Richter's syndrome with identical immunoglobulin gene rearrangements

A 75-year-old man was admitted to our hospital because of hepatosplenomegaly, generalized lymphadenopathy and lymphocytosis in February, 1989. The leukocyte counts were 93,200/microliters with 95% small lymphocytes which expressed surface membrane immunoglobulin (SmIg) M, D and kappa. Histological finding of the cervical lymph node was diffuse small cell lymphoma. A diagnosis of chronic lymphocytic leukemia (CLL) was made. He was followed up without chemotherapy. In January, 1990, he was re-admitted because of progressively enlarged lymph nodes and increased white blood cell counts, up to 183,200/microliters with 98% lymphocytes. He was treated with vincristine, cyclophosphamide, prednisolone. The leukocyte counts decreased to 5,000/microliters and lymph node swelling decreased in size. In April, 1990, generalized lymphadenopathy re-appeared. The biopsied lymph node specimen showed diffuse large cell non-Hodgkin lymphoma (NHL-DL). The lymph node cells were found to express SmIgM and kappa. The diagnosis of Richter's syndrome was made. DNA analysis using Southern blot method revealed identical immunoglobulin heavy and kappa chain gene rearrangements in the two neoplasms. These findings suggest that the CLL cells and the NHL-DL cells originate from the same clone in this case.     

Serum immunoglobulin free light chain and heavy/light chain measurements in POEMS syndrome

POEMS (polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes) syndrome is a rare plasma cell dyscrasia. Nearly all patients present with a λ-restricted monoclonal gammopathy. Most patients with POEMS syndrome have been reported to have a normal serum free light chain ratio (sFLC-R), but the underlying mechanism is still unclear. We assessed the serum free light chains in 83 patients with newly diagnosed POEMS syndrome. The clinical and laboratory data associated with this disorder were collected to identify factors affecting sFLC-R. Fifty-six patients (67 %) showed elevated serum free λ light chains, but only 11 patients (13 %) had an abnormal sFLC-R. A comparison of patients with and without abnormal sFLC-Rs indicated that the latter group had more common splenomegaly and worse renal function. However, the introduction of an extended renal range for sFLC-R did not dramatically improve the diagnostic value of sFLC-R in these patients. Further analyses identified a correlation between the serum free κ light chain and the uninvolved immunoglobulin in patients with an IgAλ clone, implying that the activation of polyclonal immunoglobulin production could mask the presumed skewing of the sFLC-R induced by the underlying monoclonal gammopathy. Therefore, a serum heavy/light chain (sHLC) assay was performed in a subset of patients with stored serum samples available, and the prevalence of abnormal sHLC ratios was high in these patients. In summary, the overproduction of polyclonal immunoglobulin accounts for the high frequency of normal sFLC-R in patients with POEMS syndrome. The sHLC assay may provide unique information about this disorder.     

Clonal immunoglobulin heavy chain and T-cell receptor γ gene rearrangements in primary gastric lymphoma

AIM:To study the diagnostic value of immunoglobulin heavy chain(IgH)and T-cell receptorγ (TCR-γ)gene monoclonal rearrangements in primary gastric lymphoma(PGL).METHODS:A total of 48 patients with suspected PGL at our hospital were prospectively enrolled in this study from January 2009 to December 2011.The patients were divided into three groups(a PGL group,a gastric linitis plastica group,and a benign gastric ulcer group)based on the pathological results(gastric mucosal specimens obtained by endoscopy or surgery)and follow-up.Endoscopic ultrasonography(EUS)and EUSguided biopsy were performed in all the patients.The tissue specimens were used for histopathological examination and for IgH and TCR-γ gene rearrangement polymerase chain reaction analyses.RESULTS:EUS and EUS-guided biopsy were successfully performed in all 48 patients.In the PGL group(n=21),monoclonal IgH gene rearrangements were detected in 14(66.7%)patients.A positive result for each set of primers was found in 12(57.1%),8(38.1%),and 4(19.0%)cases using FR1/JH,FR2/JH,and FR3/JH primers,respectively.Overall,12(75%)patients with mucosal-associated lymphoid tissue lymphoma(n=16)and 2(40%)patients with diffuse large B-cell lymphoma(n=5)were positive for monoclonal IgH gene rearrangements.No patients in the gastric linitis plastica group(n=17)and only one(10%)patient in the benign gastric ulcer group(n=10)were positive for a monoclonal IgH gene rearrangement.No TCRgene monoclonal rearrangements were detected.The sensitivity of monoclonal IgH gene rearrangements was 66.7%for a PGL diagnosis,and the specificity was96.4%.In the PGL group,8(100%)patients with stage IIE PGL(n=8)and 6(46.1%)patients with stage IE PGL(n=13)were positive for monoclonal IgH gene rearrangements.CONCLUSION:IgH gene rearrangements may be associated with PGL staging and may be useful for the diagnosis of PGL and for differentiating between PGL and gastric linitis plastica.     

