Translocations of 14q32 and deletions of 13q14 are common chromosomal abnormalities in systemic amyloidosis

Systemic monoclonal immunoglobulin light chain amyloidosis (AL) is associated with clonal plasma cell dyscrasias that are often subtle and non-proliferating. AL shares numerical chromosomal changes with multiple myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS). Illegitimate translocations involving the immunoglobulin heavy chain gene (IGH) at 14q32 and deletions of the long arm of chromosome 13, [del(13q)], commonly occur in MM, MGUS and plasma cell leukaemia. In AL IGH rearrangements have been identified but, to date, there are no reports of del(13q). In this study of 32 patients with AL, 24 with systemic and eight with localized disease, translocations involving IGH and del(13q) were found using dual-colour interphase fluorescence in situ hybridization (FISH). IGH translocations were observed in 11 patients (37% overall and in 46% with systemic disease), of which nine had the IGH/CCND1 fusion from t(11;14)(q13;q32). Two showed IGH translocations other than the t(11;14) or t(4;14)(p16;q32). In one of these patients a breakpoint within the constant region of IGH between Calpha1 and Calpha2 was indicated. In the second a deletion covering Calpha1 and Calpha2 accompanied the translocation. Ten patients (27% overall and 33% of those with systemic disease) showed del(13q). The gain or loss of IGH and CCND1 signals provided evidence of numerical chromosomal changes in three patients.     

The human CD40 gene lies within chromosome 20q deletions associated with myeloid malignancies

Deletions of chromosome 20q are associated with myeloid malignancies and have been previously shown to arise in a multipotent progenitor of both myeloid and B cells. However, B-cell differentiation from the abnormal progenitor was impaired. The CD40 antigen is a surface glycoprotein which is expressed in B cells and haemopoietic stem cells and is important for B-cell growth and development. Following the recent mapping of CD40 to chromosome 20q we sought to determine its position relative to 20q deletions. Analysis of lymphoblastoid cell lines carrying 20q deletions placed CD40 within a 19–21 cM interval which is almost coincidental with the common deleted region defined by previous analysis of patient samples. Our results raise the possibility that genetic alteration of this locus may contribute to the pathogenesis of myeloid disorders associated with 20q deletions.     

Molecular detection of deletions involving band q14 of chromosome 13 in retinoblastomas.

DNA fragments from a locus spanning 29 kilobases within chromosome band 13q14 detected deletions in 3 retinoblastomas out of 37 such tumors examined. Somatically occurring, homozygous deletions spanning at least 25 kilobases were detected in retinoblastomas from two unrelated patients. These deletions are bounded by the esterase D locus proximally. In a third patient, both tumor cells and leukocytes have a deletion of one chromosome 13 homolog, with one end of the deletion localized to a 1.55-kilobase fragment within the cloned region. It is likely that the cloned locus is within a few hundred kilobases of the retinoblastoma gene (i.e., the locus governing predisposition to such tumors) and that the deletions detected also involve the retinoblastoma gene. Further, it may be possible to base a successful approach to the isolation of the retinoblastoma gene on this assumed physical proximity of the two loci.     

Syndecan-1 in B lymphoid malignancies

Syndecans are heparan sulfate-bearing proteoglycans that are found on the surface of most cells. Syndecan-1 is expressed predominantly on epithelia, but is also present on pre-B cells and plasma cells. The syndecans act to bind various effector molecules via their heparan sulfate chains, including both soluble and insoluble molecules within the extracellular milieu. These interactions promote cell adhesion to extracellular matrix and to adjacent cells. In addition, the syndecans can bind to and affect the biological activity of a number of heparin-binding growth factors. Thus, syndecan-1 can play a dramatic role in regulating cell behavior. In this review we discuss the expression of syndecan-1 on malignant B lymphoid cells as well as specific structure-function relationships of the molecule. Emphasis is placed on the important role that syndecan-1 has in regulating the growth of B lymphoid malignancies, particularly multiple myeloma.     

BL22 and lymphoid malignancies

BL22 is a recombinant immunotoxin containing a truncated form of the bacterial toxin Pseudomonas exotoxin A attached to an Fv fragment of an anti-CD22 monoclonal antibody. Its mechanism of action involves binding to CD22, being internalized into the target cell by endocytosis, being processed to generate a free toxin fragment which is translocated into the cytoplasm, and finally induction of cell death by catalytic inactivation of elongation factor 2. In phase-I testing BL22 was very active in chemoresistant hairy-cell leukemia (HCL), with 19 (61%) of 31 patients achieving complete remission (CR). The low blood counts (cytopenias) which are characteristic of HCL improved in all complete and partial responders. Dose-limiting toxicity in HCL was due to a reversible hemolytic uremic syndrome (HUS), observed only during cycles 2 or 3. Already under way are a phase-II trial in HCL and phase-I trials in chronic lymphocytic leukemia (CLL) and acute lymphocytic leukemia (ALL) administering BL22 in a modified protocol in an effort to prevent HUS.     

