha also stimulated DNA synthesis in all three cell lines; however, the magnitude of the response was generally lower than that observed in cultures containing IL-6. Transforming growth factor-beta, by contrast, uniformly inhibited the growth of all three cell lines. IL-1alpha and IL-1beta induced the proliferation of the DP-6 cells, but had minimal effects on the KAS-6/1 and KP-6 cells. Interferon (IFN)-alpha stimulated DNA synthesis in the KAS-6/1 cells, but inhibited the proliferation of the DP-6 and KP-6 cells. By comparison, IFN-gamma induced the growth of the KAS-6/1 and DP-6 cells, but inhibited the KP-6 cells. The gp130-associated cytokines, IL-11, leukemia inhibitory factor and oncostatin M, stimulated the growth of the KAS-6/1 cells, but had minimal effects on the DP-6 and KP-6 cells. The cell lines were also analyzed for IL-6 expression. RT-PCR analysis demonstrated that all three cell lines expressed IL-6 mRNA. However, when culture supernatants were tested using a sensitive IL-6 ELISA or IL-6 bioassay only the DP-6 and KP-6 cells were shown to be secreting biologically active IL-6. In summary, although all three of these cell lines were established from myeloma patients, the heterogeneity observed between these cell lines was considerable and may reflect, as well as provide tools to study, the heterogeneity observed in clinical disease.

Fluorescent in situ hybridization studies of lymphocytes and neutrophils in chronic granulocytic leukemia.

Using immunomagnetic cell separation and fluorescent in situ hybridization (FISH), we studied nine patients who had chronic granulocytic leukemia (CGL) for the proportion of interphase nuclei with Mbcr/abl fusion in a direct preparation of the bone marrow and also in the mononuclear cell (MNC), neutrophil, and B- and T-cell fractions of the peripheral blood. In five untreated patients, conventional cytogenetics revealed 97% to 100% Philadelphia chromosome (Ph)+ metaphases. In three of these five patients, FISH studies on bone marrow direct preparations and peripheral blood MNCs indicated that an Mbcr/abl fusion occurred in 62% to 69% of the cells. We observed 69% to 88% of nuclei with Mbcr/abl fusion within the neutrophil fractions. In contrast, the values were 12% to 39% within the T-cell fractions in the four patients we studied. B-cell fractions were studied in three patients, and only one had an abnormal value (58%). In the four patients receiving alpha-interferon therapy, the degree of conventional cytogenetic remission correlated best with the degree of FISH remission observed in the peripheral blood neutrophil fraction. Our results are in agreement with earlier studies in that both B and T lymphocytes may be involved with the clonal process in CGL. The FISH-based detection of Mbcr/abl fusion in the peripheral blood neutrophil compartment provided the best estimate for the proportion of Ph metaphases determined by conventional cytogenetics.