Application of self-quenched JH consensus primers for real-time quantitative PCR of IGH gene to minimal residual disease evaluation in multiple myeloma.

Monitoring multiple myeloma patients for relapse requires sensitive methods to measure minimal residual disease and to establish a more precise prognosis. The present study aimed to standardize a real-time quantitative polymerase chain reaction (PCR) test for the IgH gene with a JH consensus self-quenched fluorescence reverse primer and a VDJH or DJH allele-specific sense primer (self-quenched PCR). This method was compared with allele-specific real-time quantitative PCR test for the IgH gene using a TaqMan probe and a JH consensus primer (TaqMan PCR). We studied nine multiple myeloma patients from the Spanish group treated with the MM2000 therapeutic protocol. Self-quenched PCR demonstrated sensitivity of >or=10(-4) or 16 genomes in most cases, efficiency was 1.71 to 2.14, and intra-assay and interassay reproducibilities were 1.18 and 0.75%, respectively. Sensitivity, efficiency, and residual disease detection were similar with both PCR methods. TaqMan PCR failed in one case because of a mutation in the JH primer binding site, and self-quenched PCR worked well in this case. In conclusion, self-quenched PCR is a sensitive and reproducible method for quantifying residual disease in multiple myeloma patients; it yields similar results to TaqMan PCR and may be more effective than the latter when somatic mutations are present in the JH intronic primer binding site.

Characterization of IGH locus breakpoints in multiple myeloma indicates a subset of translocations appear to occur in pregerminal center B cells.

Translocations in myeloma are thought to occur solely in mature B cells in the germinal center through class switch recombination (CSR). We used a targeted captured technique followed by massively parallel sequencing to determine the exact breakpoints in both the immunoglobulin heavy chain (IGH) locus and the partner chromosome in 61 presentation multiple myeloma samples. The majority of samples (62%) have a breakpoint within the switch regions upstream of the IGH constant genes and are generated through CSR in a mature B cell. However, the proportion of CSR translocations is not consistent between cytogenetic subgroups. We find that 100% of t(4;14) are CSR-mediated; however, 21% of t(11;14) and 25% of t(14;20) are generated through DH-JH recombination activation gene-mediated mechanisms, indicating they occur earlier in B-cell development at the pro-B-cell stage in the bone marrow. These 2 groups also generate translocations through receptor revision, as determined by the breakpoints and mutation status of the segments used in 10% and 50% of t(11;14) and t(14;20) samples, respectively. The study indicates that in a significant number of cases the translocation-based etiological events underlying myeloma may arise at the pro-B-cell hematological progenitor cell level, much earlier in B-cell development than was previously thought.

The Clinical Impact and Molecular Biology of del(17p) in Multiple Myeloma Treated with Conventional or Thalidomide-Based Therapy

Hemizygous deletion of 17p (del(17p)) has been identified as a variable associated with poor prognosis in myeloma, although its impact in the context of thalidomide therapy is not well described. The clinical outcome of 85 myeloma patients with del(17p) treated in a clinical trial incorporating both conventional and thalidomide-based induction therapies was examined. The clinical impact of deletion, low expression, and mutation of TP53 was also determined. Patients with del(17p) did not have inferior response rates compared to patients without del(17p), but, despite this, del(17p) was associated with impaired overall survival (OS) (median OS 26.6 vs. 48.5 months, P <0.001). Within the del(17p) group, thalidomide induction therapy was associated with improved response rates compared to conventional therapy, but there was no impact on OS. Thalidomide maintenance was associated with impaired OS, although our analysis suggests that this effect may have been due to confounding variables. A minimally deleted region on 17p13.1 involving 17 genes was identified, of which only TP53 and SAT2 were underexpressed. TP53 was mutated in <1% in patients without del(17p) and in 27% of patients with del(17p). The higher TP53 mutation rate in samples with del(17p) suggests a role for TP53 in these clinical outcomes. In conclusion, del(17p) defined a patient group associated with short survival in myeloma, and although thalidomide induction therapy was associated with improved response rates, it did not impact OS, suggesting that alternative therapeutic strategies are required for this group. (C) 2011 Wiley-Liss, Inc.

