Management of patients with germline predisposition to haematological malignancies considered for allogeneic blood and marrow transplantation: Best practice consensus guidelines from the UK Cancer Genetics Group (UKCGG), CanGene-CanVar, NHS England Genomic Laboratory Hub (GLH) Haematological Malignancies Working Group and the British Society of Blood and Marrow Transplantation and cellular therapy (BSBMTCT).

Germline predisposition to haematological cancers is increasingly being recognised. Widespread adoption of high-throughput and whole genome sequencing is identifying large numbers of causative germline mutations. Constitutional pathogenic variants in six genes (DEAD-box helicase 41 [DDX41], ETS variant transcription factor 6 [ETV6], CCAAT enhancer binding protein alpha [CEBPA], RUNX family transcription factor 1 [RUNX1], ankyrin repeat domain containing 26 [ANKRD26] and GATA binding protein 2 [GATA2]) are particularly significant in increasing the risk of haematological cancers, with variants in some of these genes also associated with non-malignant syndromic features. Allogeneic blood and marrow transplantation (BMT) is central to management in many haematological cancers. Identification of germline variants may have implications for the patient and potential family donors. Beyond selection of an appropriate haematopoietic stem cell donor there may be sensitive issues surrounding identification and counselling of hitherto asymptomatic relatives. If BMT is needed, there is frequently a clinical urgency that demands a rapid integrated multidisciplinary approach to testing and decision making involving haematologists in collaboration with Clinical and Laboratory Geneticists. Here, we present best practice consensus guidelines arrived at following a meeting convened by the UK Cancer Genetics Group (UKCGG), the Cancer Research UK (CRUK) funded CanGene-CanVar research programme (CGCV), NHS England Genomic Laboratory Hub (GLH) Haematological Oncology Malignancies Working Group and the British Society of Blood and Marrow Transplantation and Cellular Therapy (BSBMTCT).

Germline predisposition to haematological malignancies: Best practice consensus guidelines from the UK Cancer Genetics Group (UKCGG), CanGene-CanVar and the NHS England Haematological Oncology Working Group.

The implementation of whole genome sequencing and large somatic gene panels in haematological malignancies is identifying an increasing number of individuals with either potential or confirmed germline predisposition to haematological malignancy. There are currently no national or international best practice guidelines with respect to management of carriers of such variants or of their at-risk relatives. To address this gap, the UK Cancer Genetics Group (UKCGG), CanGene-CanVar and the NHS England Haematological Oncology Working Group held a workshop over two days on 28-29th April 2022, with the aim of establishing consensus guidelines on relevant clinical and laboratory pathways. The workshop focussed on the management of disease-causing germline variation in the following genes: DDX41, CEBPA, RUNX1, ANKRD26, ETV6, GATA2. Using a pre-workshop survey followed by structured discussion and in-meeting polling, we achieved consensus for UK best practice in several areas. In particular, high consensus was achieved on issues regarding standardised reporting, variant classification, multidisciplinary team working and patient support. The best practice recommendations from this meeting may be applicable to an expanding number of other genes in this setting.

Constitutional and somatic rearrangement of chromosome 21 in acute lymphoblastic leukaemia.

Changes in gene dosage are a major driver of cancer, known to be caused by a finite, but increasingly well annotated, repertoire of mutational mechanisms. This can potentially generate correlated copy-number alterations across hundreds of linked genes, as exemplified by the 2% of childhood acute lymphoblastic leukaemia (ALL) with recurrent amplification of megabase regions of chromosome 21 (iAMP21). We used genomic, cytogenetic and transcriptional analysis, coupled with novel bioinformatic approaches, to reconstruct the evolution of iAMP21 ALL. Here we show that individuals born with the rare constitutional Robertsonian translocation between chromosomes 15 and 21, rob(15;21)(q10;q10)c, have approximately 2,700-fold increased risk of developing iAMP21 ALL compared to the general population. In such cases, amplification is initiated by a chromothripsis event involving both sister chromatids of the Robertsonian chromosome, a novel mechanism for cancer predisposition. In sporadic iAMP21, breakage-fusion-bridge cycles are typically the initiating event, often followed by chromothripsis. In both sporadic and rob(15;21)c-associated iAMP21, the final stages frequently involve duplications of the entire abnormal chromosome. The end-product is a derivative of chromosome 21 or the rob(15;21)c chromosome with gene dosage optimized for leukaemic potential, showing constrained copy-number levels over multiple linked genes. Thus, dicentric chromosomes may be an important precipitant of chromothripsis, as we show rob(15;21)c to be constitutionally dicentric and breakage-fusion-bridge cycles generate dicentric chromosomes somatically. Furthermore, our data illustrate that several cancer-specific mutational processes, applied sequentially, can coordinate to fashion copy-number profiles over large genomic scales, incrementally refining the fitness benefits of aggregated gene dosage changes.

Genetics in myeloma: genetic technologies and their application to screening approaches in myeloma.

