Multiple Myeloma Presenting in Patients Younger than 50 Years of Age: A Single Institution Experience.

INTRODUCTION: Multiple myeloma (MM) is uncommon in persons younger than 50 years of age. The presenting features in this age group are unclear. METHODS: We analyzed a cohort of patients <50 years of age with MM treated in our center. RESULTS: Twenty-three patients at a median age of 41.5 years (range 27-49) were analyzed. Patients presented at International Staging System (ISS) I-II (79%), had high frequency of bone lytic lesions (89%), extramedullary disease (26%), light-chain myeloma (45%), and translocation t(11;14) (68%). The subpopulation of patients carrying t(11;14) were younger (p = 0.025). This subgroup had higher bone marrow infiltration of plasma cells (75 vs. 47.5%), higher incidence of proteinuria (2.9 vs. 0.19 g/day), and poorer response to therapy: 85.7% of patients achieving complete/very good partial remission after induction therapy did not have t(11;14). A trend toward inferior progression-free survival (PFS) was observed in patients with t(11; 14) compared to patients without this translocation (median PFS 18 and 36 months, respectively). DISCUSSION/CONCLUSION: Translocation t(11; 14) seems to be more prevalent in young myeloma patients. Young myeloma patients and especially those who harbor translocation t(11; 14) may represent a distinct clinical entity that confers a poor outcome.

Newly diagnosed multiple myeloma patients carrying monoallelic deletion of the whole locus of immunoglobulin heavy chain gene have a better prognosis compared to those with t(4;14) and t(14;16).

The current study evaluated the prognostic significance of the monoallelic deletion of the whole locus of the immunoglobulin heavy-chain (w_del(IGH)) gene compared to translocations t(4;14) and t(14;16) among newly diagnosed multiple myeloma (MM) patients. We retrospectively analyzed clinical (age, gender, and staging) and laboratory data at diagnosis and the overall survival (OS) of 255 newly diagnosed MM patients carrying w_del(IGH) or translocations t(4;14) or t(14;16). Bone marrow samples were examined by morphological and sequential interphase fluorescense in situ hybridization analyses. Among 255 patients, 117 (45.8%) had w_del(IGH), 99 (38.8%) had t(4;14), and 39 (15.3%) had t(14;16). Mean age was 61.6 ± 11.6 years. Groups did not differ significantly in age, gender, or lactate dehydrogenase levels. Patients in the w_del(IGH) group presented more frequently at International Staging System stage I than at stage II/III. Patients in the w_del(IGH) group had significantly fewer additional chromosomal aberrations (1.58) than the other two groups (2.3 and 2.13 in the del(IGH), t(14;16) and t(4;14) groups, respectively, P < 0.0001). Furthermore, the w_del(IGH) group had significantly longer estimated median OS (9.47 years) compared to those with translocations t(14;16) (3.02 years, P = 0.002) or t(4;14) (4.18 years, P = 0.001), respectively. These findings suggest a potential prognostic significance of monoallelic deletion of IGH among these patients. Additional studies are needed to better understand the nature and mechanism of this prognostic factor.

Sustained Response to Imatinib in a Pediatric Patient with Concurrent Myeloproliferative Disease and Lymphoblastic Lymphoma Associated with a CCDC88C-PDGFRB Fusion Gene.

BACKGROUND: The WHO defined myeloid and lymphoid neoplasms (MLN) with eosinophilia associated with PDGFRB, PDGFRA, FGFR1 rearrangements as a new entity in 2016. PDGFRB-rearranged MLN sensitive to imatinib were described in adult patients. We report the first pediatric patient with PDGFRB-rearranged myeloproliferative disorder associated with T-lymphoblastic lymphoma bearing the t(5; 14)(q33;q32) translocation who was successfully treated with imatinib only. Methods/Aims: Analysis of bone marrow and peripheral blood cells by fluorescent in situ hybridization identified the PDGFRB partner as CCDC88C. Whole genome sequencing of the patient's DNA identified the exact junction site, confirmed by PCR amplification and Sanger sequencing. A real-time quantitative PCR assay was designed to quantify the fused CCDC88C-PDGFRB product. RESULTS: A 2.5-year-old boy was diagnosed with myeloproliferative disorder and eosinophilia associated with lymphoblastic lymphoma both bearing the CCDC88C-PDGFRB fusion. Imatinib therapy resulted in rapid clinical, hematological, and cytogenetic response. Molecular response to treatment was monitored by a real-time PCR assay specific for the CCDC88C- PDGFRB fusion. CONCLUSION: This is the first description of MLN with eosinophilia in the pediatric age group. Response to treatment with imatinib only was monitored by specific quantitative PCR assay with sustained remission lasting 5.5 years from diagnosis.

