Detection of inherited chromosomally integrated HHV-6 (ciHHV-6) in a marker chromosome.

OBJECTIVES: Inherited chromosomally integrated human herpesvirus-6 (ciHHV-6) is characterised by the complete HHV-6 genome integration into the host germ line genome and is vertically transmitted with a Mendelian inheritance. By now, the only relationship between ciHHV-6 and diseases seems to be with angina pectoris. METHODS: We report a case of an 82-year-old man diagnosed with diffuse large B-cell lymphoma (DLBCL) on October 2014. To substantiate the suspicion of ciHHV-6, we analysed peripheral blood mononuclear cells, bone marrow biopsy and pleural effusion-derived mesothelial cells with PCR, RT-PCR and FISH. RESULTS: Virological routine screening by PCR showed the absence of HHV-8 and EBV infections, while the presence of HHV-6 DNA (ie, U22, U42 and U94 HHV-6 genes), with a viral load of about 1.0 genome per cell, strongly suggests ciHHV-6. The RT-PCR showed the positivity only for the immediate-early U94, at low levels of transcription (100±15 transcripts/1 µg RNA). FISH analysis reported a case of inherited ciHHV-6 in 17p chromosome region and, for the first time, in a marker chromosome. CONCLUSIONS: This is the first case of inherited ciHHV-6 in a marker chromosome, possibly elucidating the role of this abnormality in the biology of DLBCL.

Can genome array screening replace FISH as a front-line test in multiple myeloma?

Multiple myeloma (MM) is a malignant disorder characterized by neoplastic transformation of mature B cells in the bone marrow (BM), accompanied by complex genetic changes. The disease is heterogeneous at both the clinical and genomic levels. Molecular genetics and genomic investigations have demonstrated that disease evolution is associated with an accumulation of specific aberrations, mostly genome imbalances, which not only shed light on the disease pathogenesis but also allow risk assessment and treatment monitoring. We used a catalogue version of the Agilent 8x60K oligo-array with immuno-magnetically isolated CD138(+) cells from BM samples of 50 patients with myeloma to evaluate the merit of array comparative genomic hybridization (aCGH) as a diagnostic tool. We demonstrate the ability of aCGH to detect clonal imbalances to a level well below established clinically significant thresholds. aCGH, combined with target enrichment and complemented with tests for IGH rearrangements offers a cost neutral alternative to multiprobe fluorescence in situ hybridization screening. While we recognize the limitations of the standard version of the 8x60k array we demonstrate the value of aCGH as a first tier test in the diagnostic workup of MM. The array technology enables high-risk disease stratification with the added benefit of providing whole genome data to assist in establishing clinically relevant predicative markers.

Secondary plasma cell leukaemia (PCL) with plasmablastic morphology.

A 71-year-old female with relapsed IgA lambda myeloma developed progressive cytopenia. The peripheral blood film showed 5% blastoid cells. Flow cytometry analysis was indicative of plasma cells. The bone marrow smear was packed with plasmablasts. Target CD138-cell FISH and molecular karyotyping identified a complex genome. NGS identified high-risk mutations. Bone marrow histology confirmed myeloma with no evidence of acute leukaemia. The patient was diagnosed with plasmablastic progression of myeloma and secondary PCL. Secondary PCL patients have a poor prognosis. It is essential to recognize this subtype and explore a novel treatment approach.

Chronic myeloid leukaemia (CML) presenting in B-lymphoblastic crisis: a diagnostic challenge.

We report the case of a 75-year-old female presented with lethargy, Hb 93 g/L, WBC 64 x 109/L, platelet 110 x 109/L. Blood film showed blasts, myelocytes, metamyelocytes, neutrophils. Quantitative PCR detected p210 BCR::ABL1 transcript in sorted CD19+ cells, and sorted CD19- cells. Bone marrow smear was packed with blasts. Flow cytometry and bone marrow histology revealed B-lymphoblasts. The patient was diagnosed with CML Blymphoblastic crisis. CML presenting in B-lymphoblastic crisis could resemble features of de novo Ph+ B-ALL, which makes the diagnosis challenging. These patients have inferior outcomes; therefore, it is important to distinguishing CML B -lymphoblastic crisis from de novo Ph+ B-ALL. Positive BCR::ABL1 in both CD19+ and CD19- sorted cell populations support the diagnosis of CML B-lymphoblastic crisis in this case.

