Addressing heterogeneity of individual blood cancers: the need for single cell analysis.

Cancer heterogeneity is a significant factor in response to treatment and escape leading to relapse. Within an individual cancer, especially blood cancers, there exists multiple subclones as well as distinct clonal expansions unrelated to the clinically detected, dominant clone. Over time, multiple subclones and clones undergo emergence, expansion, and extinction. Although sometimes this intra-clonal and inter-clonal heterogeneity can be detected and/or quantified in tests that measure aggregate populations of cells, frequently, such heterogeneity can only be detected using single cell analysis to determine its frequency and to detect minor clones that may subsequently emerge to become drug resistant and dominant. Most genetic/genomic tests look at the pooled tumor population as a whole rather than at its individual cellular components. Yet, minor clones and cancer stem cells are unlikely to be detected against the background of expanded major clones. Because selective pressures are likely to govern much of what is seen clinically, single cell analysis allows identification of otherwise cryptic compartments of the malignancy that may ultimately mediate progression and relapse. Single cell analysis can track intra- or inter-clonal heterogeneity and provide useful clinical information, often before changes in the disease are detectable in the clinic. To a very limited extent, single cell analysis has already found roles in clinical care. Because inter- and intra-clonal heterogeneity likely occurs more frequently than can be currently appreciated on a clinical level, future use of single cell analysis is likely to have profound clinical utility.

Single-Cell Analysis and Next-Generation Immuno-Sequencing Show That Multiple Clones Persist in Patients with Chronic Lymphocytic Leukemia.

The immunoglobulin heavy chain (IGH) gene rearrangement in chronic lymphocytic leukemia (CLL) provides a unique molecular signature; however, we demonstrate that 26/198 CLL patients (13%) had more than one IGH rearrangement, indicating the power of molecular technology over phenotypic analysis. Single-cell PCR analysis and next-generation immuno-sequencing identified IGH-defined clones. In 23% (18/79) of cases whose clones carried unmutated immunoglobulin heavy chain variable (IGHV) genes (U-CLL), IGH rearrangements were bialleic with one productive (P) and one non-productive (NP) allele. Two U-CLL were biclonal, each clone being monoallelic (P). In 119 IGHV-mutated (M-CLL) cases, one had biallelic rearrangements in their CLL (P/NP) and five had 2-4 distinct clones. Allelic exclusion was maintained in all B-clones analyzed. Based on single-cell PCR analysis, 5/11 partner clones (45%) reached levels of >5x10(9) cells/L, suggesting second CLL clones. Partner clones persisted over years. Conventional IGH characterization and next-generation sequencing of 13 CLL, 3 multiple myeloma, 2 Waldenstrom's macroglobulinemia and 3 age-matched healthy donors consistently identified the same rearranged IGH sequences. Most multiple clones occurred in M-CLL, perhaps indicative of weak clonal dominance, thereby associating with a good prognosis. In contrast, biallelic CLL occurred primarily in U-CLL thus being associated with poor prognosis. Extending beyond intra-clonal diversity, molecular analysis of clonal evolution and apparent subclones in CLL may also reflect inter-clonal diversity.

Aberrant posttranscriptional processing of hyaluronan synthase 1 in malignant transformation and tumor progression.

It is becoming increasingly apparent that splicing defects play a key role in cancer, and that alterations in genomic splicing elements promote aberrant splicing. Alternative splicing increases the diversity of the human transcriptome and increases the numbers of functional gene products. However, dysregulation that leads to aberrant pre-mRNA splicing can contribute to cancer. Hyaluronan (HA), known to be an important component of cancer progression, is synthesized by hyaluronan synthases (HASs). In cancer cells, hyaluronan synthase 1 (HAS1) pre-mRNA is abnormally spliced to generate a family of aberrant splice variants (HAS1Vs) that synthesize extracellular and intracellular HA. HAS1Vs are clinically relevant, being found almost exclusively in malignant cells. Expression of aberrant HAS1Vs predicts poor survival in multiple myeloma. In this review, we summarize the unusual properties of HAS1Vs and their relationship to cancer. HAS1Vs form heterogeneous multimers with normally spliced HAS1 as well as with each other and with HAS3. Aberrant variants of HAS1 synthesize HA. Extracellular HA synthesized by HAS1Vs is likely to promote malignant spread. We speculate that synthesis of intracellular HA plays a fundamental and early role in oncogenesis by promoting genetic instability and the emergence of viable cancer variants that lead to aggressive disease.

Frequent occurrence of highly expanded but unrelated B-cell clones in patients with multiple myeloma.