BACKGROUND: Despite advances in the treatment of multiple myeloma (MM), it remains an incurable malignant disease. Myeloma genetics is intrinsically complex, but it offers an opportunity to categorize the disease and apply a personalized medicine approach. AREAS OF AGREEMENT: Research into the genetics of myeloma is moving at a fast pace and is highlighting areas and patient cohorts likely to benefit from specific treatment. Targeting residual disease is likely to be crucial to improved clinical outcome. AREAS OF CONTROVERSY: Patients in clinical trials are more likely to receive genetic diagnosis than non-trial patients, for whom access is ad hoc and dependent upon regional commissioning arrangements. AREAS TIMELY FOR DEVELOPING RESEARCH: Relating genetics to potential treatment pathways will become crucial for improved myeloma outcomes. Universal access to standardized genetic testing will facilitate modern personalized treatments.

Cytogenetics of long-term survivors of ETV6-RUNX1 fusion positive acute lymphoblastic leukemia.

This study describes the cytogenetics of 33 children with ETV6-RUNX1 positive acute lymphoblastic leukemia (ALL) who had been in continuous complete remission for a minimum of 8.8 years [median event-free survival (EFS) 10.9 years]. The results were compared with a published series of 16 fusion positive patients treated on the same childhood ALL trial, who had relapsed (median EFS, 2.3 years). Interphase fluorescence in situ hybridization (FISH) at diagnosis showed deletion of the second ETV6 signal from all fusion positive cells in 45% of the long-term survivors but in none of the relapsed patients, whereas patients with mixed populations with retained or lost second signals were more frequent among those who had relapsed (69%) than the long-term survivors (21%). Interphase populations with two fusion signals in 18% of the long-term survivors and 31% of relapsed patients were smaller in the long-term survivors (median, 4% of total cells) than in the relapsed patients (median, 84%). The additional copy of chromosome 21 in 30% of long-term survivors and in 69% of relapsed patients was a derived chromosome 21 in 20% and 55% of patients, respectively. Metaphase FISH for 26 long-term survivors and 15 relapsed patients revealed complex karyotypes in both groups. Variant translocations involved different chromosome arms between the long-term survivors and relapsed patients. It appears that the two groups have some distinguishing cytogenetic features at the time of diagnosis, which may provide pointers to relapse that are worthy of more detailed study.

Translocation (6;17)(q23;q11.2): a novel cytogenetic abnormality in congenital acute myeloid leukemia?

Congenital leukemia occurring within 4 weeks of birth is extremely rare and, excluding transient neonatal myeloproliferation associated with Down syndrome, makes up approximately 1% of childhood leukemias. It is usually seen as acute myeloid leukemia (AML), most frequently French-American-British (FAB) types M4 and M5. Recurrent cytogenetic abnormalities have been reported in this group, and in approximately one third of cases the MLL gene at 11q23 is involved. These patients generally have a poor prognosis. We present a case of congenital leukemia (AML FAB type M1) with an acquired translocation between chromosomes 6 and 17.

Reprogramming in inter-species embryonal carcinoma-somatic cell hybrids induces expression of pluripotency and differentiation markers.

Somatic cell reprogramming holds great promise for the development of novel cellular therapeutics. A number of sources of reprogramming potential have been identified, including oocytes, embryonic germ (EG) cells and embryonic stem (ES) cells. However, each of these sources of reprogramming factors is problematic, since they are either not freely available or have special growth requirements. Embryonal carcinoma (EC) cells are another source of pluripotent cells that, unlike ES and EG cells, do not usually require special growth conditions. Since they share many of the key characteristics of ES cells, such as pluripotency, EC cells may provide a readily amenable alternative source of reprogramming factors and could serve as a model for ES cells in this respect. Here we show that mouse EC cells can also function as donors of reprogramming factors. PEG-mediated fusion between murine EC cells (P19) and the cells of a human T-lymphoma cell line (CEM-GFP) resulted in inter-species hybrid colony formation. Colonies of hybrid cells displayed heterogeneity in cellular morphology as well as in their pattern of human gene expression. Expression of two human transcription factors characteristic of undifferentiated pluripotent stem cells, Oct-4 and Sox-2, was detected in the hybrid cells, demonstrating activation of endogenous human markers of pluripotency. Simultaneously, down-regulation of CD45, a marker present in lymphocytic cells, was observed in some hybrids. The detection of human specific markers of differentiation, such as nestin, lamininbeta1, and collagen IValpha1, indicates that fusion resulted in reprogramming of the human cells to reflect the differentiation potential of the murine EC partner.

Detection of cryptic MLL insertions using a commercial dual-color fluorescence in situ hybridization probe.

Involvement of the MLL gene located at chromosome region 11q23 is a frequent occurrence in both acute myelocytic leukemia and acute lymphoblastic leukemia. More than 30 loci have now been associated with MLL, usually by reciprocal translocation. Deletions, insertions, and more complex rearrangements of MLL are rarely seen. We present three cases of AML M5 showing no cytogenetic evidence of 11q23 rearrangement, in which a commercial MLL dual-color fluorescence in situ hybridization probe revealed a nonstandard abnormal signal pattern, suggesting cryptic insertion of the MLL gene into its partner gene site.

