Overexpression of EZH2 in multiple myeloma is associated with poor prognosis and dysregulation of cell cycle control.

Myeloma is heterogeneous at the molecular level with subgroups of patients characterised by features of epigenetic dysregulation. Outcomes for myeloma patients have improved over the past few decades except for molecularly defined high-risk patients who continue to do badly. Novel therapeutic approaches are, therefore, required. A growing number of epigenetic inhibitors are now available including EZH2 inhibitors that are in early-stage clinical trials for treatment of haematological and other cancers with EZH2 mutations or in which overexpression has been correlated with poor outcomes. For the first time, we have identified and validated a robust and independent deleterious effect of high EZH2 expression on outcomes in myeloma patients. Using two chemically distinct small-molecule inhibitors, we demonstrate a reduction in myeloma cell proliferation with EZH2 inhibition, which leads to cell cycle arrest followed by apoptosis. This is mediated via upregulation of cyclin-dependent kinase inhibitors associated with removal of the inhibitory H3K27me3 mark at their gene loci. Our results suggest that EZH2 inhibition may be a potential therapeutic strategy for the treatment of myeloma and should be investigated in clinical studies.

Bi-allelic inactivation is more prevalent at relapse in multiple myeloma, identifying RB1 as an independent prognostic marker.

The purpose of this study is to identify prognostic markers and treatment targets using a clinically certified sequencing panel in multiple myeloma. We performed targeted sequencing of 578 individuals with plasma cell neoplasms using the FoundationOne Heme panel and identified clinically relevant abnormalities and novel prognostic markers. Mutational burden was associated with maf and proliferation gene expression groups, and a high-mutational burden was associated with a poor prognosis. We identified homozygous deletions that were present in multiple myeloma within key genes, including CDKN2C, RB1, TRAF3, BIRC3 and TP53, and that bi-allelic inactivation was significantly enriched at relapse. Alterations in CDKN2C, TP53, RB1 and the t(4;14) were associated with poor prognosis. Alterations in RB1 were predominantly homozygous deletions and were associated with relapse and a poor prognosis which was independent of other genetic markers, including t(4;14), after multivariate analysis. Bi-allelic inactivation of key tumor suppressor genes in myeloma was enriched at relapse, especially in RB1, CDKN2C and TP53 where they have prognostic significance.

Clinical value of molecular subtyping multiple myeloma using gene expression profiling.

Using a data set of 1217 patients with multiple myeloma enrolled in Total Therapies, we have examined the impact of novel therapies on molecular and risk subgroups and the clinical value of molecular classification. Bortezomib significantly improved the progression-free survival (PFS) and overall survival (OS) of the MMSET (MS) subgroup. Thalidomide and bortezomib positively impacted the PFS of low-risk (LoR) cases defined by the GEP70 signature, whereas high-risk (HiR) cases showed no significant changes in outcome. We show that molecular classification is important if response rates are to be used to predict outcomes. The t(11;14)-containing CD-1 and CD-2 subgroups showed clear differences in time to response and cumulative response rates but similar PFS and OS. Furthermore, complete remission was not significantly associated with the outcome of the MAF/MAFB (MF) subgroup or HiR cases. HiR cases were enriched in the MF, MS and proliferation subgroups, but the poor outcome of these groups was not linked to subgroup-specific characteristics such as MAF overexpression per se. It is especially important to define risk status if HiR cases are to be managed appropriately because of their aggressive clinical course, high rates of early relapse and the need to maintain therapeutic pressure on the clone.

Molecular delineation of deletions on 2q37.3 in three cases with an Albright hereditary osteodystrophy-like phenotype.

A minority of the reported cases of terminal 2q37 deletion clinically resemble Albright hereditary osteodystrophy (AHO)/pseudopseudohypoparathyroidism and have only mild-to-moderate mental retardation. Our molecular and cytogenetic fluorescence in situ hybridization (FISH) findings on an additional three patients further reduce the size of the minimal critical region deleted in this syndrome to about 3 Mb. This region includes the G-protein-coupled receptor 35 (GPR35), glypican 1 (GPC1), and serine/threonine protein kinase 25 (STK25) genes on 2q37.3. We have further defined several polymorphic variants within the coding region and flanking regions of GPR35 gene, which could potentially be useful for rapid detection of GPR35 gene deletion. We postulate that the absence of GPR35 may, at least partly, account for the phenotypic resemblance to the AHO. We also believe that the deletion of GPR35 could be responsible for the entity brachydactyly mental retardation syndrome (OMIM #600430), which was coined based on the above minority of patients with terminal 2q37 deletion. We recommend that every patient with AHO phenotype should undergo 2q subtelomeric FISH screen and subsequently a molecular study on the GPR35 gene. GPC1 and/or STK25 haploinsufficiency may also contribute to the AHO-like phenotype.