Association and clonal distribution of trisomy 12 and 13q14 deletions in chronic lymphocytic leukaemia

The coexistence of trisomy 12 and deletions of chromosome 13 (13q12-q32) has rarely been observed in chronic lymphocytic leukaemia (CLL). Fluorescence in situ hybridization (FISH) performed on 600 consecutive CLL patients revealed the association of trisomy 12 and 13q14 deletion, of at least one of the three markers analysed (RB1, D13S319 and D13S25), in 55 cases (9% of 600 and 46% of 120 trisomy 12 cases). Trisomy 12 and isolated RB1 deletion were seen in 14/120 cases, trisomy 12 and D13S319/D13S25 deletion with diploid RB1 in 19/118, and trisomy 12 and deletion encompassing the three 13q markers studied in 22/118 cases. The heterogenous distribution of trisomy 12 and 13q deletions within the neoplastic B cells suggests that they are secondary rather than primary events in CLL leukaemogenesis.     

Reovirus therapy of lymphoid malignancies

Reoviruses infect cells that manifest an activated Ras-signaling pathway, and have been shown to effectively destroy many different types of neoplastic cells, including those derived from brain, breast, colon, ovaries, and prostate. In this study, we investigated the reovirus as a potential therapeutic agent against lymphoid malignancies. A total of 9 lymphoid cell lines and 27 primary human lymphoid malignancies, as well as normal lymphocytes and hematopoietic stem/progenitor cells, were tested for susceptibility to reovirus infection. For in vitro studies, the cells were challenged with reovirus (serotype 3 Dearing), and viral infection was assessed by cytopathic effects, viability, viral protein synthesis, and progeny virus production. We present evidence of efficient reovirus infection and cell lysis in the diffuse large B-cell lymphoma cell lines and Burkitt lymphoma cell lines Raji and CA46 but not Daudi, Ramos, or ST486. Moreover, when Raji and Daudi cell lines were grown subcutaneously in severe combined immunodeficient/nonobese diabetic (SCID/NOD) mice and subsequently injected with reovirus intratumorally or intravenously, significant regression was observed in the Raji-induced, but not the Daudi-induced, tumors, which is consistent with the in vitro results. Susceptibility to reovirus infection was also detected in 21 of the 27 primary lymphoid neoplasias tested but not in the normal lymphocytes or hematopoietic stem/progenitor cells. Our results suggest that reovirus may be an effective agent against several types of human lymphoid malignancies.     

TEL and KIP1 define the smallest region of deletions on 12p13 in hematopoietic malignancies

Unbalanced translocations as well as interstitial deletions of the short arm of chromosome 12 [del(12p)] are found as recurring chromosomal changes in a broad spectrum of hematopoietic malignancies. These changes result in the hemizygous deletion of genetic material from 12p. We mapped a yeast artificial chromosome containing the TEL gene, a cosmid contig containing part of TEL and a P1 contig containing the KIP1 gene to 12p13. These probes were used for fluorescence in situ hybridization to analyze samples from 47 patients with various hematologic malignancies who had unbalanced translocations (25 patients) leading to loss of 12p or deletions (22 patients) involving 12p13. The patients had acute lymphoblastic leukemia (8 cases), myelodysplastic syndrome (MDS; 11 cases), acute myeloid leukemia (AML; 10 cases), myeloproliferative disorders (4 cases), therapy-related MDS or AML (7 cases), non-Hodgkin's lymphoma (2 cases), and other hematopoietic malignancies (5 cases). All three probes were hemizygously detected in 26 cases and were completely retained in only 9 cases. In 12 cases probes for one of the two genes were deleted, allowing us to map the smallest region of overlap of these deletions to a small genomic region that is bordered on the telomeric side by the TEL gene and on the centromeric side by KIP1. The genomic distance between TEL and KIP1 is estimated to be about 1 to 2 Mbp.     

