Partner Chromosomes Research Articles

The enigma of KMT2A gene in hematological disorders

Abstract Introduction/Objective Presence of distinct MLL rearrangements is an independent dismal prognostic factor. Rearrangements of the histone lysine [K]- MethylTransferase 2A gene (KMT2A) on chromosome 11q23, formerly known as the mixed-lineage leukemia (MLL) gene, are found in 10% and 5% of adult and children with acute leukemia respectively. Chromosome 11 breakpoints are typically localized to band 11q23.3, frequently altering the proto- oncogene KMT2A/MLL whereas partner chromosomes involved in translocation may vary. More than 80 different gene partners involved in this fusion have been described, although the majority of leukemias results from 6 common partner genes. KMT2A gene rearrangement are associated with poor prognosis in both Lymphoid and Myeloid leukemia. Methods/Case Report A retrospective study conducted in Cytogenetic laboratory of The Indus hospital, Karachi. All karyotype cases (age 1-60 year) with chromosome 11 aberrations were included from October 2020 to December 2022. Karyotype and FISH performed using G-banding and break a part probe for MLL respectively. Results (if a Case Study enter NA) Total of 923 cases received for karyotyping out of which 32(3.4%) cases were reported for 11q23 abnormalities. The median age was 5 (1.25-12.5) years, majority 15 (47%) were 1-10 years, 9(28%) were >10 years and 7(22%) were <1 years. M:F ratio was 1.9:1. B-ALL were 19(59.4%), AML 8(25%), and 5 (15.6%) were others. FISH performed in 23(72%) cases, 21 (91%) showed concordance with karyotype. The t(9;11) was the commonest 7(22%), followed by t(4;11) and (1;11). Chromosome 9 was the most common partner identified in B-ALL. Loss of 11q23 observed in 3 (13%) cases. Conclusion The findings underscore the heterogeneity of partner chromosomes involved in KMT2A gene rearrangements and emphasize the importance of employing both FISH and karyotyping for comprehensive detection. The high concordance rate between the two methods supports their complementary roles in clinical diagnostics. Understanding the specific translocations and partner chromosomes associated with MLL gene rearrangements contributes valuable insights for prognostication and targeted therapeutic strategies in acute leukemia. FISH and karyotype showed 91% concordance to detect this aberration.

Olfactory receptor genes and chromosome 11 structural aberrations: Players or spectators?

The largest multi-gene family in metazoans is the family of olfactory receptor (OR) genes. Human ORs are organized in clusters over most chromosomes and seem to include >0.1% of the human genome. Because 369 out of 856 OR genes are mapped on chromosome 11 (HSA11), we sought whether they mediate structural rearrangements involving this chromosome. To this aim, we analyzed 220 specimens collected during diagnostic procedures involving structural rearrangements of chromosome 11. A total of 222 chromosomal abnormalities were included, consisting of inversions, deletions, translocations, duplications, and one insertion, detected by conventional chromosome analysis and/or FISH and array-CGH. We verified by bioinformatics and statistical approaches the occurrence of breakpoints in cytobands with or without OR genes. We found that OR genes are not involved in chromosome 11 reciprocal translocations, suggesting that different DNA motifs and mechanisms based on homology or non-homology recombination can cause chromosome 11 structural alterations. We also considered the proximity between the chromosomal territories of chromosome 11 and its partner chromosomes involved in the translocations by using the deposited Hi-C data concerning the possible occurrence of chromosome interactions. Interestingly, most of the breakpoints are located in regions highly involved in chromosome interactions. Further studies should be carried out to confirm the potential role of chromosome territories’ proximity in promoting translocations, to help understand mechanisms underlying genome structural variation possibly associated to genetic diseases, but also fundamental to promote genome evolution.