Abnormal immunoglobulin synthesis in monoclonal immunoglobulin light chain and light and heavy chain deposition disease.

The Congo red-binding fibrils of AL amyloidosis are the most common form of monoclonal immunoglobulin tissue deposition (MIDD). Nonetheless, the less structured deposits found in light chain deposition disease (LCDD) and the similar, but distinct, deposits of light and heavy chain deposition disease (LHCDD) and heavy chain deposition disease (HCDD) can produce significant clinical pathology. Analyses of immunoglobulin synthesis by bone marrow cells obtained from 7 patients with LCDD and LHCDD demonstrated the production of excess light chains in all and the presence of incomplete light chains or heavy chain fragments in 5, regardless of the presence of an intact monoclonal protein or related subunit in the serum or urine. Our data indicate that, as is the case with the fibrillar deposits of AL amyloid, the non-fibrillar forms of monoclonal Ig deposition (LCDD and LHCDD) can be associated with the presence of immunoglobulin fragments in bone marrow cells. In some instances these appeared to be synthetic in origin, although rapid intracellular proteolysis or a combination of both could not be excluded. In either case the fragments may be more susceptible to tissue deposition, with subsequent organ compromise, than intact Ig chains.     

Lymphoma with multi gene rearrangement on the level of immunoglobulin heavy chain,light chains,and T-cell receptor β chain

A unique case with diffuse mixed malignant lymphoma was investigated for gene rearrangement on the level of T-cell receptor (TCR), heavy chain immunoglobulin (Ig), and both light chains. Cell phenotype was examined with immunofluorescence techniques using antibodies against surface immunoglobulins (Slg) and the kappa and lambda light chains. Monoclonal antibodies were used against CD3, CD4, CD5, CD8, CD10, CD19, CD22, HLA-DR, and TdT. Gene rearrangement analysis for monoclonality determination was carried out with restricted DNA (EcoR I and Hind III) hybridized with one of the following ³²P-labelled probes: T-cell receptor (TCR |gb), immunoglobulin heavy chain (JH), k light chain, and |gl light chain. Phenotyping of the cell population from the excised lymph node (LN) revealed the presence of 66% B-cells and 35% T-cells. Most of the B cells (94%) expressed μ heavy chain only. Expression of both light chains was negligible (k = 7% and λ = 2%). Gene rearrangement, which indicates monoclonality, was positive on the level of TCR, Ig heavy chain, and both light chains. The data obtained suggests a neoplastic transforming event in lymphoid stem cells, which preceded the subsequent differentiation process into either B or T lymphoma. Am. J. Hematol. 56:219–223, 1997. © 1997 Wiley-Liss, Inc.     

Richter syndrome with two B cell clones possessing different surface immunoglobulins and immunoglobulin gene rearrangements

A patient with two populations of B cell malignancy in the bone marrow is reported. One population consisted of mature small lymphocytes, expressing surface IgM + D, lambda and proliferating very slowly. The other population consisted of abnormal large lymphoid cells, expressing surface IgM, K and proliferating actively. We considered the former as chronic lymphocytic leukemia cells and the latter as malignant lymphoma cells. Therefore, this case was considered as Richter syndrome. The analysis of immunoglobulin heavy chain gene rearrangement showed the different patterns between the two populations. It suggested that the two populations arose from the different origins. We discussed the genetic relationship between the two clones of Richter syndrome referring to other reported cases.     