Clonal diversity in multiple myeloma (MM) includes both MM-related and MM-unrelated clonal expansions which are subject to dominance exerted by the MM clone. Here we show evidence for the existence of minor but highly expanded unrelated B-cell clones in patients with MM defined by their complementary determining region 3 (CDR3) peak. We further characterize these clones over the disease and subsequent treatment. Second clones were identified by their specific IgH-VDJ sequences that are distinct from those of dominant MM clones. Clonal frequencies were determined through semi-quantitative PCR, quantitative PCR and single-cell polymerase chain reaction of the clone-specific sequence. In 13/74 MM patients, more than one dominant CDR3 peak was identified with 12 patients (16%) being truly biclonal. Second clones had different frequencies, were found in different locations and were found in different cell types from the dominant MM clone. Where analysis was possible, they were shown to have chromosomal characteristic distinct from those of the MM clone. The frequency of the second clone also changed over the course of the disease and often persisted despite treatment. Molecularly-defined second clones are infrequent in monoclonal gammopathy of undetermined significance (MGUS, 1/43 individuals or 2%), suggesting that they may arise at relatively late stages of myelomagenesis. In further support of our findings, biclonal gammopathy and concomitant MM and CLL (chronic lymphocytic leukemia) were confirmed to originate from two unrelated clones. Our data supports the idea that the clone giving rise to symptomatic myeloma exerts clonal dominance to prevent expansion of other clones. MM and second clones may arise from an underlying niche permissive of clonal expansion. The clinical significance of these highly expanded but unrelated clones remains to be confirmed. Overall, our findings add new dimensions to evaluating related and unrelated clonal expansions in MM and the impact of disease evolution and treatment on clonal diversity.

Alteration of introns in a hyaluronan synthase 1 (HAS1) minigene convert Pre-mRNA [corrected] splicing to the aberrant pattern in multiple myeloma (MM): MM patients harbor similar changes.

Aberrant pre-mRNA splice variants of hyaluronan synthase 1 (HAS1) have been identified in malignant cells from cancer patients. Bioinformatic analysis suggests that intronic sequence changes can underlie aberrant splicing. Deletions and mutations were introduced into HAS1 minigene constructs to identify regions that can influence aberrant intronic splicing, comparing the splicing pattern in transfectants with that in multiple myeloma (MM) patients. Introduced genetic variations in introns 3 and 4 of HAS1 as shown here can promote aberrant splicing of the type detected in malignant cells from MM patients. HAS1Vd is a novel intronic splice variant first identified here. HAS1Vb, an intronic splice variant previously identified in patients, skips exon 4 and utilizes the same intron 4 alternative 3'splice site as HAS1Vd. For transfected constructs with unaltered introns 3 and 4, HAS1Vd transcripts are readily detectable, frequently to the exclusion of HAS1Vb. In contrast, in MM patients, HAS1Vb is more frequent than HAS1Vd. In the HAS1 minigene, combining deletion in intron 4 with mutations in intron 3 leads to a shift from HAS1Vd expression to HAS1Vb expression. The upregulation of aberrant splicing, exemplified here by the expression of HAS1Vb, is shown here to be influenced by multiple genetic changes in intronic sequences. For HAS1Vb, this includes enhanced exon 4 skipping and increased usage of alternative 3' splice sites. Thus, the combination of introduced mutations in HAS1 intron3 with introduced deletions in HAS1 intron 4 promoted a shift to an aberrant splicing pattern previously shown to be clinically significant. Most MM patients harbor genetic variations in intron 4, and as shown here, nearly half harbor recurrent mutations in HAS1 intron 3. Our work suggests that aberrant intronic HAS1 splicing in MM patients may rely on intronic HAS1 deletions and mutations that are frequent in MM patients but absent from healthy donors.

In a patient with biclonal Waldenstrom macroglobulinemia only one clone expands in three-dimensional culture and includes putative cancer stem cells.

A small percentage of cases of Waldenstrom macroglobulinemia (WM) present with biclonality, defined here as the rearrangement of two distinct VDJ gene segments. Here we investigated the expansion of two clones from a patient with WM expressing molecularly detectable clonotypic gene rearrangements, one V(H)3 and one V(H)4. Biclonality was determined in blood and bone marrow mononuclear cells using real-time quantitative PCR (RQ-PCR). V(H)4 expressing cells but not V(H)3 expressing cells underwent clonal expansion in 3-D culture of reconstructed WM bone marrow. After 3-D culture, secondary culture in a colony forming unit assay, and RQ-PCR, only the V(H)4 clone was shown to harbor a subpopulation with characteristics of cancer stem cells, including proliferative quiescence, self-regeneration, and the ability to generate clonotypic progeny, suggesting that the V(H)4, but not the V(H)3, clone is clinically significant. Enrichment of potential WM stem cells in 3-D cultures holds promise for monitoring their response to treatment and for testing new therapies.