Deletions of CDKN2C in Multiple Myeloma: Biological and Clinical Implications

Purpose: Deletions of chromosome 1 have been described in 7% to 40% of cases of myeloma with inconsistent clinical consequences. CDKN2C at 1p32.3 has been identified in myeloma cell lines as the potential target of the deletion. We tested the clinical impact of 1p deletion and used high-resolution techniques to define the role of CDKN2C in primary patient material.Experimental Design: We analyzed 515 cases of monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), and newly diagnosed multiple myeloma using fluorescence in situ hybridization (FISH) for deletions of CDKN2C. In 78 myeloma cases, we carried out Affymetrix single nucleotide polymorphism mapping and U133 Plus 2.0 expression arrays. In addition, we did mutation, methylation, and Western blotting analysis.Results: By FISH we identified deletion of 1p32.3 (CDKN2C) in 3 of 66 MGUS (4.5%), 4 of 39 SMM (10.3%), and 55 of 369 multiple myeloma cases (15%). We examined the impact of copy number change at CDKN2C on overall survival (OS), and found that the cases with either hemizygous or homozygous deletion of CDKN2C had a worse OS compared with cases that were intact at this region (22 months versus 38 months; P = 0.003). Using gene mapping we identified three homozygous deletions at 1p32.3, containing CDKN2C, all of which lacked expression of CDKN2C. Cases with homozygous deletions of CDKN2C were the most proliferative myelomas, defined by an expression-based proliferation index, consistent with its biological function as a cyclin-dependent kinase inhibitor.Conclusions: Our results suggest that deletions of CDKN2C are important in the progression and clinical outcome of myeloma.

Gene mapping and expression analysis of 16q loss of heterozygosity identifies WWOX and CYLD as being important in determining clinical outcome in multiple myeloma.

We performed fluorescent in situ hybridization (FISH) for 16q23 abnormalities in 861 patients with newly diagnosed multiple myeloma and identified deletion of 16q [del(16q)] in 19.5%. In 467 cases in which demographic and survival data were available, del(16q) was associated with a worse overall survival (OS). It was an independent prognostic marker and conferred additional adverse survival impact in cases with the known poor-risk cytogenetic factors t(4;14) and del(17p). Gene expression profiling and gene mapping using 500K single-nucleotide polymorphism (SNP) mapping arrays revealed loss of heterozygosity (LOH) involving 3 regions: the whole of 16q, a region centered on 16q12 (the location of CYLD), and a region centered on 16q23 (the location of the WW domain-containing oxidoreductase gene WWOX). CYLD is a negative regulator of the NF-kappaB pathway, and cases with low expression of CYLD were used to define a "low-CYLD signature." Cases with 16q LOH or t(14;16) had significantly reduced WWOX expression. WWOX, the site of the translocation breakpoint in t(14;16) cases, is a known tumor suppressor gene involved in apoptosis, and we were able to generate a "low-WWOX signature" defined by WWOX expression. These 2 genes and their corresponding pathways provide an important insight into the potential mechanisms by which 16q LOH confers poor prognosis.

Demonstration of changes in plasma cell subsets in multiple myeloma.

Increases in free light chain (FLC) production are associated with disease progression in multiple myeloma (MM). Using a double immunofluorescence staining method to produce a differential count of plasma cells in bone marrow, single populations were demonstrated, containing intact monoclonal immunoglobulins (M-Igs) in 74% and FLCs only in 8% of cases. However, 18% contained a mixture of both cell populations. Progression from cells making intact M-Ig to cells restricted to FLC only production occurred in individual cases during the course of their disease. The presence of FLC only cells was associated with shortened survival.

Minimal residual disease monitoring in multiple myeloma: a comparison between allelic-specific oligonucleotide real-time quantitative polymerase chain reaction and flow cytometry.