Fluorescence in situ hybridisation (FISH) in histologically challenging conjunctival melanocytic lesions.

BACKGROUND: Even in experienced hands, the classification of some melanocytic lesions of the conjunctiva remains challenging. In skin pathology, the recent application of fluorescence in situ hybridisation (FISH) has been demonstrated to be of use for the analysis and diagnosis of ambiguous melanocytic neoplasms of the skin. This study set out to evaluate this method on seven prospective conjunctival cases that were histologically equivocal. METHODS: 18 unequivocal retrospective melanocytic controls were exposed to FISH. Commercially available probes assessing copy numbers of RREB1 (6p25), MYB (6q23) and CCND1 (11q13) genes compared with CEP6 (a chromosome six centromeric reference point) were used. After control verification, seven prospective, equivocal cases were identified and exposed to FISH. RESULTS: There was complete correlation between FISH result and the control section histopathology report. Control cases of melanoma cases were all positive for FISH and control benign lesions were negative. Of the seven equivocal cases, five were positive and classed as invasive melanoma or melanoma-in situ, one was negative and one tetraploid, classed as negative (these last two cases were classed as naevi with careful clinical observation). CONCLUSIONS: FISH is very useful in classifying equivocal conjunctival melanocytic lesions, especially those with atypical junctional activity and naevoid melanocytic proliferations of the conjunctiva.

Five members of the CEBP transcription factor family are targeted by recurrent IGH translocations in B-cell precursor acute lymphoblastic leukemia (BCP-ALL).

CCAAT enhancer-binding protein (CEBP) transcription factors play pivotal roles in proliferation and differentiation, including suppression of myeloid leukemogenesis. Mutations of CEBPA are found in a subset of acute myeloid leukemia (AML) and in some cases of familial AML. Here, using cytogenetics, fluorescence in situ hybridization (FISH), and molecular cloning, we show that 5 CEBP gene family members are targeted by recurrent IGH chromosomal translocations in BCP-ALL. Ten patients with t(8;14)(q11;q32) involved CEBPD on chromosome 8, and 9 patients with t(14;19)(q32;q13) involved CEBPA, while a further patient involved CEBPG, located 71 kb telomeric of CEBPA in chromosome band 19q13; 4 patients with inv(14)(q11q32)/t(14;14)(q11;q32) involved CEBPE and 3 patients with t(14;20)(q32;q13) involved CEBPB. In 16 patients the translocation breakpoints were cloned using long-distance inverse-polymerase chain reaction (LDI-PCR). With the exception of CEBPD breakpoints, which were scattered within a 43-kb region centromeric of CEBPD, translocation breakpoints were clustered immediately 5' or 3' of the involved CEBP gene. Except in 1 patient with t(14;14)(q11;q32), the involved CEBP genes retained germ-line sequences. Quantitative reverse transcription (RT)-PCR showed overexpression of the translocated CEBP gene. Our findings implicate the CEBP gene family as novel oncogenes in BCP-ALL, and suggest opposing functions of CEBP dysregulation in myeloid and lymphoid leukemogenesis.

Folate-sensitive fragile site FRA10A is due to an expansion of a CGG repeat in a novel gene, FRA10AC1, encoding a nuclear protein.

Fragile sites appear visually as nonstaining gaps on chromosomes that are inducible by specific cell culture conditions. Expansion of CGG/CCG repeats has been shown to be the molecular basis of all five folate-sensitive fragile sites characterized molecularly so far, i.e., FRAXA, FRAXE, FRAXF, FRA11B, and FRA16A. In the present study we have refined the localization of the FRA10A folate-sensitive fragile site by fluorescence in situ hybridization. Sequence analysis of a BAC clone spanning FRA10A identified a single, imperfect, but polymorphic CGG repeat that is part of a CpG island in the 5'UTR of a novel gene named FRA10AC1. The number of CGG repeats varied in the population from 8 to 13. Expansions exceeding 200 repeat units were methylated in all FRA10A fragile site carriers tested. The FRA10AC1 gene consists of 19 exons and is transcribed in the centromeric direction from the FRA10A repeat. The major transcript of approximately 1450 nt is ubiquitously expressed and codes for a highly conserved protein, FRA10AC1, of unknown function. Several splice variants leading to alternative 3' ends were identified (particularly in testis). These give rise to FRA10AC1 proteins with altered COOH-termini. Immunofluorescence analysis of full-length, recombinant EGFP-tagged FRA10AC1 protein showed that it was present exclusively in the nucleoplasm. We show that the expression of FRA10A, in parallel to the other cloned folate-sensitive fragile sites, is caused by an expansion and subsequent methylation of an unstable CGG trinucleotide repeat. Taking advantage of three cSNPs within the FRA10AC1 gene we demonstrate that one allele of the gene is not transcribed in a FRA10A carrier. Our data also suggest that in the heterozygous state FRA10A is likely a benign folate-sensitive fragile site.