Translocations involving 13q14 without associated deletion in chronic lymphoid leukaemia target DLEU2

Burkitt lymphoma (BL) and Diffuse Large B‐Cell Lymphoma (DLBCL) account for most cases of non‐Hodgkin lymphoma (NHL) in childhood. We report the clinical characteristics, outcome and prognostic factors in children with BL or DLBCL treated according to the Associazione Italiana Ematologia Oncologia Pediatrica (AIEOP) LNH‐97 protocol. Patients aged up to 18 years that were newly diagnosed with BL/DLBCL were included in the study. Therapy consisted of pre‐phase followed by 2–6 high‐dose chemotherapy courses tailored according to lactate dehydrogenase (LDH) value and disease stage. A total of 442 patients (379 BL, 63 DLBCL) were enrolled between 1997 and 2014, of whom 18 failed to achieve remission, 6 experienced treatment‐related death, 2 developed second malignancy and 20 relapsed. At a median follow‐up time of 5 years, overall survival was 93% (±1%) and event‐free survival was 90% (±1%). LDH value above the median value had an independently negative prognostic value (P < 0·0001). However, in the subgroup of 128 patients in which minimal disseminated disease (MDD) was analysed, MDD‐positivity became the only unfavourable prognostic factor for progression‐free survival. Tailored chemotherapy could be extremely effective with limited toxicity. Identification of MDD as a hallmark of a higher risk of treatment failure may provide a target population for treatment intensification by anti‐CD20.     

Plasma-based detection of clonality in lymphoid malignancies

Objectives: Plasma has been found to be enriched with tumor-specific DNA, RNA, and protein in patients with hematologic disease. We assessed the utility of plasma as a DNA source for detection of genetic abnormalities in patients with suspected B- or T-cell lymphoproliferative disorders. Methods: DNA was extracted from paired peripheral blood (PB) cells and plasma for polymerase chain reaction (PCR)-based detection of immunoglobulin heavy chain (IgH) and T-cell receptor gamma chain (TCR-γ) rearrangements, and B-cell leukemia/lymphoma (BCL)-1/IgH and BCL-2/IgH translocations. Results: Concordance between plasma and PB cell analysis was 100% for IgH (n = 57), TCR-γ (n = 57), and BCL-1/IgH (n = 37) rearrangements, and 94% (60/64) for BCL-2/IgH; four of 11 plasma samples positive for BCL-2/IgH tested negative in paired cells. No plasma or PB cell samples from 195 healthy donors showed genetic abnormalities. Conclusions: These findings indicate that plasma is a reliable sample type for detection of abnormalities associated with B- and T-cell lymphoproliferative disorders, providing sensitivity equal to or greater than that of PB cells.     

Over-expression of cyclin D1 in chronic B-cell malignancies with abnormality of chromosome 11q13

Accurate identification of B-cell chronic malignancies is sometimes uncertain, despite careful cytologic and immunophenotypic evaluation. Cytogenetics and molecular biology studies may therefore prove useful, because some of these disorders are associated with nonrandom abnormalities, such as the t(11;14)(q13;q32) translocation and bcl-1 rearrangement mainly observed in mantle-cell lymphoma (MCL). We studied the expression of cyclin D1 in malignant lymphoid cells from the peripheral blood of 32 patients with various B-cell chronic lympho-proliferative disorders, using Northern blot (NB) and RNA in situ hybridization (ISH). Cytogenetic analysis was informative in 18 cases, and most of the missing karyotype data were from typical B-CLL cases where a t(11;14) is unlikely to be found. Over-expression of cyclin D1 mRNA was observed by both NB and ISH in four samples (MCL: two cases; lymphoplasmacytic lymphoma: one case, unclassified B-cell chronic disorder: one case). In each of these cases there was an abnormality of chromosome 11q13, either a t(11;14)(q13;q32) translocation (three cases) or a del(11)(q13) without evidence of chromosome 14 involvement (one case). Cytogenetic and gene rearrangement studies are not available in all institutions and have some technical pitfalls. Because of its close association with bcl-1 rearrangement and/or t(11;14), the demonstration of cyclin D1 mRNA over-expression either by NB, or, more conveniently, by ISH, may represent additional information which could be of help for the identification of B-cell malignancies.     