BACKGROUND AND OBJECTIVES: Minimal residual disease (MRD) studies are useful in multiple myeloma (MM). However, the definition of the best technique and clinical utility are still unresolved issues. The aim of this study was to analyze and compare the clinical utility of MRD studies in MM with two different techniques: allelic-specific oligonucleotide real-time quantitative PCR (ASO-RQ-PCR), and flow cytometry (FCM). DESIGN AND METHODS: Bone marrow samples from 32 MM patients who had achieved complete response after transplantation were evaluated by ASO-RQ-PCR, using TaqMan technology, and multiparametric FCM. RESULTS: ASO-RQ-PCR was only applicable in 75% of patients for a variety of technical reasons, while FCM was applicable in up to 90%. Therefore, simultaneous PCR/FCM analysis was possible in only 24 patients. The number of residual tumor cells identified by both techniques was very similar (mean=0.29%, range=0.001-1.61%, correlation coefficient=0.861). However, RQ-PCR was able to detect residual myelomatous cells in 17 patients while FCM only did so in 11; thus, 6 cases were FCM negative but PCR positive, all of them displaying a very low number of clonal cells (median=0.014%, range=0.001-0.11). Using an MRD threshold of 0.01% (10(-4)) two risk groups with significantly different progression-free survival could be identified by either PCR (34 vs. 15m, p=0.04) or FCM (27 vs. 10m, p=0.05). INTERPRETATION AND CONCLUSIONS: Although MRD evaluation by ASO-RQ-PCR is slightly more sensitive and specific than FCM, it is applicable in a lower proportion of MM patients and is more time-consuming, while both techniques provide similar prognostic information.

Molecular characteristics and gene segment usage in IGH gene rearrangements in multiple myeloma.

BACKGROUND AND OBJECTIVES: Analysis of IgH rearrangements in B-cell malignancies has provided clinical researchers with a wide range of information during the last few years. However, only a few studies have contributed to the characterization of these features in multiple myeloma (MM), and they have been focused on the analysis of the expressed IgH allele only. Comparison between the expressed and the non-functional IgH alleles allows further characterizion of the selection processes to which pre-myeloma cells are submitted. DESIGN AND METHODS: We analyzed a cohort of 84 untreated MM patients in order to characterize their functional VDJH and non-functional DJH rearrangements. The pattern of mutations and gene segment usage for both types of rearrangements was analyzed by polymerase chain reaction and sequencing. RESULTS: VH3 and VH1 family members were over- and under-represented, respectively. VH3-30 and VH3-15 segments were the most frequently used, whereas VH4-34 was found only in non-functional or heavily mutated VDJH rearrangements. DH2 and DH3 family members were over-represented in both VDJH and DJH repertoires, while the DH1 family was under-represented only in the productive VDJH rearrangements. Finally, DH3-22 and DH2-21 gene segments were found to be over-represented in the functional repertoire while segments commonly used by less mature B-cell malignancies, such as DH6-19 or DH3-3, were under-represented. INTERPRETATION AND CONCLUSIONS: Data reported here help to identify the clonogenic MM cell as a post-germinal center B cell that has undergone selection processes during the germinal center reaction.

Incomplete DJH rearrangements of the IgH gene are frequent in multiple myeloma patients: immunobiological characteristics and clinical implications.

DH-JH rearrangements of the Ig heavy-chain gene (IGH) occur early during B-cell development. Consequently, they are detected in precursor-B-cell acute lymphoblastic leukemias both at diagnosis and relapse. Incomplete DJH rearrangements have also been occasionally reported in mature B-cell lymphoproliferative disorders, but their frequency and immunobiological characteristics have not been studied in detail. We have investigated the frequency and characteristics of incomplete DJH as well as complete VDJH rearrangements in a series of 84 untreated multiple myeloma (MM) patients. The overall detection rate of clonality by amplifying VDJH and DJH rearrangements using family-specific primers was 94%. Interestingly, we found a high frequency (60%) of DJH rearrangements in this group. As expected from an immunological point of view, the vast majority of DJH rearrangements (88%) were unmutated. To the best of our knowledge, this is the first systematic study describing the incidence of incomplete DJH rearrangements in a series of unselected MM patients. These results strongly support the use of DJH rearrangements as PCR targets for clonality studies and, particularly, for quantification of minimal residual disease by real-time quantitative PCR using consensus JH probes in MM patients. The finding of hypermutation in a small proportion of incomplete DJH rearrangements (six out of 50) suggests important biological implications concerning the process of somatic hypermutation. Moreover, our data offer a new insight in the regulatory development model of IGH rearrangements.