Quercetin alters the DNA damage response in human hematopoietic stem and progenitor cells via TopoII- and PI3K-dependent mechanisms synergizing in leukemogenic rearrangements.

Quercetin (Que) is an abundant flavonoid in the human diet and high-concentration food supplement with reported pro- and anti-carcinogenic activities. Topoisomerase II (TopoII) inhibition and subsequent DNA damage induction by Que was implicated in the mixed lineage leukemia gene (MLL) rearrangements that can induce infant and adult leukemias. This notion raised concerns regarding possible genotoxicities of Que in hematopoietic stem and progenitor cells (HSPCs). However, molecular targets mediating Que effects on DNA repair relevant to MLL translocations have not been defined. In this study we describe novel and potentially genotoxic Que activities in suppressing non-homologous end joining and homologous recombination pathways downstream of MLL cleavage. Using pharmacological dissection of DNA-PK, ATM and PI3K signalling we defined PI3K inhibition by Que with a concomitant decrease in the abundance of key DNA repair genes to be responsible for DNA repair inhibition. Evidence for the downstream TopoII-independent mutagenic potential of Que was obtained by documenting further increased frequencies of MLL rearrangements in human HSPCs concomitantly treated with Etoposide and Que versus single treatments. Importantly, by engaging a tissue engineered placental barrier, we have established the extent of Que transplacental transfer and hence provided the evidence for Que reaching fetal HSPCs. Thus, Que exhibits genotoxic effects in human HSPCs via different mechanisms when applied continuously and at high concentrations. In light of the demonstrated Que transfer to the fetal compartment our findings are key to understanding the mechanisms underlying infant leukemia and provide molecular markers for the development of safety values.

Translocation t(11;14) in newly diagnosed patients with multiple myeloma: Is it always favorable?

The most common translocation in multiple myeloma (MM) is t(11;14)(q13;q32). According to several studies, this translocation represents a unique subset of patients with relatively favorable outcomes. Using combined analyses of morphology and fluorescence in situ hybridization (I-FISH), we examined the co-occurrence rates of t(11;14) with seven chromosomal aberrations (CAs), del(13q), del(17p), del(1p), gain(1q), multiple gains(1q), del(16q), and del(IGH), and assessed the effect of the different combinations on patient outcomes, with overall survival (OS) as the main outcome measure. Bone marrow samples and clinical data from 212 patients with MM with t(11;14) were analyzed. At least two additional CAs were found in 35% (75/205) of patients and a strong correlation between specific CAs. The occurrence of three CAs [multiple gains of (1q) (HR = 6.94, P = 0.001), del(1p) (HR = 4.47, P = 0.008), and del(IGH) (HR = 2.38, P = 0.002)] exerted a profoundly deleterious effect on median OS when compared with patients with t(11;14) only. Del(17p) and del(13q) have also exerted a deleterious effect albeit to a lesser extent (HR = 2.05, P = 0.07 and HR = 1.81, P = 0.03, respectively). When compared with t(11;14) alone, the addition of certain CAs lead to worse outcomes. These findings may have important clinical and biological implications. Patients with coexisting adverse lesions and t(11;14) may be considered at high risk and managed accordingly. © 2016 Wiley Periodicals, Inc.

Fluorescence lifetime imaging of DAPI-stained nuclei as a novel diagnostic tool for the detection and classification of B-cell chronic lymphocytic leukemia.