Toll-like receptors in lymphoid malignancies: Double-edged sword

Toll-like receptors (TLRs) are the best characterized pattern recognition receptors (PRRs), which play an essential role in the recognition of invading pathogens via specific microbial molecular motifs, comprising a bridge between the innate and adaptive immune responses. Toll-like receptors expression is determined in both normal immune cells and malignant cells, with a distinctive pattern compared to each other, rendering them plausible targets for cancer therapy. Improved molecular profiling of lymphoid malignancies may give new insights into pathogenesis of these cancers and pave the way for novel therapeutic agents, including TLR agonists. In the current review, we summarize the immunopathogenic roles of TLRs in B cell and T cell lymphomas, acute lymphoblastic leukemia, multiple myeloma, and chronic lymphocytic leukemia, as well as the results of studies on TLR ligands and their future implications to manage these hematologic malignancies.     

Significance of minimal residual disease in lymphoid malignancies

Modern treatment protocols lead to complete remission (CR) in a considerable proportion of patients with lymphoproliferative disorders. However, many of these patients ultimately relapse, implying that achievement of a clinical CR is compatible with significant amounts of residual malignant cells. Cytogenetic, molecular and immunological techniques that are more sensitive than morphology are increasingly used to assess and quantify minimal residual disease (MRD). Immunological marker analysis allows the detection of aberrant or unusual immunophenotypes, PCR techniques target fusion regions of chromosome aberrations and clone-specific immunoglobulin and T-cell receptor gene rearrangements. The rationale underlying MRD studies is to improve measurement of treatment response, to provide independent prognostic information and to optimise therapeutic strategies. In acute lymphoblastic leukemia (ALL), the MRD based evaluation of initial response to front-line therapy emerged as a highly relevant diagnostic tool, particularly in childhood ALL, where MRD has been shown to be an independent prognostic factor allowing a precise risk group classification. In patients with chronic lymphocytic leukemia (CLL) and non-Hodgkin's lymphoma (NHL) the prognostic significance of MRD is still a matter of debate, as the majority of patients remain MRD positive after conventional treatment. This phenomenon has changed with the implementation of new treatment modalities, such as application of monoclonal antibodies, where a significant proportion of patients with NHL converts to MRD negativity and experiences prolonged remission. Whether this molecular remission will translate into a superior overall survival is currently the goal of ongoing prospective studies.     

Somatic deletions in hereditary breast cancers implicate 13q21 as a putative novel breast cancer susceptibility locus

A significant proportion of familial breast cancers cannot be explained by mutations in the BRCA1 or BRCA2 genes. We applied a strategy to identify predisposition loci for breast cancer by using mathematical models to identify early somatic genetic deletions in tumor tissues followed by targeted linkage analysis. Comparative genomic hybridization was used to study 61 breast tumors from 37 breast cancer families with no identified BRCA1 or BRCA2 mutations. Branching and phylogenetic tree models predicted that loss of 13q was one of the earliest genetic events in hereditary cancers. In a Swedish family with five breast cancer cases, all analyzed tumors showed distinct 13q deletions, with the minimal region of loss at 13q21-q22. Genotyping revealed segregation of a shared 13q21 germ-line haplotype in the family. Targeted linkage analysis was carried out in a set of 77 Finnish, Icelandic, and Swedish breast cancer families with no detected BRCA1 and BRCA2 mutations. A maximum parametric two-point logarithm of odds score of 2.76 was obtained for a marker at 13q21 (D13S1308, theta = 0.10). The multipoint logarithm of odds score under heterogeneity was 3.46. The results were further evaluated by simulation to assess the probability of obtaining significant evidence in favor of linkage by chance as well as to take into account the possible influence of the BRCA2 locus, located at a recombination fraction of 0.25 from the new locus. The simulation substantiated the evidence of linkage at D13S1308 (P < 0.0017). The results warrant studies of this putative breast cancer predisposition locus in other populations.     

Liver biopsy immunotyping to characterize lymphoid malignancies

To determine the feasibility of liver biopsy immunotyping to characterize hepatic lymphoid malignancies, we employed a panel of monoclonal antibodies on snap-frozen hepatic tissue from 18 patients. Six patients proved to have a histologic diagnosis of lymphoid malignancy. By using free avidin and biotin-blocking reagents to block endogenous biotin, followed by standard immunochemistry, immunotyping was successful in all six cases. Serial section typing allowed delineation of complex B cell phenotypes. Furthermore, architecture was preserved allowing discernment of disease patterns (e.g., sinusoidal, hairy cell leukemia vs. portal, follicular small-cleaved cell lymphoma. Unexpectedly, we found striking expression of common ALL antigen in normal bile canaliculi, which may prove of diagnostic or therapeutic relevance. This study establishes the utility of immunohistochemical techniques applied to hepatic biopsies as a valuable adjunct to histologic diagnosis as well as a tool in revealing the immunobiology of the liver.