DNA rearrangements in MPC-11 immunoglobulin heavy chain class-switch variants.

Immunoglobulin heavy chain class switching has been observed in vitro. In the IgG2b-producing MPC-11 mouse myeloma cell line, IgG2a-producing cells arise at a high frequency. In some cases, switch variants producing normal-sized (Mr 55,000) gamma 2a heavy chains have arisen spontaneously from a mutagen-induced "intermediate" (ICR 9.7.1) that produces an unusually large (Mr 75,000) heavy chain. Other switch variants have been isolated directly from the parent cell line. The expressed and unexpressed gamma 2b genes of MPC-11 can be distinguished in restriction endonuclease digests of total genomic DNA so that DNA rearrangements detected in MPC-11 variants can be directly associated with one or the other of these two genes. We describe here DNA rearrangements occurring on the expressed heavy chain chromosome of several MPC-11 gamma 2a switch variants and on the expressed chromosome of the ICR 9.7.1 intermediate. Our data indicate that all of these variants express the parental heavy chain variable region (VH) gene, supporting previous protein studies. We provide mapping data for the expressed gene of both ICR 9.7.1 and one of the IgG2a-producing variant cell lines (ICR 9.9.2.1) derived from it and discuss the advantages of an in vitro switching system for examining the dynamics of the immunoglobulin heavy chain class switch.     

Diffuse large B-cell lymphoma expressing surface immunoglobulin heavy chain (Ig alpha) and lacking light chains

A 59-year-old woman with goiter complained of nausea, vomiting and weight loss in April 2000. She underwent an endoscopic examination and was admitted to our hospital because gastric biopsy specimens revealed that she had diffuse large B-cell lymphoma. A thyroid biopsy also detected the diffuse infiltration of lymphoma cells, which were positive for CD19, CD20, CD38 and HLA-DR. Although the cells expressed surface immunoglobulin a chain, they lacked expressions of the kappa and lambda light chains. Chromosomal analysis of the thyroid cells showed 47, XX, t(2 ; 3)(q31 ; q13), + 3, t(8 ; 22)(q24 ; q11). After five courses of biweekly CHOP chemotherapy, she received autologous peripheral blood stem cell transplantation in October 2000. Currently, she has maintained complete remission for more than 4 years.     

neu protooncogene fused to an immunoglobulin heavy chain gene requires immunoglobulin light chain for cell surface expression and oncogenic transformation.

The protein encoded by the neu protooncogene (human gene symbol NGL for neuro/glioblastoma-derived) is a member of the surface receptor/tyrosine kinase family. Though its structure suggests that it can transduce a transmembrane signal, neither its extracellular ligand nor its critical intracellular substrates are known. To explore the functional properties of the protein encoded by neu, we created a fusion gene that joins the cytoplasmic domain of neu to the extracellular portion of an immunoglobulin heavy chain. The localization of the fusion polypeptide can then be controlled by coexpression with immunoglobulin light chain. In the absence of light chain, the heavy chain-neu polypeptide is expressed intracellularly and has no transforming activity. By contrast, in the presence of light chain the fusion polypeptide is expressed at the cell surface and produces tumorigenic foci. Thus, transformation apparently requires expression at the cell surface, where the neu intracellular domain can interact with components that are localized to the plasma membrane. The fusion protein is active in cellular transformation when the transmembrane domain is derived either from neu or from immunoglobulin, indicating that the neu transmembrane domain is not specifically required for transformation, although neu activation in tumors is known to result from a point mutation in this region. The extracellular immunoglobulin heavy and light chain domains of the fusion protein form a functional binding site that allows antigen to modulate its activity, reversing the transforming effect.     

Polymorphism in immunoglobulin heavy chains suggesting gene conversion.

