Response to Therapy, Treatment Intolerance and Tyrosine Kinase Inhibitor Cessation Eligibility in a Real-World Cohort of Chronic Myeloid Leukaemia Patients.

Tyrosine kinase inhibitor (TKI) therapy has revolutionised chronic myeloid leukaemia (CML) management, it is however associated with significant side effects and economic burden. Recent studies have demonstrated that treatment free remission is possible in certain patients. The aim of this study was to characterise a real-world population in terms of response to therapy, treatment intolerance and potential eligibility for stopping treatment. Included were 105 CML patients diagnosed in Northern Ireland from March 2009-February 2018. Response to treatment was defined as per the 2009 and 2013 European Leukaemia Net guidelines. Potential for treatment cessation was assessed as per the 2017 UK Interim Expert Opinion on Discontinuing Tyrosine Kinase Inhibitor Treatment in Clinical Practice for Treatment-Free Remission in Chronic Myeloid Leukaemia. Our cytogenetic data cohort had a 12-month complete cytogenetic response rate of 66% and the molecular data cohort had a 12-month major molecular response rate of 38%. Of those commenced on 2nd line TKI therapy 81% achieved an optimal response at 12 months. Twenty-two patients developed intolerance and required a change in TKI therapy. The commonest side effects were gastro-intestinal upset (18%), transaminitis (16%) and fluid retention (16%). In our cohort, 20% were considered eligible to stop TKI therapy. The commonest reason for ineligibility was insufficient duration of therapy (25%). We observed that 1st and 2nd line TKI therapy are effective but problems with failure and intolerance persist. Additionally, this study identifies a cohort of patients who may attempt TKI cessation using the UK Interim Expert Opinion report on TKI therapy discontinuation.

Gene expression profiling identifies different sub-types of retinoblastoma.

BACKGROUND: Mutation of the RB1 gene is necessary but not sufficient for the development of retinoblastoma. The nature of events occurring subsequent to RB1 mutation is unclear, as is the retinal cell-of-origin of this tumour. METHODS: Gene expression profiling of 21 retinoblastomas was carried out to identify genetic events that contribute to tumorigenesis and to obtain information about tumour histogenesis. RESULTS: Expression analysis showed a clear separation of retinoblastomas into two groups. Group 1 retinoblastomas express genes associated with a range of different retinal cell types, suggesting derivation from a retinal progenitor cell type. Recurrent chromosomal alterations typical of retinoblastoma, for example, chromosome 1q and 6p gain and 16q loss were also a feature of this group, and clinically they were characterised by an invasive pattern of tumour growth. In contrast, group 2 retinoblastomas were found to retain many characteristics of cone photoreceptor cells and appear to exploit the high metabolic capacity of this cell type in order to promote tumour proliferation. CONCLUSION: Retinoblastoma is a heterogeneous tumour with variable biology and clinical characteristics.

Molecular analysis of single colonies reveals a diverse origin of initial clonal proliferation in B-precursor acute lymphoblastic leukemia that can precede the t(12;21) translocation.

The pathogenesis of pediatric B-precursor acute lymphoblastic leukemia is largely unknown, and even with nonrandom chromosomal translocations present, the precise order of clonal molecular events is undefined. We developed an in vitro system using cytokines interleukin (IL)-3, IL-7, IL-10, and FMS-like tyrosine kinase 3 ligand with CD40 ligand-expressing fibroblasts to obtain single blast colonies from which clonal immunoglobulin heavy chain (IgH), T-cell receptor delta gene rearrangements, and, in t(12;21)-positive cases, TEL-AML1 fusion transcripts could be simultaneously PCR amplified. The proliferation of early tumor progenitors increased subclone detection enabling us, in seven diagnostic samples, to determine the stage of differentiation at which each leukemia occurred. Four were derived from the stage before initiation of IgH rearrangement, one during recombination of variable, joining, and diversity segments of the heavy chain gene VDJ(H), and two after completion of IgH rearrangement. Furthermore, analysis of a t(12;21)-positive leukemia with unusually late onset, identified both TEL-AML1-positive and -negative colonies carrying a clonal T-cell receptor delta rearrangement, inferring the presence of clonal expansion before the occurrence of the t(12;21). In contrast, in a typical, early onset t(12;21)-positive leukemia, the t(12;21) appeared to be the first clonal event. In both leukemias, the t(12;21) occurred before recombination of variable, joining and diversity segments of the heavy chain gene VDJ.