Amplified segment in the 'Down syndrome critical region' on HSA21 shared between Down syndrome and euploid AML-M0 excludes RUNX1, ERG and ETS2.

Children with Down syndrome have a 20- to 50-fold increased risk of acute lymphocytic or myeloid leukaemia. Whole or partial gains of chromosome 21 have been described in multiple childhood leukaemias, and have recently been reported as a likely primary event in B-precursor-acute lymphoblastic leukaemia. It is unclear which amplified gene(s) on chromosome 21 play a key role in leukaemia progression. We describe a minimal amplified segment within the so-called 'Down syndrome critical region' shared between two cases of AML-M0; a Down syndrome, and a constitutionally normal individual. Interestingly, the amplified region does not include the oncogenes RUNX1, ETS2 and ERG.

Differentiating inherited human herpesvirus type 6 genome from primary human herpesvirus type 6 infection by means of dried blood spot from the newborn screening card.

To differentiate active human herpesvirus type 6 (HHV-6) infection from inherited HHV-6 (iHHV-6), we analyzed dried blood spots from archived newborn screening cards in 3 patients with high HHV-6 DNA copy numbers. Two patients were positive for HHV-6 DNA as neonates suggesting iHHV-6. In 1 patient, the absence of HHV-6 DNA excluded iHHV-6.

2-Phenylacetylenesulfonamide (PAS) induces p53-independent apoptotic killing of B-chronic lymphocytic leukemia (CLL) cells.

We studied the actions of 2-phenylacetylenesulfonamide (PAS) on B-chronic lymphocytic leukemia (CLL) cells. PAS (5-20 microM) initiated apoptosis within 24 hours, with maximal death at 48 hours asassessed by morphology, cleavage of poly(ADP-ribose) polymerase (PARP), caspase 3 activation, and annexin V staining. PAS treatment induced Bax proapoptotic conformational change, Bax movement from the cytosol to the mitochondria, and cytochrome c release, indicating that PAS induced apoptosis via the mitochondrial pathway. PAS induced approximately 3-fold up-regulation of proapoptotic Noxa protein and mRNA levels. In addition, Noxa was found unexpectedly to be bound to Bcl-2 in PAS-treated cells. PAS treatment of CLL cells failed to up-regulate p53, suggesting that PAS induced apoptosis independently of p53. Furthermore, PAS induced apoptosis in CLL isolates with p53 gene deletion in more than 97% of cells. Normal B lymphocytes were as sensitive to PAS-induced Noxa up-regulation and apoptosis as were CLL cells. However, both T lymphocytes and bone marrow hematopoietic progenitor cells were relatively resistant to PAS. Our data suggest that PAS may represent a novel class of drug that induces apoptosis in CLL cells independently of p53 status by a mechanism involving Noxa up-regulation.

Deletions of immunoglobulin heavy chain and T cell receptor gene regions are uniquely associated with lymphoid blast transformation of chronic myeloid leukemia.

BACKGROUND: Chronic myelogenous leukemia (CML) results from the neoplastic transformation of a haematopoietic stem cell. The hallmark genetic abnormality of CML is a chimeric BCR/ABL1 fusion gene resulting from the Philadelphia chromosome rearrangement t(9;22)(q34;q11). Clinical and laboratory studies indicate that the BCR/ABL1 fusion protein is essential for initiation, maintenance and progression of CML, yet the event(s) driving the transformation from chronic phase to blast phase are poorly understood. RESULTS: Here we report multiple genome aberrations in a collection of 78 CML and 14 control samples by oligonucleotide array comparative genomic hybridization. We found a unique signature of genome deletions within the immunoglobulin heavy chain (IGH) and T cell receptor regions (TCR), frequently accompanied by concomitant loss of sequences within the short arm regions of chromosomes 7 and 9, including IKZF1, HOXA7, CDKN2A/2B, MLLT3, IFNA/B, RNF38, PAX5, JMJD2C and PDCD1LG2 genes. CONCLUSIONS: None of these genome losses were detected in any of the CML samples with myeloid transformation, chronic phase or controls, indicating that their presence is obligatory for the development of a malignant clone with a lymphoid phenotype. Notably, the coincidental deletions at IGH and TCR regions appear to precede the loss of IKZF1 and/or p16 genes in CML indicating a possible involvement of RAG in these deletions.