Extramedullary granulopoiesis mimicking recurrent lymphoma after prolonged administration of human recombinant granulocyte colony-stimulating factor.

Human recombinant granulocyte colony-stimulating factor (G-CSF) has become a treatment of choice for neutropenia of diverse etiologies. We describe a 71-year-old man who, while receiving G-CSF for graft failure after peripheral blood stem cell transplant, developed dramatic extramedullary granulopoiesis that mimicked recurrent lymphoma.

Modified giemsa-11 staining protocol for chromosomes of human and hybrid cells.

Giemsa-11 or G-11 is a specialized staining technique utilized to (1) differentiate heterochromatic regions of human chromosomes, (2) identify the presence of human chromosomes in human-rodent hybrid cells, and (3) identify human-rodent translocation products in hybrid cells. Earlier procedures, though useful, are problematic and may fail to yield results due to inadequate differentiation between light and dark staining regions. The improved protocol presented here is easy, reliable, and applicable in both clinical and research situations. A discussion of the biology of the staining process is also given.

Delineation of 14q32.3 deletion syndrome.

A patient with a 14q32.3 terminal band deletion and cat cry is reported. Review of four other 14q32.3 deletion cases suggests the possible presence of a recognisable 14q32.3 terminal deletion syndrome, which is characterised by (1) apparently postnatal onset of small head size in comparison to body size, (2) high forehead with lateral hypertrichosis, (3) epicanthic folds, (4) broad nasal bridge, (5) high arched palate, (6) single palmar crease, and (7) mild to moderate developmental delay. Although none of the above seven features in unique to this syndrome, and indeed are quite common in other chromosomal disorders or genetic syndromes, patients with a terminal 14q32.3 deletion do show a recognisable facial gestalt. Interestingly, unlike ring chromosome 14, the 14q32.3 terminal deletion has rarely been reported, possibly because it is harder to detect, and an optimal chromosome preparation is required for its identification.

Interstitial 6q deletion and Prader-Willi-like phenotype.

A third case of an interstitial deletion of the long arm of chromosome 6 with clinical features mimicking Prader-Willi syndrome (PWS) is presented. Although preliminary clinical evaluation in each case suggested PWS, further review revealed that the features in all three cases are not completely compatible with the characteristic findings in Prader-Willi syndrome. Furthermore, the deletions in the three cases do not show a consistent region of overlap. Consequently, no particular band or region in 6q can be defined as associated with obesity. However, our findings confirm the suggestion of Villa et al. in 1995, that individuals with a PWS phenotype who are cytogenetically and molecularly negative for a deletion of 15q11-q13 should be examined for a deletion of 6q.

CD8+ polylobated T-cell leukemia/lymphoma. A case report with immunophenotypic, ultrastructural, gene rearrangement, karyotypic, and DNA content analysis and autopsy description.

Polylobated lymphoma is a morphologic variant of malignant lymphoma characterized by large pleomorphic neoplastic cells with polylobated nuclei. We report an unusual case in a 57-year-old man with a 9-year history of an antecedent low-grade peripheral T-cell lymphoma with dermal involvement. The polylobated lymphoma expressed the CD2, 8, 45, and DR surface antigens and had a clonally rearranged T-cell receptor beta-chain gene. The DNA content analysis indicated that most of the neoplasm was DNA diploid and tetraploid, with a high S phase. Cytogenetic analysis demonstrated the presence of a clone with an abnormal karyotype 45, X, -Y, -1, -10, -10, -17, -19, +5 mar. Serology and polymerase chain reaction analysis showed no evidence of retroviral infection (human immunodeficiency virus types 1 and 2 and human T-cell lymphotropic virus types I and II). We review the literature on polylobated T-cell lymphoma.

Endometrial adenocarcinoma without prior hormone replacement in a diabetic patient with gonadal dysgenesis.