B-cell chronic lymphocytic leukaemia (B-CLL) and B-cell precursor acute lymphoblastic leukaemia (B-ALL) are the most common type of leukaemia in adults and children, respectively. Today, fluorescence in situ hybridization (FISH) is the standard for detecting chromosomal aberrations that reflect adverse and favorable outcome. This study revealed a new, simple, and fast diagnostic tool to detect pathological cells by measuring and imaging the fluorescence lifetime (FLT) using FLT imaging microscopy (FLIM) of the peripheral blood (PB) cells of B-CLL samples that were labeled with the DNA binder, DAPI. The FLT of DAPI in healthy individuals was found to be 2.66 ± 0.12 ns. In contrast, PB cells of B-CLL and BM cells of B-ALL patients were characterized by a specific group distribution of the FLT values. The FLT of DAPI was divided into four subgroups, relative to 2.66 ns: short+, normal, prolonged, and prolonged+. These alterations could be related to different chromatin arrangements of B-CLL and B-ALL interphase nuclei. Notably, extremely long FLT of nuclear DAPI correlate with the presence of extra chromosome 12, while moderate increases compared to normal characterize the deletion of p53. Such correlations potentially enable a FLT-based rapid automatic diagnosis and classification of B-CLL even when the frequency of genetic and chromosomal abnormalities is low. © 2016 International Society for Advancement of Cytometry.

Severe eosinophilia in children: a diagnostic dilemma.

The differential diagnosis of hypereosinophilia includes both primary (clonal and idiopathic) and secondary medical conditions. Here we raise the awareness of physicians to the unusual causes of hypereosinophilic states and describe the molecular assays used in the diagnosis of hypereosinophilia. Two unusual cases of hypereosinophilia in children that were initially misdiagnosed are reported. T-cell receptor gene rearrangement, skewed X inactivation, fluorescence in situ hybridization analysis, and chromosomal karyotyping were used to reach the final correct diagnosis. Both patients displayed significant eosinophilia and were initially misdiagnosed as having parasitic infection. Nonspecific T-cell clonal expansion was diagnosed in 1 patient based on the clonality of the T-cell receptor variable γ-chain and the skewed chromosome inactivation. The second patient was diagnosed with B-lineage acute lymphoblastic leukemia with a translocation (5;14) (q13;q32) that is well known to be associated with hypereosinophilia. The level of awareness to clonal expansion of WBC subsets which can cause hypereosinophilia should be high when evaluating a patient with extreme eosinophilia. Advanced molecular assays to detect clonal expansion should be used to exclude aberrant clonal processes in such patients.

Treatment with interferon alpha prior to discontinuation of imatinib in patients with chronic myeloid leukemia.

Imatinib (IM) is the current first line treatment for chronic myeloid leukemia (CML). However, the disease will progress in the majority of patients pausing IM. IFN-α may intensify the response and increase the percentage of patients maintaining remission after IM cessation. Eleven patients with stable (≥ 2 years) complete cytogenetic responses (CCyR) on IM therapy were recruited to the study. They were administered Peg-IFN-α for 9 months before and for 3 months following IM discontinuation. During the 12 months of Peg-IFN-α therapy the remission status improved in five (45%) of the patients. Six (55%) of the patients experienced cytogenetic relapses at a median period of 8 months (range 2-33) after IM withdrawal. All six patients regained CCyR following IM restart. With a median follow up of 47 months (range 35-50), five (45%) out of the 11 studied patients maintain cytogenetic response off IM therapy. The role of Peg-IFN-α in patients pausing IM is to be further evaluated.

Characterizing T cells in SCID patients presenting with reactive or residual T lymphocytes.

INTRODUCTION: Patients with severe combined immunodeficiency (SCID) may present with residual circulating T cells. While all cells are functionally deficient, resulting in high susceptibility to infections, only some of these cells are causing autoimmune symptoms. METHODS: Here we compared T-cell functions including the number of circulating CD3(+) T cells, in vitro responses to mitogens, T-cell receptor (TCR) repertoire, TCR excision circles (TREC) levels, and regulatory T cells (Tregs) enumeration in several immunodeficinecy subtypes, clinically presenting with nonreactive residual cells (MHC-II deficiency) or reactive cells. The latter includes patients with autoreactive clonal expanded T cell and patients with alloreactive transplacentally maternal T cells. RESULTS: MHC-II deficient patients had slightly reduced T-cell function, normal TRECs, TCR repertoires, and normal Tregs enumeration. In contrast, patients with reactive T cells exhibited poor T-cell differentiation and activity. While the autoreactive cells displayed significantly reduced Tregs numbers, the alloreactive transplacentally acquired maternal lymphocytes had high functional Tregs. CONCLUSION: SCID patients presenting with circulating T cells show different patterns of T-cell activity and regulatory T cells enumeration that dictates the immunodeficient and autoimmune manifestations. We suggest that a high-tolerance capacity of the alloreactive transplacentally acquired maternal lymphocytes represents a toleration advantage, yet still associated with severe immunodeficiency.