p53-mediated apoptosis of CLL cells: evidence for a transcription-independent mechanism.

The p53 protein plays a key role in securing the apoptotic response of chronic lymphocytic leukemia (CLL) cells to genotoxic agents. Transcriptional induction of proapoptotic proteins including Puma are thought to mediate p53-dependent apoptosis. In contrast, recent studies have identified a novel nontranscriptional mechanism, involving direct binding of p53 to antiapoptotic proteins including Bcl-2 at the mitochondrial surface. Here we show that the major fraction of p53 induced in CLL cells by chlorambucil, fludarabine, or nutlin 3a was stably associated with mitochondria, where it binds to Bcl-2. The Puma protein, which was constitutively expressed in a p53-independent manner, was modestly up-regulated following p53 induction. Pifithrin alpha, an inhibitor of p53-mediated transcription, blocked the up-regulation of Puma and also of p21(CIP1). Surprisingly, pifithrin alpha dramatically augmented apoptosis induction by p53-elevating agents and also accelerated the proapoptotic conformation change of the Bax protein. These data suggest that direct interaction of p53 with mitochondrial antiapoptotic proteins including Bcl-2 is the major route for apoptosis induction in CLL cells and that p53's transcriptional targets include proteins that impede this nontranscriptional pathway. Therefore, strategies that block up-regulation of p53-mediated transcription may be of value in enhancing apoptosis induction of CLL cells by p53-elevating drugs.

A high circulating copy number of HHV-6 due to chromosomal integration in a child with acute lymphoblastic leukemia.

We report a case of a 3.5-year-old female with a very high copy number of human herpesvirus 6 (HHV-6) detected by PCR in blood during acute lymphoblastic leukemia induction therapy. The patient was unsuccessfully treated with antiviral drugs. HHV-6 genome was shown to be constitutively integrated into chromosome 22q-tel, likely to be inherited from the mother who was found to carry high HHV-6 copy number. This case highlights the importance of excluding HHV-6 chromosomal integration before diagnosing HHV-6 infection or reactivation in immunocompromised patients.

EZH2-Deficient T-cell Acute Lymphoblastic Leukemia Is Sensitized to CHK1 Inhibition through Enhanced Replication Stress.

Loss-of-function mutations of EZH2, the enzymatic component of PRC2, have been associated with poor outcome and chemotherapy resistance in T-cell acute lymphoblastic leukemia (T-ALL). Using isogenic T-ALL cells, with and without CRISPR/Cas9-induced EZH2-inactivating mutations, we performed a cell-based synthetic lethal drug screen. EZH2-deficient cells exhibited increased sensitivity to structurally diverse inhibitors of CHK1, an interaction that could be validated genetically. Furthermore, small-molecule inhibition of CHK1 had efficacy in delaying tumor progression in isogenic EZH2-deficient, but not EZH2 wild-type, T-ALL cells in vivo, as well as in a primary cell model of PRC2-mutant ALL. Mechanistically, EZH2 deficiency resulted in a gene-expression signature of immature T-ALL cells, marked transcriptional upregulation of MYCN, increased replication stress, and enhanced dependency on CHK1 for cell survival. Finally, we demonstrate this phenotype is mediated through derepression of a distal PRC2-regulated MYCN enhancer. In conclusion, we highlight a novel and clinically exploitable pathway in high-risk EZH2-mutated T-ALL. SIGNIFICANCE: Loss-of-function mutations of PRC2 genes are associated with chemotherapy resistance in T-ALL, yet no specific therapy for this aggressive subtype is currently clinically available. Our work demonstrates that loss of EZH2 activity leads to MYCN-driven replication stress, resulting in increased sensitivity to CHK1 inhibition, a finding with immediate clinical relevance.This article is highlighted in the In This Issue feature, p. 890.