Incomplete DJH rearrangements as a novel tumor target for minimal residual disease quantitation in multiple myeloma using real-time PCR.

The hypervariable regions of immunoglobulin heavy-chain (IgH) rearrangements provide a specific tumor marker in multiple myeloma (MM). Recently, real-time PCR assays have been developed in order to quantify the number of tumor cells after treatment. However, these strategies are hampered by the presence of somatic hypermutation (SH) in VDJH rearrangements from multiple myeloma (MM) patients, which causes mismatches between primers and/or probes and the target, leading to a nonaccurate quantification of tumor cells. Our group has recently described a 60% incidence of incomplete DJH rearrangements in MM patients, with no or very low rates of SH. In this study, we compare the efficiency of a real-time PCR approach for the analysis of both complete and incomplete IgH rearrangements in eight MM patients using only three JH consensus probes. We were able to design an allele-specific oligonucleotide for both the complete and incomplete rearrangement in all patients. DJH rearrangements fulfilled the criteria of effectiveness for real-time PCR in all samples (ie no unspecific amplification, detection of less than 10 tumor cells within 10(5) polyclonal background and correlation coefficients of standard curves higher than 0.98). By contrast, only three out of eight VDJH rearrangements fulfilled these criteria. Further analyses showed that the remaining five VDJH rearrangements carried three or more somatic mutations in the probe and primer sites, leading to a dramatic decrease in the melting temperature. These results support the use of incomplete DJH rearrangements instead of complete somatically mutated VDJH rearrangements for investigation of minimal residual disease in multiple myeloma.

The detection of contaminating clonal cells in apheresis products is related to response and outcome in multiple myeloma undergoing autologous peripheral blood stem cell transplantation.

In the present paper, we report on the use of the heteroduplex PCR technique to detect the presence of clonally rearranged VDJ segments of the heavy chain immunoglobulin gene (VDJH) in the apheresis products of patients with multiple myeloma (MM) undergoing autologous peripheral blood stem cell (APBSC) transplantation. Twenty-three out of 31 MM patients undergoing APBSC transplantation with VDJH segments clonally rearranged detected at diagnosis were included in the study. Samples of the apheresis products were PCR amplified using JH and VH (FRIII and FRII) consensus primers and subsequently analyzed with the heteroduplex technique, and compared with those obtained at diagnosis. 52% of cases yielded positive results (presence of clonally rearranged VDJH segments in at least one apheresis). The presence of positive results in the apheresis products was not related to any pretransplant characteristics with the exception of response status at transplant. Thus, while no one patient with positive apheresis products was in complete remission (CR), negative immunofixation, before the transplant, five cases (46%) with negative apheresis were already in CR at transplant (P = 0.01). The remaining six cases with heteroduplex PCR negative apheresis were in partial remission before transplant. Patients with clonally free products were more likely to obtain CR following transplant (64% vs 17%, P= 0.02) and a longer progression-free survival, (40 months in patients transplanted with polyclonal products vs 20 with monoclonal ones, P = 0.03). These results were consistent when the overall survival was considered, since it was better in those patients with negative apheresis than it was in those with positive (83% vs 36% at 5 years from diagnosis, P= 0.01). These findings indicate that the presence of clonality rearranged VDJH segments is related to the response and outcome in MM transplanted patients.

De novo methylation of tumor suppressor gene p16/INK4a is a frequent finding in multiple myeloma patients at diagnosis.