Complete heavy (H) chain variable region (V region; amino acids 1-118) sequences have been determined for three phosphocholine (PCho)-binding monoclonal antibodies of CBA mouse strain origin. Two of these were found to differ from the sequence of the BALB/c T15 germline VH segment (segment of the V region that includes amino acids 1-95) at four positions but were identical to the allelic form of T15 (C3) found in C57BL. The third VH segment, HP101.6G6 (6G6), was clearly the product of a second, related VH gene, probably the allele of the BALB/c V11 gene, a second member of the P-Cho VH gene family. Thus, more than one VH gene is capable of encoding heavy chains of PCho-binding antibodies. The 6G6 VH segment differs from VII at seven positions; four of these distinguishing amino acids are encoded in other membranes of the PCho VH gene family. We postulate that the origin of the 6G6 VH sequence can most easily be explained by a process of gene conversion occurring between the least three members of the PCho VH family.     

Clonal evolution as judged by immunoglobulin heavy chain gene rearrangements in relapsing precursor-B acute lymphoblastic leukemia.

Oligoclonality and ongoing clonal evolution are common features in patients with precursor-B (pre-B) acute lymphoblastic leukemia (ALL), as judged by immunoglobulin heavy chain (IgH) gene rearrangement analysis. These features are considered to be results of secondary rearrangements after malignant transformation or emergence of new tumor clones. In the present study we analyzed the IgH gene rearrangement status in 18 cases with relapsing pre-B ALL using variable heavy chain (V(H)) gene family specific polymerase chain reaction (PCR) amplification and single stranded conformation polymorphism (SSCP) analysis. Clonal IgH rearrangements were displayed in all leukemias but one, and altered rearrangement patterns occurred in five cases (29%), which were selected for detailed nucleotide sequence analysis. In one case, multiple subclones at diagnosis were suggested to be derived from a progenitor clone through joining of different V(H) germline gene segments to a pre-existing D-J(H) complex (V(H) to D-J(H) joining). Evidence for V(H) gene replacement with identical N-sequences at the V(H)-D junction and a common D-J(H) region was observed in one case. Diversification at the V(H)-D junction consisting of heterogeneous N-sequences were observed in one case. This molecular modification of the V(H)-D region could fit a hypothesized "open-and-shut" mechanism. Nevertheless, despite these ongoing events at least one IgH rearrangement remained unchanged throughout the disease in most patients, indicating that the immunoglobulin heavy chain locus can be a suitable marker for detection of minimal residual disease (MRD).     

Multiple productive immunoglobulin heavy chain gene rearrangements in chronic lymphocytic leukemia are mostly derived from independent clones

In chronic lymphocytic leukemia, usually a monoclonal disease, multiple productive immunoglobulin heavy chain gene rearrangements are identified sporadically. Prognostication of such cases based on immunoglobulin heavy variable gene mutational status can be problematic, especially if the different rearrangements have discordant mutational status. To gain insight into the possible biological mechanisms underlying the origin of the multiple rearrangements, we performed a comprehensive immunogenetic and immunophenotypic characterization of 31 cases with the multiple rearrangements identified in a cohort of 1147 patients with chronic lymphocytic leukemia. For the majority of cases (25/31), we provide evidence of the co-existence of at least two B lymphocyte clones with a chronic lymphocytic leukemia phenotype. We also identified clonal drifts in serial samples, likely driven by selection forces. More specifically, higher immunoglobulin variable gene identity to germline and longer complementarity determining region 3 were preferred in persistent or newly appearing clones, a phenomenon more pronounced in patients with stereotyped B-cell receptors. Finally, we report that other factors, such as TP53 gene defects and therapy administration, influence clonal selection. Our findings are relevant to clonal evolution in the context of antigen stimulation and transition of monoclonal B-cell lymphocytosis to chronic lymphocytic leukemia.     