Molecular cytogenetic characterization of two non-MYCN amplified neuroblastoma cell lines with complex t(11;17).

The pediatric tumor neuroblastoma is characterized by a very variable, and at times unpredictable, pattern of clinical behavior, ranging from a benign localized tumor to an aggressive malignancy with poor prognosis. Standard clinical and pathological assessments do not always differentiate reliably between tumor subtypes and, therefore, genetic markers are now playing an increasingly important role in treatment decisions. MYCN oncogene amplification, for example, provides a useful marker of poor prognosis. However, less than one-half of all patients who present with, or who later develop, metastatic disease show MYCN amplification. Consequently, the identification of characteristic patterns of genetic alteration in the remaining tumors is of importance. In this report, we describe two new cell lines that we have established from metastatic, non-MYCN amplified, advanced stage neuroblastomas. These cell lines show a number of features in common, including unbalanced translocation between 11q and 17q, loss of 3p, 4p and 11q and gain of 17q. Therefore, they provide a valuable resource for the characterization of genetic pathways leading to aggressive tumor growth in non-MYCN amplified neuroblastomas.

Neuroblastomas with chromosome 11q loss and single copy MYCN comprise a biologically distinct group of tumours with adverse prognosis.

Neuroblastoma is a heterogeneous tumour and its effective clinical management is dependent on accurate prognostic evaluation. In approximately 25% of patients amplification of the MYCN oncogene is known to be associated with a poor outcome. In order to identify additional molecular markers with prognostic potential in non-MYCN-amplified neuroblastomas, we looked for a correlation between clinical outcome and loss of heterozygosity (LOH) on four chromosomes that frequently show alteration in neuroblastoma (chromosomes 3, 4, 11 and 14). Chromosome 11q loss (with frequent parallel loss of chromosomes 3p, 4p and/or 14q) was found exclusively in tumours without MYCN amplification and was significantly associated with poor event-free survival. The 2-year event-free survival rate for 11q LOH cases was 30%, compared to 34% for MYCN-amplified cases and 100% for cases without these abnormalities. While 11q LOH was associated predominantly with advanced-stage disease, 2 cases with low-stage disease and 11q LOH both suffered relapses. We conclude that chromosome 11q loss defines a biologically distinct group of tumours without MYCN amplification that appear to have potential for aggressive metastatic growth. Thus this genetic alteration may be an important new prognostic marker in neuroblastoma.

Exclusion of candidate genes and chromosomal regions in familial neuroblastoma.

Two families with recurrence of neuroblastoma one Italian and one British with three and two affected children respectively were genotyped using polymorphic markers on chromosome 1 spanning the p32-p36 region frequently deleted in neuroblastoma tumor cells. Linkage to this region was excluded by haplotype inspection and negative lod scores. Furthermore, the exclusion of genes involved in neurocristopathies sometimes associated with neuroblastoma was carried out by typing the Italian family with polymorphic markers located in or near the corresponding genes. Finally, linkage analysis in the two families showed negative lod scores for markers spanning the 16p12-13 chromosomal region where a locus for familial neuroblastoma has been recently mapped. Our findings indicate that different genes are involved in the pathogenesis of familial neuroblastoma.

Clonal diversity of Ig and T-cell receptor gene rearrangements in childhood B-precursor acute lymphoblastic leukaemia.