HHV-6 DNA throughout the tissues of two stem cell transplant patients with chromosomally integrated HHV-6 and fatal CMV pneumonitis.

Two patients with the characteristic high human herpesvirus 6 (HHV-6) DNA loads in peripheral blood caused by chromosomally integrated (CI) virus received a haematopoietic stem cell transplant (HSCT) from a donor without CI HHV-6. Both patients died in consequence of cytomegalovirus (CMV) pneumonitis. At autopsy, high amounts of CMV DNA were detected in lungs but at much lower levels in other organs. In contrast HHV-6 DNA was detected at high levels throughout the organs with the exception of donor-derived haematopoietic tissue. In individuals with chromosomal integration, HHV-6 DNA is found in every tissue of recipient origin indicating inheritance through the germ line.

Human herpesvirus 6 integrates within telomeric regions as evidenced by five different chromosomal sites.

Fluorescent in situ hybridization (FISH) was used to investigate the chromosomal integration sites of human herpesvirus 6 (HHV-6) in phytohemagglutinin-stimulated leukocytes and B lymphocytes from Epstein-Barr virus transformed lymphoblastoid cell lines (LCLs). Five different chromosomal integration sites were found in nine individuals. Only one site was identified in each individual, each site was in the vicinity of the telomeric region and was on either the p or q arm of only one of the two chromosome homologues. The sites were 9q34.3, 10q26.3, 11p15.5, 17p13.3, and 19q 13.4, of which three have not been previously identified. For 9q34.3 the site of integration was further mapped using a locus-specific probe for 9q34.3 together with a pan-telomeric probe and both co-localized with the HHV-6 signal. Similarly an arm-specific telomeric probe for 19q co-localized with the HHV-6 signal. It was therefore concluded that the site of integration is actually within the telomere. The number of viral DNA copies/cell was calculated in blood, LCL cells and hair follicles and was one or more in every case for each of the nine individuals. This result was confirmed by FISH where 100% of cells gave an HHV-6 signal. These findings add to previous reports suggesting that integrated HHV-6 DNA is found in every cell in the body and transmitted vertically. Finally, including our data, worldwide seven different chromosomal sites of HHV-6 integration have now been identified. Large epidemiological studies of chromosomal integration are required to identify further telomeric sites, geographical or racial variation and possible clinical consequences.

Deletions adjacent to BCR and ABL1 breakpoints occur in a substantial minority of chronic myeloid leukemia patients with masked Philadelphia rearrangements.

Deletions at the t(9;22) breakpoint regions, found in 15% of chronic myeloid leukemia patients (CML) with an overt Philadelphia (Ph) translocation, are associated with an adverse disease prognosis in patients receiving interferon-alpha therapy. The incidence of deletions has been shown to vary for different cytogenetic subgroups of CML, with a significantly higher incidence of deletion in patients with a variant Ph translocation. To date, however, the frequency of such deletions in the subgroup of CML patients in whom the BCR/ABL1 fusion arises via submicroscopic chromosomal insertion (masked Ph) has not been investigated. We report the evaluation of 14 patients with masked Ph-positive CML for the presence of deletions extending 3' from BCR and 5' from ABL1 using two triple-color BCR/ABL probes. Deletions were identified in 3 patients (21%), encompassing sequences 5' to ABL1 in two of these and sequences 3' to BCR in the remaining patient, thus demonstrating that the phenomenon is a significant feature of the masked Ph CML subgroup. Furthermore, our findings are consistent with the notion that loss of genomic material is a potential side effect of any DNA breakage event at the 9q34.1 and 22q11.2 chromosomal regions, regardless of the subsequent mechanism of chromosomal rearrangement.