Serum immunoglobulin free light chain measurements and heavy chain isotype usage provide insight into disease biology in patients with POEMS syndrome

POEMS (polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, skin changes) syndrome is a rare paraneoplastic syndrome in which nearly all patients have a monoclonal lambda restricted plasma cell disorder. We investigated whether patients with POEMS have abnormal serum immunoglobulin free light chain (FLC) ratios. Fifty patients with newly diagnosed POEMS syndrome were assessed. Cystatin C levels were measured to discern whether subclinical renal insufficiency could account for FLC elevations in the presence of a normal FLC ratio. Forty‐five patients (90%) had elevated lambda FLC; however, only nine (18%) had abnormal FLC ratios. The rise in serum FLC of POEMS patients appeared to be multifactorial—both a function of subclinical renal insufficiency and polyclonal activation of medullary and extramedullary plasma cells. Those patients expressing a clonal IgA were more likely to have clonal plasmacytosis observed on iliac crest biopsy than those with IgG. In summary, serum immunoglobulin profiles are unique in POEMS syndrome as compared with other plasma cell disorders. Am. J. Hematol. 2010. © 2010 Wiley‐Liss, Inc.     

Synthesis of immunoglobulin mu chain gene products precedes synthesis of light chains during B-lymphocyte development.

Immunoglobulin (Ig) gene expression has been followed during the later stages of development of the murine fetal liver. Biosynthetic labeling and immunoprecipitation were used to isolate Ig-related polypeptides from fetal and neonatal livers. By examination of the specific immune precipitates, the earliest detectable Ig was shown to consist only of mu heavy chain. At about the time of birth, when light chain synthesis became evident, separation of surface Ig-positive cells from surface Ig-negative cells by using anti-Ig-coated dishes showed that cells lacking surface Ig (pre-B lymphocytes) synthesized only mu chains. Thus, commencement of light chain synthesis was closely coordinated with the appearance of surface Ig. Ig RNA species were examined by electrophoretic fractionation and hybridization with cloned Ig DNA sequences. The sizes and amounts of Ig mRNA were found to correlate with the pattern of mu and light chain protein biosynthesis. mu chain RNA species appeared earlier in gestation than light chain RNA did, and only after birth did light chain sequences reach levels equivalent to those of mu chain. Cell populations enriched in pre-B lymphocytes also contained an excess of mu over light chain mRNA.     

Rearrangement patterns of immunoglobulin heavy chain (IgH) and light chain genes in acute lymphoblastic leukemia and chronic myelocytic leukemia lymphoid crisis cells showing oligoclonal IgH gene rearrangements.

We investigated leukemic cells with multiple immunoglobulin heavy chain (IgH) gene rearrangements from nine B-precursor cell acute lymphoblastic leukemia (ALL) patients and three chronic myelocytic leukemia lymphoid crisis (CML.Ly-BC) patients in order to determine detailed recombination patterns of the variable (V), diversity (D), and joining (J) region genes. Southern blot study, using DNA probes for DQ52 and 5'D region genes, was useful to distinguish VDJ recombination from DJ recombination at the level of each allele. Leukemic cells from seven out of eight CD10-positive ALL patients showed biallelic VDJ recombinations. Rearrangements of Ig kappa genes were found in only one case. Leukemic cells from all of the CML.Ly-BC patients had a DJ/(V)DJ IgH genotype. These findings suggest that the multiple IgH gene rearrangements in B-precursor cell ALL occurred as a consequence of continuing V-(V)DJ rearrangements after neoplastic transformation, and were closely related to the stage of bone marrow B-precursor cell differentiation. Multiple IgH gene rearrangements in CML.Ly-BC might take place earlier in the process of IgH gene rearrangements than is the case in B-precursor cell ALL. In this sense, the genotypic oligoclonality observed in ALL and CML.Ly-BC should be regarded not as 'true', but as 'pseudo' oligoclonal leukemia.     