Inherited and acquired variations in the hyaluronan synthase 1 (HAS1) gene may contribute to disease progression in multiple myeloma and Waldenstrom macroglobulinemia.

To characterize genetic contributions toward aberrant splicing of the hyaluronan synthase 1 (HAS1) gene in multiple myeloma (MM) and Waldenstrom macroglobulinemia (WM), we sequenced 3616 bp in HAS1 exons and introns involved in aberrant splicing, from 17 patients. We identified a total of 197 HAS1 genetic variations (GVs), a range of 3 to 24 GVs/patient, including 87 somatic GVs acquired in splicing regions of HAS1. Nearly all newly identified inherited and somatic GVs in MM and/or WM were absent from B chronic lymphocytic leukemia, nonmalignant disease, and healthy donors. Somatic HAS1 GVs recurred in all hematopoietic cells tested, including normal CD34(+) hematopoietic progenitor cells and T cells, or as tumor-specific GVs restricted to malignant B and plasma cells. An in vitro splicing assay confirmed that HAS1 GVs direct aberrant HAS1 intronic splicing. Recurrent somatic GVs may be enriched by strong mutational selection leading to MM and/or WM.

Analysis of clonotypic switch junctions reveals multiple myeloma originates from a single class switch event with ongoing mutation in the isotype-switched progeny.

Multiple myeloma (MM) is a cancer of plasma cells (PCs) expressing immunoglobulin heavy chain (IgH) postswitch isotypes. The discovery of earlier stage cells related to postswitch PCs, called preswitch clonotypic IgM (cIgM) cells led to the hypothesis that cIgM cells may be MM progenitors, replenishing the tumor throughout malignancy. cIgM cells may do this by undergoing class switch recombination (CSR), a process detectable in postswitch PCs as multiple IgH switch junctions associated with a single clonotypic IgH V/D/J. We addressed this with a specific clonotypic-switch polymerase chain reaction (PCR), informative for 32 of 41 cases. Here we made 2 significant discoveries: (1) in all cases, we detected only a single clonotypic switch fragment that persists over time (1-7.6 years), and (2) we detected ongoing mutation upstream of the switch junction in 5 of 6 patients, often targeting the intronic enhancer, a key control region in IgH expression. The presence of a single, unchanging clonotypic switch junction suggests that cIgM cells are not MM-PC progenitors; rather, postswitch PCs arise from a single cIgM cell, and MM-PC progenitors reside in the postswitch population. Furthermore, mutations revealed here provide a new marker to identify MM-PC progenitors and aggressive clones that evolve throughout malignancy.

Establishment of BCWM.1 cell line for Waldenström's macroglobulinemia with productive in vivo engraftment in SCID-hu mice.

A significant impairment in understanding the biology and advancing therapeutics for Waldenstrom's macroglobulinemia (WM) has been the lack of a representative cell line and animal model. We, therefore, report on the establishment of the BCWM.1 cell line, which was derived from the long-term culture of CD19(+) selected bone marrow lymphoplasmacytic cells isolated from an untreated patient with WM. BCWM.1 cells morphologically resemble lymphoplasmacytic cells (LPC) and propagate in RPMI-1640 medium supplemented with 10% fetal bovine serum. Phenotypic characterization by flow cytometric analysis demonstrated typical WM LPC characteristics: CD5(-), CD10(-), CD19(+), CD20(+), CD23(+), CD27(-), CD38(+), CD138(+), CD40(+), CD52(+), CD70(+), CD117(+), cIgM(+), cIgG(-), cIgA(-), ckappa(-), clambda(+), as well as the survival proteins APRIL and BLYS, and their receptors TACI, BCMA and BAFF-R. Enzyme-linked immunosorbent assay studies demonstrated secretion of IgMlambda and soluble CD27. Karyotypic and multicolor fluorescence in situ hybridization studies did not demonstrate cytogenetic abnormalities. Molecular analysis of BCWM.1 cells confirmed clonality by determination of IgH rearrangements. Inoculation of BCWM.1 cells in human bone marrow chips implanted in severe combined immunodeficient-hu mice led to rapid engraftment of tumor cells and serum detection of human IgM, lambda, and soluble CD27. These studies support the use of BCWM.1 cells as an appropriate model for the study of WM, which in conjunction with the severe combined immunodeficient-hu mouse model may be used as a convenient model for studies focused on both WM pathogenesis and development of targeted therapies for WM.