The majority of paediatric B precursor acute lymphoblastic leukaemias in children are derived from a single transformed haematopoietic cell with complete or partial VDJ recombination within the immunoglobulin heavy chain gene. A high frequency of patients also show rearrangements within TCRdelta and TCRgamma loci and in up to 40% of children there is an excess of immune system gene rearrangements compared with the number of identified alleles of immune system genes, suggesting the presence of multiple leukaemic subclones -clonal diversity. It has been observed by us and other investigators that in individual patients the pattern of immune system gene rearrangements often changes between presentation and relapse. In order to explore the possibility that clonal diversity plays a biological role during disease progression we optimised methods for subclone detection and analysed the prognostic significance of clonal diversity among 75 children with B precursor-ALL. Our results suggest that clonal diversity plays a role in disease progression as patients with oligoclonal disease showed a significantly shorter disease free survival than patients with monoclonal disease. This trend was of particular importance in the 'standard risk' group of ALL where aggressive disease could not be recognised by other means. In addition, generation of independent subclones from an early, non-rearranged tumour progenitor appears to be a common feature among leukaemias with aggressive clinical behaviour. We speculate on the type of genetic factors which may participate both in the generation of subclones and also in wider genomic instability and which are likely to be required for the aggressive clinical phenotype in children with ALL.

Pediatric oncology and hematology in Birmingham, England.

The hematology and oncology service at Birmingham Children's Hospital was established in the late 1960s and now is one of the largest in the United Kingdom. It provides comprehensive care for the entire range of childhood malignancies, coagulation disorders, and hemoglobinopathies and other hematological disorders, and undertakes bone marrow transplant and megatherapy/peripheral blood stem cell procedures. Research includes clinical trials of treatments of childhood cancers; molecular biology studies on leukemia, Hodgkin's disease, neuroblastoma, and sarconas; childhood cancer epidemiology, and geographical and racial incidence; and treatment of hemophilia and molecular investigation of coagulation disorders. These activities involve collaboration with local, national, and international research groups.

Clonal diversity of Ig and T-cell-receptor gene rearrangements identifies a subset of childhood B-precursor acute lymphoblastic leukemia with increased risk of relapse.

Current prognostic indicators such as age, sex, and white blood cell count (WBC) fail to identify all children with more aggressive forms of B-precursor acute lymphoblastic leukemia (ALL), and a proportion of patients without poor prognostic indicators still relapse. Results obtained from an analysis of 65 pediatic B-precursor ALL patients indicated that subclone formation leading to clonal diversity, as detected by Ig and T-cell receptor (TCR) gene rearrangements, may represent a very useful prognostic indicator, independent of age, sex, and WBC. Disease-free survival was significantly shorter in those patients showing clonal diversity at presentation. Furthermore, clonal diversity was detected not only in the majority of high-risk patients who relapsed but was also associated with a high probability of relapse in standard-risk patients. Sixty-five percent (13/20) of standard-risk patients who also showed clonal diversity subsequently relapsed, whereas the percentage of relapses among standard-risk patients without clonal diversity was much lower at 19% (7/36). Continued clonal evolution during disease progression is an important feature of aggressive B-precursor ALL. All 5 patients with clonal diversity who were followed up in our study showed a change in the pattern of clonality between presentation and relapse. This implies an important role for clonal diversity as a mechanism of disease progression through the process of clonal variation and clonal selection.

Construction of a transcription map around the gene for ataxia telangiectasia: identification of at least four novel genes.

We have constructed YAC, PAC, and cosmid contigs in the ataxia-telangiectasia gene region and used the assembled clones to isolate expressed sequences by exon trapping and hybridization selection. In the interval between D11S1819 and D11S2029, exons and cDNAs for potentially 13 different genes were identified. Three of these genes, F37, K28, and 6.82, are large novel genes expressed in a variety of different tissues. K28 shows sequence homology to the Rab GTP binding protein family and gene 6.82 homology to the rabbit vasopressin activated calcium mobilizing receptor, while gene F37 has no homology to any known sequence in the database. Three further clones, exon 6.41 and cDNAs K22 and E74, from the interval between D11S1819 and D11S2029, appear to be expressed endogenous retrovirus sequences. The fourth large novel genes, E14, together with two further possible novel genes, E13 and E3, was identified from exons and cDNAs in the more telomeric 300-kb interval between markers D11S2029 and D11S2179. These are in addition to the genes for mitochondrial acetoacetyl-CoA-acetyltransferase (ACAT) and the ATM gene in the same region. Genes E3, E13, and E14 do not show homology to any known genes. K28, 6.82, ACAT, and ATM all appear to have the same transcriptional orientation toward the telomere.