The p16 gene competes with cyclin D for binding to CDK4/CDK6 and therefore inhibits CDK4/6 complex kinase activity, resulting in dephosphorylation of pRb and related G1 growth arrest. Inactivation of this gene has been involved in a variety of tumors by different mechanisms: homozygous/hemyzygous deletions, point mutations and methylation of a 5' CpG island into exon E1alpha of the p16 gene. Homozygous deletions have been rarely found in multiple myeloma (MM) and no point mutations have been reported. Two recent studies have reported a high prevalence of methylation in the exon E1alpha of the p16 gene, but included only a small number of cases. We have analyzed the methylation pattern of exon E1alpha of the p16 gene in 101 untreated MM and five primary plasma cell leukemias (PCL). A PCR assay, relying on the inability of some restriction enzymes to digest methylated sequences, was used to analyze the methylation status. Southern blot analysis was used to confirm these results. Forty-one of 101 MM patients (40.5%) as well as four of the five (80%) primary PCL patients had shown methylation of the exon E1alpha. Our study confirms that hypermethylation of the p16 gene is a frequent event in MM. Leukemia (2000) 14, 183-187.

Heteroduplex PCR analysis of rearranged immunoglobulin genes for clonality assessment in multiple myeloma.

BACKGROUND AND OBJECTIVE: Molecular analysis by PCR of monoclonally rearranged immunoglobulin (Ig) genes can be used for diagnosis in B-cell lymphoproliferative disorders (LPD), as well as for monitoring minimal residual disease (MRD) after treatment. This technique has the risk of false-positive results due to the "background" amplification of similar rearrangements derived from polyclonal B-cells. This problem can be resolved in advance by additional analyses that discern between polyclonal and monoclonal PCR products, such as the heteroduplex analysis. A second problem is that PCR frequently fails to amplify the junction regions, mainly due to somatic mutations frequently present in mature (post-follicular) B-cell lymphoproliferations. The use of additional targets (e.g. Ig light chain genes) can avoid this problem. DESIGN AND METHODS: We studied the specificity of heteroduplex PCR analysis of several Ig junction regions to detect monoclonal products in samples from 84 MM patients and 24 patients with B cell polyclonal disorders. RESULTS: Using two distinct VH consensus primers (FR3 and FR2) in combination with one JH primer, 79% of the MM displayed monoclonal products. The percentage of positive cases was increased by amplification of the Vlamda-Jlamda junction regions or kappa(de) rearrangements, using two or five pairs of consensus primers, respectively. After including these targets in the heteroduplex PCR analysis, 93% of MM cases displayed monoclonal products. None of the polyclonal samples analyzed resulted in monoclonal products. Dilution experiments showed that monoclonal rearrangements could be detected with a sensitivity of at least 10(-2) in a background with >30% polyclonal B-cells, the sensitivity increasing up to 10(-3) when the polyclonal background was

Multiple training interventions significantly improve reproducibility of PET/CT-based lung cancer radiotherapy target volume delineation using an IAEA study protocol

BACKGROUND AND PURPOSE: To assess the impact of a standardized delineation protocol and training interventions on PET/CT-based target volume delineation (TVD) in NSCLC in a multicenter setting.

MATERIAL AND METHODS: Over a one-year period, 11 pairs, comprised each of a radiation oncologist and nuclear medicine physician with limited experience in PET/CT-based TVD for NSCLC from nine different countries took part in a training program through an International Atomic Energy Agency (IAEA) study (NCT02247713). Teams delineated gross tumor volume of the primary tumor, during and after training interventions, according to a provided delineation protocol. In-house developed software recorded the performed delineations, to allow visual inspection of strategies and to assess delineation accuracy.

RESULTS: Following the first training, overall concordance indices for 3 repetitive cases increased from 0.57±0.07 to 0.66±0.07. The overall mean surface distance between observer and expert contours decreased from -0.40±0.03 cm to -0.01±0.33 cm. After further training overall concordance indices for another 3 repetitive cases further increased from 0.64±0.06 to 0.80±0.05 (p=0.01). Mean surface distances decreased from -0.34±0.16 cm to -0.05±0.20 cm (p=0.01).

CONCLUSION: Multiple training interventions improve PET/CT-based TVD delineation accuracy in NSCLC and reduces interobserver variation.