Molecular characterization of Waldenstrom's macroglobulinemia reveals frequent occurrence of two B-cell clones having distinct IgH VDJ sequences.

PURPOSE: Malignant B lineage cells in Waldenstrom's macroglobulinemia (WM) express a unique clonotypic IgM VDJ. The occurrence of biclonal B cells and their clonal relationships were characterized. EXPERIMENTAL DESIGN: Bone marrow and blood from 20 WM patients were analyzed for clonotypic VDJ sequences, clonal B-cell frequencies, and the complementary determining region 3 profile. RESULTS: Two different clonotypic VDJ sequences were identified in 4 of 20 WM. In two cases, partner clones had different VDJ rearrangements, with one clonotypic signature in bone marrow and a second in blood. For both cases, the bone marrow clone was hypermutated, whereas the blood clone was germ line or minimally mutated. In two other cases, partner clones shared a common VDJ rearrangement but had different patterns of somatic mutations. They lacked intraclonal diversity and were more abundant in bone marrow than in blood. VDJ mutation profiles suggested they arose from a common IgM progenitor. Single-cell analysis in one case indicated the partner clones were reciprocally expressed, following rules of allelic exclusion. CONCLUSIONS: The existence of two B-cell clones having distinct VDJ sequences is common in WM, suggesting that frequent transformation events may occur. In two cases, the partner clones had distinct tissue distributions in either blood or bone marrow, were of different immunoglobulin isotypes, and in one case exhibited differential response to therapy. The contributions of each clone are unknown. Their presence suggests that WM may involve a background of molecular and cellular events leading to emergence of one or more malignant clones.

Impaired class switch recombination (CSR) in Waldenstrom macroglobulinemia (WM) despite apparently normal CSR machinery.

Analysis of clonotypic isotype class switching (CSR) in Waldenström macroglobulinemia (WM) and IgM monoclonal gammopathy of undetermined significance (MGUS) reveals a normal initial phase of B-cell activation as determined by constitutive and inducible expression of activation-induced cytidine deaminase (AID). Switch mu (Smu) analysis shows that large deletions are not common in WM or IgM MGUS. In CD40L/IL-4-stimulated WM cultures from 2 patients, we observed clonotypic IgG exhibiting intraclonal homogeneity associated with multiple hybrid Smu/Sgamma junctions. This suggests CSR had occurred within WM cells. Nevertheless, the estimated IgG/IgM-cell frequency was relatively low (1/1600 cells). Thus, for the majority of WM B cells, CSR does not occur even when stimulated in vitro, suggesting that the WM cell is constitutively unable to or being prevented from carrying out CSR. In contrast to WM, the majority of IgM MGUS clones exhibit intraclonal heterogeneity of IgH VDJ. Furthermore, most IgM MGUS accumulate more mutations in the upstream Smu region than do WM, making them unlikely WM progenitors. These observations suggest that switch sequence analysis may identify the subset of patients with IgM MGUS who are at risk of progression to WM.

Identification of clonotypic IgH VDJ sequences in multiple myeloma.

In multiple myeloma (MM) the rearranged immunoglobulin heavy chain (IgH) variable, diversity, and joining (VDJ) DNA sequence of malignant plasma cells (PCs) serves as a marker for cells in the MM clone. This clonotypic sequence can be isolated from MM PCs by reverse transcriptase polymerase chain reaction (RT-PCR) with consensus primers that amplify the rearranged IgH repertoire. This chapter focuses on the key steps in determining patient-specific clonotypic sequences, including bulk RT-PCR using purified bone marrow mononuclear cell (BMMC) RNA, single-cell RT-PCR using RNA from PCs sorted by flow cytometry, IgH sequence alignments using IMGT or V BASE, and patient-specific primer design. In a test panel of several MM patient BMMCs, primers specific for the proposed sequence must amplify IgH from only the original patient. Furthermore, the proposed IgH sequence is not confirmed as clonotypic until these primers generate positive amplifications in the majority of single PCs from the original patient. This two-part test ensures that the proposed IgH sequence satisfies the definition of the clonotypic sequence as the most frequent, unique IgH sequence in an MM patient PC sample. With this patient-specific MM marker, a better understanding of transformed PCs and their B-lineage predecessors can be developed.

Origins of Waldenstrom's macroglobulinemia: does it arise from an unusual B-cell precursor?