Identification and analysis of expression of human VACM-1, a cullin gene family member located on chromosome 11q22-23.

We have localized the human homolog of the rabbit vasopressin-activated calcium-mobilizing receptor VACM-1 to a region close to the gene for ataxia telangiectasia ATM on chromosome 11q22-23. We have determined the complete amino acid sequence of the human Hs-VACM-1 protein, which is 780 amino acids long. The human and rabbit sequences are highly conserved, differing at only seven amino acids. Northern analysis of the human gene showed expression in a wide range of human tissues. The Hs-VACM-1 gene has homology with the Caenorhabditis elegans gene Ce-cul-5, a member of a family of cullin genes that are involved in cell cycle regulation and that might, when mutated, contribute to tumor progression.

Mutations revealed by sequencing the 5' half of the gene for ataxia telangiectasia.

Ataxia telangiectasia is a recessive disorder in which patients show a progressive cerebellar degeneration leading to ataxia, abnormal eye movements and deterioration of speech. Other features include ocular telangiectasia, high serum AFP levels, immunodeficiency, growth retardation and an increased predisposition to some tumours, particularly T cell leukaemia and lymphoma. We report the 1348 amino acid sequence of the N-terminal half of the A-T gene product which, together with the previously published C-terminal half, completes the sequence of the A-T protein. No homologies with other genes have been found within the N-terminal half of the A-T protein. We have also identified six mutations affecting the N-terminal half of the protein. One of these mutations was found to be associated with a haplotype that is common to four apparently unrelated families of Irish descent. All the patients so far examined for both A-T alleles were shown to be compound heterozygotes. None of these mutations affected a putative promoter region which may direct divergent transcription of both the A-T gene and a novel gene E14. The ability to recognise mutations across the entire coding sequence of the A-T gene provides a practical advantage to A-T families since a DNA based prenatal diagnosis will be possible in families where the mutations are identified irrespective of the level of radiosensitivity in these families.

Analysis of 133 meioses places the genes for nevoid basal cell carcinoma (Gorlin) syndrome and Fanconi anemia group C in a 2.6-cM interval and contributes to the fine map of 9q22.3.

Four disease genes (NBCCS, ESS1, XPAC, FACC) map to 9q22.3-q31. A fine map of this region was produced by linkage and haplotype analysis using 12 DNA markers. The gene for nevoid basal cell carcinoma syndrome (NBCCS, Gorlin) has an important role in congenital malformations and carcinogenesis. Phase-known recombinants in a study of 133 meioses place NBCCS between (D9S12/D9S151) and D9S176. Haplotype analysis in a two-generation family suggests that NBCCS lies in a smaller interval of 2.6 cM centromeric to D9S287. These flanking markers will be useful clinically for gene tracking. Recombinants also map FACC (Fanconi anemia, group C) to the same region, between (D9S196/D9S197) and D9S287. The recombination rate between (D9S12/D9S151) and D9S53 in males is 8.3% and 13.2% in females, giving a sex-specific male:female ratio of 1:1.6 and a sex-averaged map distance of 10.4 cM. No double recombinants were detected, in agreement with the apparently complete level of interference predicted from the male chiasmata map.

Cancer and DNA processing disorders.

Defects in cloned DNA repair genes are now associated with particular human disorders in which an important feature is a predisposition to cancer. Recently some repair genes have been implicated in other aspects of DNA metabolism such as transcription initiation. In addition mutations in a single gene can give rise to phenotypes recognised clinically as different disorders. These newly appreciated complexities, amongst others, will eventually help us to understand the development of the complete clinical phenotype in a range of 'DNA processing disorders'.

Paired STSs amplified from radiation hybrids, and from associated YACs, identify highly polymorphic loci flanking the ataxia telangiectasia locus on chromosome 11q22-23.