Nonstochastic pairing of immunoglobulin heavy and light chains expressed by chronic lymphocytic leukemia B cells is predicated on the heavy chain CDR3

We analyzed the immunoglobulin (Ig) variable heavy (IGHV) and variable light chain genes used by leukemia cells of 258 unrelated patients with chronic lymphocytic leukemia (CLL) found to express unmutated Ig heavy chains (IgH) encoded by a 51p1 allele of IGHV1-69 among 1846 CLL patients examined. We found each had at least 98% homology to an identified germline IGKV or IGLV gene. Within the 258 IgH, we identified heavy chain CDR3 (HCDR3) motifs encoded by certain unmutated IGHD and IGHJ genes with restricted reading frames. Frequent and restricted use of particular IGKV and IGLV genes revealed nonstochastic pairing of disparate Ig light chains (IgL) with IgH that had restricted HCDR3 motifs designated CLL69A, -B, -C, and -D. Eighty-six percent (19/22) of CLL cases that expressed motif CLL69B encoded by IGHD2-2/IGHJ6 had distinctive IgL encoded by IGKV1-39. Similarly, 83% (5/6) of samples with motif CLL69D encoded by IGHD2-2/IGHJ6 expressed IGKV3-11, 100% (25/25) with motif CLL69A encoded by IGHD3-16/IGHJ3 used IGKV3-20, and 77% (10/13) with motif CLL69C encoded by IGHD3-3/IGHJ6 expressed IGLV3-9. This study reveals nonstochastic pairing of IgH with particular IgL that is predicated upon Ig HCDR3 structure, providing compelling evidence for selection of antibodies expressed in CLL by conventional antigens.     

Monosomy 7, leukaemia and immunoglobulin heavy chain gene rearrangement

We describe the haematological findings and clinical course of a 15-year-old male who presented with a spontaneous acute lymphoblastic leukaemia. The lymphoid origin of the leukaemia was supported by cell surface antigen studies and immunoglobulin heavy chain gene analysis. Bone marrow karyotype was simple monosomy 7 and the lymphoblasts expressing the myeloid associated antigen CD 33. Both of these features have been previously shown to indicate a poor prognosis. The findings in this patient support a previous hypothesis that monosomy 7 can arise at the stem cell level.     

Exon shuffling generates an immunoglobulin heavy chain gene.

From endonuclease EcoRI partial libraries of DNAs from mouse embryo and MOPC 141, a gamma 2b-producing myeloma, clones were isolated by using a DNA fragment carrying the gamma 2b constant (C) region gene as a hybridization probe. One clone from MOPC 141 contained a heavy chain variable (V) gene and the C gamma 2b gene, as demonstrated by R-loop mapping. The V gene and C gene in this clone were separated by a 3.9-kilobase intron. The characterization of this clone as well as the embryonic clones suggest that at least two recombination events occurred to create the gamma 2b gene in MOPC 141. One of the events is analogous to the V-J joining previously demonstrated in the light chain genes, which brings the major part of the V gene next to a short coding sequence (J). The other event we refer to as "C mu-C gamma 2b switch recombination" because a portion of the intron between the V gene and C gene of the rearranged gamma 2b gene is derived from the 5' flanking sequence of the embryonic C mu gene. A model suggesting how the phenomenon of switch seen in lymphocytes may occur is presented.     

PCR analysis of immunoglobulin heavy chain and TCR gene rearrangements in diagnosis of lymphoproliferative disorders on formalin-fixed, paraffin-embedded tissues

To confirm a diagnosis of malignant lymphomas it is imperative to distinguish between reactive and neoplastic proliferation. The PCR (polymerase chain reaction) is a method that can be used for detection of clonal rearrangements of the immunoglobulin heavy chain (IgH) and T-cell receptor (TCR) genes. This study summarizes the outcomes of PCR analysis of IgH and TCR gene rearrangements in 91 bioptic cases of lymphoproliferative disorders. In the class of B lymphomas we detected clonal IgH rearrangement in nearly 83% of cases and in class of T lymphomas in 81% of cases. We can affirm that PCR analysis of B and T cell clonality on DNA extracted from the whole section of formalin-fixed, paraffin-embedded tissue is very suitable for routinely elaborate this. Its influence on the diagnostics of morphological unclear cases in particular, is crucial and is useful in establishing a diagnosis of lymphoid neoplasias in specimens in which histological and immunophenotypic studies are inconclusive.