Clonotypic B cells of Waldenstrom's macroglobulinemia (WM) are CD20+ immunoglobulin (Ig) M+ IgD+ cells that lack ongoing somatic hypermutation and class switch recombination (CSR). Only a small compartment of clonotypic B cells express activation-induced cytosine deaminase. Activation by CD40L/interleukin-4 does not stimulate WM class switching. However, we found that the mutation of switch regions essential for CSR were present in IgM monoclonal gammopathy of unknown significance (MGUS) but absent from WM B cells, suggesting the possibility that not all IgM MGUS have the potential to give rise to WM, and further strengthening the hypothesis that the target cell in transformation to WM is an unusual type of B cell.

Clonotypic IgM V/D/J sequence analysis in Waldenstrom macroglobulinemia suggests an unusual B-cell origin and an expansion of polyclonal B cells in peripheral blood.

Analysis of clonotypic immunoglobulin M (IgM) from 15 patients with Waldenstrom macroglobulinemia (WM) showed a strong preferential use of the VH3/JH4 gene families. Identification of the WM IgM V/D/J was validated using single-cell analysis, confirming its presence in most B cells. Despite the extensive hypermutated VH genes in 13 of 15 patients, statistical analysis of framework/complementary-determining region (FR/CDR) mutation patterns suggests that they might have escaped antigenic selection. Neither intraclonal diversity nor isotype switching was detectable. Membranous and secreted forms of clonotypic IgM transcripts were present in bone marrow and blood. Single-cell analysis showed that clonotypic B cells coexpress CD20, surface IgM (sIgM), and sIgD but that they lack CD138. Most B cells lacked memory marker CD27 despite their hypermutated variable regions otherwise suggestive of memory status. At diagnosis, circulating B cells in WM are largely clonotypic. However, when monoclonal IgM levels are decreased, clonotypic frequencies are substantially reduced despite elevated CD20+ cells, shown to be polyclonal by DNA sequencing and CDR3 fragment analysis. Thus, WM includes the expansion of circulating, polyclonal B cells. Overall, this work suggests that WM may originate from a largely VH3-restricted, somatically mutated, predominantly CD27(-)IgM(+)IgD+ population that cannot undergo class switching, suggestive of B cells that might have bypassed the germinal center.

The malignant clone in Waldenstrom's macroglobulinemia.

The unique IgM VDJ sequence that characterizes the malignant clone in Waldenstrom's macroglobulinemia (WM), termed clonotypic, was identified for 12 WM patients. The majority of WM patients (92%) had a clonotypic IgM from the VH3 family, with predominantly long CDR3 regions, characteristic of those found in antigen-stimulated populations. Clonotypic IgM transcripts were detected in both blood and bone marrow (BM), clearly identifying a blood-borne compartment of WM. Abnormal numbers of CD20(+) B cells were usually detectable and expressed surface IgM. In most cases these cells also expressed surface IgD. Most WM patients lacked detectable CD138(+) plasma cells in either blood or BM. Longitudinal analysis suggests that phenotypic identification of B cells in blood of WM patients is insufficient for monitoring disease. Although serum IgM had decreased and clonotypic transcripts were very weak for one patient, the number of CD20(+) B cells increased dramatically. The lack of clonotypic transcripts suggests that the majority of these circulating B cells were polyclonal and were not part of the WM clone, indicating that monitoring of clonotypic IgM provides the most accurate identifier of WM cells.

Abnormal expression of hyaluronan synthases in patients with Waldenstrom's macroglobulimenia.

Little is known about the biology or spread of Waldenstrom's macroglobulinemia (WM), a lymphoplasmo-proliferative disorder. Hyaluronan synthases (HASs), plasma membrane proteins, synthesize the extracellular matrix molecule hyaluronan (HA), which plays a role in malignant cell migration and the spread of many cancers. Three isoenzymes of HAS-HAS1, HAS2, and HAS3-are detected in humans. Aberrant expression of the HASs is coupled with different abnormalities. We have analyzed the expression pattern of HASs in WM patients. HAS3 was expressed in all patients and healthy donors tested, whereas the expression of HAS1 and HAS2 varied among the WM patients. Additionally, in WM patients, we have detected novel variants of HAS1, one of which was also detected in multiple myeloma (MM) patients. We speculate that HAS1 variants synthesize the intracellular HA ligand for RHAMM (a receptor for HA). RHAMM contributes to genetic instability in MM; therefore, we speculate that it may also contribute to genetic instability in WM. Furthermore, we suggest that overexpression of HAS1 and its variants in combination with HAS3 may form an HA matrix around WM cells, thus preventing their elimination by the immune system, and it promotes their migration and may facilitate the spread of disease.