A role for common fragile site induction in amplification of human oncogenes.

Oncogene amplification is an important process in human tumorigenesis, but its underlying mechanism is currently unknown. Cytogenetic analysis indicates that amplification of drug-selected genes in rodent cells is driven by recurrent breaks within chromosomal common fragile sites (CFSs), via the breakage-fusion-bridge (BFB) mechanism. Here we show that BFB cycles drive the intrachromosomal amplification of the MET oncogene in a human gastric carcinoma. Our molecular evidence includes a "ladder-like" structure and inverted repeat organization of the MET amplicons. Furthermore, we show that the breakpoints, setting the centromeric amplicon boundaries, are within the CFS FRA7G region. Upon replication stress, this region showed perturbed chromatin organization, predisposing it to breakage. Thus, in vivo induction of CFSs can play an important role in human oncogenesis.

Correlation between losses of IGH or its segments and deletions of 13q14 in t(11;14) (q13;q32) multiple myeloma.

Multiple myeloma (MM) is a malignancy of the plasma cells (PCs) characterized by a wide variety of genetic and chromosomal abnormalities. In recent years, major attention was drawn to the significance of chromosomal aberrations involving chromosome arm 13q and the IGH region on chromosome band 14q32 as a prognostic indicator in MM. In this study we applied a combined cell morphology and FISH method for the analysis of coexistence of t(11;14)(q13;q32) with deletions of the long arm of chromosome 13 (Delta13) in PCs from 51 MM patients using several probes for the 13q14, 11q13, and IGH regions. We found 15 different variants of the t(11;14) that are the consequence of different 11q13 breakpoints and various deletions of Variable (del IGH Var) or Constant (del IGH Const) IGH segments and also duplications and losses of the IGH gene on the normal nontranslocated chromosome 14 as well as IGH/Cyclin D1 (CCND1) fusion on der(14) and CCND1/IGH fusions on der(11). A strong association between Delta13 and specific variants of t(11;14) was found: variants with deletion of the IGH gene or its segments were found only in MM cases with deleted chromosome 13, while the common translocation t(11;14) was found only in the MM cases with normal chromosome arm 13q. In contrast, we did not find any association between Delta13 and deletions of the IGH gene or its segments in the MM patients with t(4;14)(p16;q32).

SMURF2 prevents detrimental changes to chromatin, protecting human dermal fibroblasts from chromosomal instability and tumorigenesis.

E3 ubiquitin ligases (E3s) play essential roles in the maintenance of tissue homeostasis under normal and stress conditions, as well as in disease states, particularly in cancer. However, the role of E3s in the initiation of human tumors is poorly understood. Previously, we reported that genetic ablation of the HECT-type E3 ubiquitin ligase Smurf2 induces carcinogenesis in mice; but whether and how these findings are pertinent to the inception of human cancer remain unknown. Here we show that SMURF2 is essential to protect human dermal fibroblasts (HDFs) from malignant transformation, and its depletion converts HDFs into tumorigenic entity. This phenomenon was associated with the radical changes in chromatin structural and epigenetic landscape, dysregulated gene expression and cell-cycle control, mesenchymal-to-epithelial transition and impaired DNA damage response. Furthermore, we show that SMURF2-mediated tumor suppression is interlinked with SMURF2's ability to regulate the expression of two central chromatin modifiers-an E3 ubiquitin ligase RNF20 and histone methyltransferase EZH2. Silencing these factors significantly reduced the growth and transformation capabilities of SMURF2-depleted cells. Finally, we demonstrate that SMURF2-compromised HDFs are highly tumorigenic in nude mice. These findings suggest the critical role that SMURF2 plays in preventing malignant alterations, chromosomal instability and cancer.