Patients with dysgenetic gonads and Turner syndrome are unlikely to develop endometrial carcinoma unless they have received unopposed estrogen replacement therapy. This case describes a 54-year-old woman with Turner syndrome and primary amenorrhea who developed adenocarcinoma of the endometrium without having received hormone replacement. Vaginal bleeding, a pelvic mass, and sepsis were the presenting symptoms. The patient also had diabetes mellitus and hypothyroidism. Polyglandular endocrine patterns are known to occur with a high frequency in these patients. The woman's chromosome studies revealed a modified 46,X,i(Xq) (isochromosome X). This is the first report of an isochromosome X patient to develop endometrial cancer without receiving estrogen replacement. The etiology of this rare case may be an increased propensity for patients with X-chromosome deletions to develop neoplasms in general, or extragonadal estrogen production.

Localization of DNA sequences to a region within Xp11.21 between incontinentia pigmenti (IP1) X-chromosomal translocation breakpoints.

Incontinentia pigmenti (IP) is an X-linked dominant disorder characterized by developmental anomalies of the tissues and organs derived from embryonic ectoderm and neuroectoderm. An IP locus, designated IP1, probably resides in Xp11.21, since five unrelated patients with nonfamilial IP have been identified who possess constitutional de novo reciprocal X;autosome translocations involving Xp11.21. We have used a series of somatic cell hybrids containing the rearranged chromosomes derived from three of the five IP1 patients, along with other hybrid cell lines, to map probes in the vicinity of the IP1 locus. Five anonymous DNA loci--DXS422, DXS14, DXS343, DXS429, and DXS370--have been mapped to a region within Xp11.21, between two IP1 X-chromosomal translocation breakpoints; the IP1 t(X;17) breakpoint is proximal (centromeric) to this region, and the IP1 t(X;13) and t(X;9) X-chromosomal breakpoints lie distal to it. While no IP1 translocation breakpoint has yet been identified by pulsed-field gel electrophoretic (PFGE) analysis, an overlap between three probes--p58-1, 7PSH3.5, and cpX210--has been detected, placing these probes within 125 kb. Four probes--p58-1, 7PSH3.5, cpX210, and 30CE2.8--have been helpful in constructing a 1,250-kb PFGE map of the region between the breakpoints; these results suggest that the IP1 X-chromosomal translocation breakpoints are separated by at least this distance. The combined somatic cell hybrid and PFGE analyses we report here favor the probe order DXS323-(IP1 t(X;13), IP1, t(X;9]-(DXS422, DXS14, DXS343, DXS429, DXS370)-(IP1 t(X;17), DXZ1). These sequences provide a starting point for identifying overlapping genomic sequences that span the IP1 translocation breakpoints; the availability of IP1 translocation breakpoints should now assist the cloning of this locus.

Molecular and cytogenetic analysis of tumors in von Recklinghausen neurofibromatosis.

Von Recklinghausen neurofibromatosis (NF1) is a common autosomal dominant disorder mapped to 17q11.2 and typically characterized by the occurrence of neural crest-derived tumors. The gene has recently been cloned using reverse genetics or "positional cloning" approaches. Its function, however, remains unknown. We have performed cytogenetic and molecular analyses on 9 malignant tumors from NF1 patients to look for loss of alleles or chromosome rearrangements involving chromosome 17 to test the hypothesis that the NF1 gene acts as a recessive "tumor suppressor" gene. Loss of alleles on this chromosome was detected for 3 of 9 malignant tumors. Two peripheral nerve sheath tumors showed allele loss at informative loci on both the long and short arms of chromosome 17. In contrast, a glioblastoma with focal gliosarcoma showed loss of heterozygosity on the short arm of chromosome 17 only, and not at loci on the long arm. One nerve sheath tumor was previously shown by direct sequence analysis to have a point mutation at the TP53 locus at 17p13. These data support a role for the TP53 gene or other genes on the short arm of chromosome 17 in at least some malignancies in NF1. Six other neurofibrosarcomas showed no allele loss at informative loci on chromosome 17. Cytogenetic analysis was performed on 7 tumors, including 2 with allele loss. The two tumors with allele loss showed abnormal karyotypes while all others were normal. Southern blot and pulsed-field gel analysis using probes within or closely linked to the NF1 locus detected no gross deletions or rearrangements in the tumors studied.(ABSTRACT TRUNCATED AT 250 WORDS)

Association of a mature B cell leukemia with a 4p+ chromosomal abnormality: derivation and characterization of a cell line.