The high resolution mapping of the ataxia telangiectasia (A-T) locus on chromosome 11q22-23 requires the generation of new polymorphic markers specifically within the segment of 11q22-23 to which the locus has been assigned. We have made use of a library of Alu-PCR clones, amplified from a radiation reduced somatic cell hybrid containing the relevant chromosome 11 segment, to generate sequence tagged sites (STS) within the 11q22-23 region and have used YAC clones to extend the loci identified by these STSs. The identification of paired polymorphisms (from Alu-PCR and the associated YAC derived clone), which are physically linked, but which show minimal linkage disequilibrium, provides a highly informative haplotype for use in genetic linkage analysis in A-T families. We describe the characterisation of 2 such polymorphic loci, D11S535 and D11S611, which map between existing flanking markers, and which provide additional information on the location of the major A-T locus.

Characterization of a B-lymphocyte t(2;14) (p11;q32) translocation from an ataxia telangiectasia patient conferring a proliferative advantage on cells in vitro.

Patients with the recessively inherited disorder ataxia telangiectasia (AT) are particularly prone to the development of both B-cell and T-cell tumours. Specific translocations involving T-cell gene rearrangements and an unknown locus 3' of IGH have been described in AT T-cell clone and tumour cells. We describe here a t(2;14)(p11;q32) translocation which was observed in nonmalignant short-term-cultured B lymphocytes from an AT patient. In vivo, the clone of cells grew from 1% to 6% of the total cell population over a period of 2 yr. Clonal translocations may therefore be associated with AT B cells, as well as AT T cells. B lymphocytes were transformed with Epstein-Barr virus, and the t(2;14) translocation cell was cellularly cloned. Using Southern filter analysis and in situ hybridisation to define more clearly the positions of the breakpoints, we show that the translocation at 14q32 involves a deletion within the IGH chain gene of at least J1, J2, DQ52, and sequences 1.5 kb 5' of DQ52 and that the breakpoint is either adjacent to the non-deleted JH sequences or upstream of these sequences, within the D or V regions, but proximal to all members of the VHII family of genes. The breakpoint at 2p11 is outside and proximal to IGK with respect to the centromere in an unknown gene. Sub-lines with an initially low proportion of translocation cells eventually became monoclonal in vitro for these cells. This suggests they have a growth advantage in vitro.

Analysis of 7 polymorphic markers at chromosome 11q22-23 in 35 ataxia telangiectasia families; further evidence of linkage.

Ataxia telangiectasia (A-T) is an autosomal recessive disorder characterised by progressive neurological degeneration, oculocutaneous telangiectasia, immunodeficiency and a high incidence of lymphoid tumours. A prerequisite to gaining a complete understanding of the basic defect that results in these features is the localization of the gene(s) involved. We report here a linkage analysis using seven polymorphic markers, which map to 11q22-23, on a sample of 35 consecutively obtained families from the British Isles showing this disorder. In a pairwise analysis, the strongest support for linkage was a lod score of 4.01 at zero recombination from Thy-1. This result supports a previous report showing linkage of the A-T gene to 11q22-23. We have also obtained evidence in a multipoint analysis for a more centromeric A-T-linked locus in the region between YNB 3.12/CJ52.208 and 2-7-1D6. This observation is also supported by inspection of the haplotypes of selected recombinants.

Rearrangement of the same chromosome regions in different SV40 transformed human skin keratinocyte lines is associated with tumourigenicity.

Twelve different human keratinocyte strains were transformed with recombinant plasmid pSV6-1 which contained an origin defective SV40 genome. When injected into athymic nude mice lines produced either squamous cell carcinomas (SCC) in all animals, SCC in some animals and epidermal cysts in others, or epidermal cysts only in all the animals. The tumourigenic capacity of the lines could be correlated with the chromosomal changes present initially in the transformed cells. Lines which produced SCC in all the animals within a short period of time all showed simultaneous loss of part of chromosomes 3p, 8p and 11p in one homologue. Lines which were not tumourigenic did not show these simultaneously appearing rearrangements. These specific rearrangements are acquired in vitro and the time taken for a recognisable tumour to appear is related to the proportion of such cells in the line. The rearrangement of the same chromosome regions in different tumourigenic cell lines suggests that genes in these regions are important in the development of squamous cell carcinoma, possibly by loss of heterozygosity, at particular loci.