Donor-derived brain tumor following neural stem cell transplantation in an ataxia telangiectasia patient.

BACKGROUND: Neural stem cells are currently being investigated as potential therapies for neurodegenerative diseases, stroke, and trauma. However, concerns have been raised over the safety of this experimental therapeutic approach, including, for example, whether there is the potential for tumors to develop from transplanted stem cells. METHODS AND FINDINGS: A boy with ataxia telangiectasia (AT) was treated with intracerebellar and intrathecal injection of human fetal neural stem cells. Four years after the first treatment he was diagnosed with a multifocal brain tumor. The biopsied tumor was diagnosed as a glioneuronal neoplasm. We compared the tumor cells and the patient's peripheral blood cells by fluorescent in situ hybridization using X and Y chromosome probes, by PCR for the amelogenin gene X- and Y-specific alleles, by MassArray for the ATM patient specific mutation and for several SNPs, by PCR for polymorphic microsatellites, and by human leukocyte antigen (HLA) typing. Molecular and cytogenetic studies showed that the tumor was of nonhost origin suggesting it was derived from the transplanted neural stem cells. Microsatellite and HLA analysis demonstrated that the tumor is derived from at least two donors. CONCLUSIONS: This is the first report of a human brain tumor complicating neural stem cell therapy. The findings here suggest that neuronal stem/progenitor cells may be involved in gliomagenesis and provide the first example of a donor-derived brain tumor. Further work is urgently needed to assess the safety of these therapies.

The SIL gene is essential for mitotic entry and survival of cancer cells.

Although mitosis is a general physiologic process, cancer cells are unusually sensitive to mitotic inhibitors. Therefore, there is an interest in the identification of novel mitotic inhibitors. Here, we report the novel discovery of the SIL gene as a regulator of mitotic entry and cell survival. The SIL gene was cloned from leukemia-associated chromosomal translocation. It encodes a cytosolic protein with an unknown function and no homology to known proteins. Previously, we observed an increased expression of SIL in multiple cancers that correlated with the expression of mitotic spindle checkpoint genes and with increased metastatic potential. Here, we show that SIL is important for the transition from the G(2) to the M phases of the cell cycle. Inducible knockdown of SIL in cancer cells in vitro delayed entrance into mitosis, decreased activation of the CDK1 (CDC2)-cyclin B complex, and induced apoptosis in a p53-independent manner. SIL is also essential for the growth of tumor explants in mice. Thus, SIL is required for mitotic entry and cancer cell survival. Because increased expression of SIL has been noted in multiple types of cancers and correlates with metastatic spread, it may be a suitable target for novel anticancer therapy.

Is fluorescence in-situ hybridization sufficient in patients with myelodysplastic syndromes and insufficient cytogenetic testing?

Chromosome banding analysis (CBA) in myelodysplastic syndromes (MDS) remains the 'gold standard' for identification of chromosomal abnormalities, while interphase fluorescence in-situ hybridization (I-FISH) is mainly used to complement CBA. This study, retrospectively, evaluated CBA and I-FISH results in 600 patients with suspected MDS and determined the effect of CBA/FISH reallocation on IPSS-R. Our result demonstrated that in 7/586 (1.2%) patients with satisfactory karyotype, I-FISH provided additional information. In 25/453 (5.5%) of the patients with normal I-FISH, CBA detected chromosomal abnormalities, and in 68/147 (46%) of the patients with abnormal I-FISH, CBA detected additional chromosomal aberrations. When 5q- aberration was alone or accompanied by additional abnormalities by I-FISH, CBA revealed a complex karyotype (16/25;64%, 35/43;81%, respectively). Our results suggest that in cases of karyotype failure, if I-FISH is used alone, patients are at risk of being misclassified into the wrong cytogenetic risk groups and a repeat sample for CBA should be attempted.

A new MALDI-TOF-based assay for monitoring JAK2 V617F mutation level in patients undergoing allogeneic stem cell transplantation (allo SCT) for classic myeloproliferative disorders (MPD).