We have found a single 4p+ chromosomal abnormality, 46,XX, -4, +der(4)t(3;4)(q13.3;p16), in a patient with an unusual B cell leukemia of mature phenotype characterized by a high white cell count, tartrate-resistant acid phosphatase-positive malignant cells, splenic white pulp proliferation, and a serum IgM monoclonal gammopathy. The malignant cells were characterized by surface expression of CD19 (B4), CD20 (B1), IgM, IgD, kappa, and HLA-DR. They were weakly positive for CD21 (B2) and negative for CD25 (interleukin-2 receptor). The malignant cells also showed clonal rearrangement of the immunoglobulin heavy chain and kappa light chain genes. A cell line, designated HCLW-3B, was derived from unstimulated peripheral blood obtained during the leukemic phase and was found to contain the same 4p+ chromosomal abnormality as well as genomic sequences of the Epstein-Barr virus nuclear antigen. A somatic cell hybrid constructed from HCLW-3B containing the derivative chromosome 4 was used to confirm that chromosome 3q was the source of the translocated material. The availability of a cell line which is clonally derived from the patient's circulating leukemia cells should permit further characterization of this translocation at the molecular level.

Incontinentia pigmenti in a male infant with Klinefelter syndrome.

Incontinentia pigmenti is a familial disorder affecting tissues derived from neuroectoderm. Statistical analysis of reported pedigrees is consistent with transmission of incontinentia pigmenti by an X-linked dominant gene with male hemizygote lethality. This report describes a male infant with the classic clinical features of this condition and a 47,XXY chromosomal constitution. These findings support the concept that incontinentia pigmenti is an X-linked dominant disorder. This case illustrates the importance of a full genetic investigation in all males with physical findings suggestive of an X-linked dominant disorder lethal in males.

A somatic cell hybrid panel to facilitate identification of DNA sequences in the vicinity of the incontinentia pigmenti locus (IP1).

Somatic cell hybrids that retain derivative X chromosomes from women with sporadic incontinentia pigmenti (IP1) and de novo X/autosomal translocations with consistent breakpoints at Xp11.21 were constructed. An assembled hybrid panel was used to physically map DNA sequences in relationship to the IP breakpoint. DSX14 was found to map to region Xp11.21----p11.1. Regional assignments of 19 X-chromosomal loci were reviewed.

Chromosome breakage and recombination at fragile sites.

Chromosomal fragile sites are points on chromosomes that usually appear as nonstaining chromosome or chromatid gaps. It has frequently been suggested that fragile sites may be involved in chromosome breakage and recombination events. We and others have previously shown that fragile sites predispose to intrachromosomal recombination as measured by sister-chromatid exchanges. These findings suggested that fragile site expression often, if not always, is accompanied by DNA strand breakage. In the present report, fragile sites are shown to predispose to deletions and interchromosomal recombination. By use of somatic cell hybrids containing either human chromosome 3 or the fragile X chromosome, deletions and translocations were induced by FUdR or aphidicolin with breakpoints at the fragile sites Xq27 or 3p14.2 (FRA3B) or at points so close to the fragile sites as to be cytogenetically indistinguishable. Southern blot analysis of DNA from a panel of chromosome 3 deletion and translocation hybrids was then utilized to detect loss or retention of markers flanking FRA3B and to corroborate the cytogenetic evidence that the breakpoints were at this fragile site. One cell line with a reciprocal translocation between human chromosome 3 (with breakpoint at 3p14.2) and a hamster chromosome showed cytogenetically that the fragile site was expressed on both derivative chromosomes, supporting the hypothesis that the fragile site represents a repeated sequence. The approach described provides a means of generating specific rearrangements in somatic cell hybrids with a breakpoint at a fragile site.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of sister chromatid exchanges at common fragile sites.

Experiments were performed to gain further insight into chromosome structure and behavior at common fragile sites by testing the hypothesis that gaps at these sites predispose to intrachromosomal recombination as measured by sister chromatid exchanges (SCEs). Human lymphocytes were concurrently treated with aphidicolin, for determination of fragile site expression, and with 5-bromodeoxy-uridine, for SCE analysis. Aphidicolin induced chromosome gaps nonrandomly, with the great majority of gaps occurring at common fragile sites. On average, 66% of gaps were accompanied by an SCE at the site of the lesion. Analysis of two specific common fragile sites at 3p14 and 16q23 showed the same pattern; that is, on average 70% of gaps at these sites were accompanied by an SCE. These results show that common fragile sites are hot spots not only for chromosomal lesions such as gaps but also for SCE formation.