JAK2 V617F mutation is found in a high proportion of MPD patients. We developed a quantitative assay for the detection of the JAK2 mutation and demonstrated its clinical utility in MPD patients who underwent SCT. Sixty percent of the patients were JAK2 V617F positive prior to the SCT (mean mutation levels 74%, range 16-98%). After the procedure, the mutation levels progressively decreased and were in correlation with the donor-recipient chimerism status (r=0.97, p<0.001). At a median follow up of 16 months (range 2-58), 9/15 patients are alive and in CR. The levels of the JAK2 V617F mutation reached 0% in all surviving patients.

SKY reveals a high frequency of unbalanced translocations involving chromosome 6 in t(12;21)-positive acute lymphoblastic leukemia.

The G-band cryptic t(12;21)(p13;q22) is the most common chromosomal rearrangement in childhood acute lymphoblastic leukemia (ALL). To investigate the nature of additional chromosomal events in this group of patients spectral karyotyping (SKY) following G-banding analysis was performed in 14 cases. From these cases six showed structural aberrations of chromosome 6, including both simple deletions and unbalanced translocations, and involved both q (n=4) and p (n=3) arms. The results show that rearrangements of 6p are also non-random events t(12;21)-positive ALL. This study illustrates the value of a combined SKY and G-banding approach in identifying novel karyotypic events in childhood ALL.

Malignant transformation of normal enterocytes following downregulation of Bak expression.

Bak is a pro-apoptotic gene, which plays an important role in the multi-step process of gastrointestinal tumorigenesis. We hypothesized that downregulation of Bak expression in normal enterocytes will result in a transformed phenotype. The nontumorigenic intestinal epithelial cell line (IEC18) was transfected with the vector pMV12-AS-bak (encoding anti-sense bak). Three clones, with Bak protein levels similar to those seen in colon cancer cell lines and significantly lower than those found in the parental cells, were further evaluated. The three clones proliferated faster, demonstrated anchorage-independent growth in soft agar and a higher saturation density and plating efficiency. Furthermore, when injected into nude mice, these cells generated tumors after approximately 2-3 weeks. The cells were more resistant to the induction of apoptosis by sulindac sulfide and sulindac sulfone but more sensitive to COX 2 inhibitors (celecoxib and nimesulide). The levels of p16, cyclin D1 and COX 2 were higher in the three transformed clones. In summary,downregulation of Bak expression in normal enterocytes contributes to abnormal growth and tumorigenesis. COX 2 inhibitors may serve as important agents in the prevention and treatment of CRC as they only inhibit the growth of malignant cells.

Chromosome 13q deletion and IgH abnormalities may be both masked by near-tetraploidy in a high proportion of multiple myeloma patients: a combined morphology and I-FISH analysis.

Ploidy status and chromosomal aberrations involving chromosome 13q and the immunoglobulin heavy chain locus (IgH) are important prognostic features in multiple myeloma (MM). However, conventional cytogenetic studies are often not reveling and determination of plasma cells (PC) ploidy status in MM is technically difficult. We have used a combined cell morphology and interphase FISH (I-FISH) analysis in 184 consecutive BM samples from 136 MM patients for the diagnosis of chromosome 13q deletion [del (13q)] and IgH abnormalities. We have found a high prevalence (37%) of near-tetraploid (NT) PC in the BM samples studied. NT status of PC was verified with DNA index (DI) measurements. del (13q) was found in 69% and a total absence of one IgH copy (loss of IgH) in 20% of NT samples. We have shown that the presence of del (13q) and loss of IgH can be masked in NT cases: in 12 NT samples originally identified as normal for del (13q) the abnormality was obscured in the majority of plasma cells due to the presence of NT. Similarly, loss of IgH was masked in four samples with a large population of NT cells. Moreover, in one case the appearance of a 100% tetraploidy during disease progression masked the presence of del (13q), originally present, and could therefore falsely appear as disappearance of this prognostic marker. In conclusion, we have shown that a combination of three abnormalities, i.e., del (13q), loss of IgH and NT, all of potential prognostic significance, can be overlooked unless NT is specifically searched for and ruled out. Therefore, we suggest that a search for NT should be added to the routine BM assessment in MM patients.