A t(4;13)(q21;q14) translocation in B-cell chronic lymphocytic leukemia causing concomitant homozygous DLEU2/miR15a/miR16-1 and heterozygous ARHGAP24 deletions

13q14 deletion is the most recurrent chromosomal aberration reported in B-CLL, having a favorable prognostic significance when occurring as the sole cytogenetic alteration. However, its clinical outcome is also related to the deletion size and number of cells with the del(13)(q14) deletion. In 10% of cases, 13q14 deletion arises following a translocation event with multiple partner chromosomes, whose oncogenic impact has not been investigated so far due to the assumption of a possible role as a passenger mutation.Here, we describe a t(4;13)(q21;q14) translocation occurring in a B-CLL case from the diagnosis to spontaneous regression. FISH and SNP-array analyses revealed a heterozygous deletion at 4q21, leading to the loss of the Rho GTPase Activating Protein 24 (ARHGAP24) tumor suppressor gene, down-regulated in the patient RNA, in addition to the homozygous deletion at 13q14 involving DLEU2/miR15a/miR16–1 genes. Interestingly, targeted Next Generation Sequencing analysis of 54 genes related to B-CLL indicated no additional somatic mutation in the patient, underlining the relevance of this t(4;13)(q21;q14) aberration in the leukemogenic process.In all tested RNA samples, RT-qPCR experiments assessed the downregulation of the PCNA, MKI67, and TOP2A proliferation factor genes, and the BCL2 anti-apoptotic gene as well as the up-regulation of TP53 and CDKN1A tumor suppressors, indicating a low proliferation potential of the cells harboring the aberration. In addition, RNA-seq analyses identified four chimeric transcripts (ATG4B::PTMA, OAZ1::PTMA, ZFP36::PTMA, and PIM3::BRD1), two of which (ATG4B::PTMA and ZFP36::PTMA) failed to be detected at the remission, suggesting a possible transcriptional remodeling during the disease course. Overall, our results indicate a favorable prognostic impact of the described chromosomal aberration, as it arises a permissive molecular landscape to the spontaneous B-CLL regression in the patient, highlighting ARHGAP24 as a potentially relevant concurrent alteration to the 13q14 deletion in delineating B-CLL disease evolution.

Is t(11;14)(q13;q32) good or bad for newly diagnosed multiple myeloma?

Is t(11;14)(q13;q32) good or bad for newly diagnosed multiple myeloma?

A Single Next Generation Sequencing Assay for Detection of Driver Mutations, Translocations and Copy Number Alterations in Patients with Multiple Myeloma

Multiple myeloma (MM) is a plasma cell malignancy characterized by several genetic abnormalities that impact risk stratification, outcome, and response to therapy; with fluorescence in situ hybridization (FISH) considered the gold-standard assay to detect the presence of chromosomal translocations and copy-number variations (CNV). Here, we evaluate the performance of a single clinically actionable targeted sequencing (NGS) assay for detecting cytogenetic abnormalities in addition to recurrent driver mutations in MM. Patient-derived bone marrow aspirates were obtained and enriched for CD138+ plasma cells. Targeted sequencing was performed using a 2.3 Mb custom capture panel covering 139 genes and regions surrounding MYC, immunoglobulin heavy chain (IgH), and immunoglobulin light chain kappa (IgK) and lambda (IgL) loci. Chromosomal translocations were identified using an in-house algorithm designed specifically for targeted capture DNA sequencing data analyses. Structural variants were called based on discordant read pairs as well as junction split reads, solving the challenges from both the overlapping paired-end reads due to the longer read lengths (150 bp) and the mapping ambiguities in the Ig regions. CNV were assessed using both on and off target reads, using a 200 kb bin size for segmentation covering the entire genome. Clinical annotations and FISH panel reports were abstracted. We sequenced 264 MM patient samples, 94% of which had available FISH information. A median of two somatic mutated genes per sample (range 0-13) was found. The most commonly mutated genes per sample were: KRAS (35%), NRAS (25%), FAM46C (15%), DIS3 (13%), BRAF (13%), ATM (11%), and TP53 (11%). Comparing NGS with FISH, we found a perfect concordance for the identification of t(4;14), t(6;14), t(14;16), and t(14;20); a 100% specificity and 97% sensitivity for t(11;14); and sensitivity >90% and an average specificity of 85% for trisomies of chromosomes 3, 7, 9, 11, and 15. The NGS approach was also able to detect gain(1q) and mono(13) with a sensitivity and specificity >90%, and del(17p) with a 60% sensitivity and 100% specificity. The reduced sensitivity for del(17p) detection was mostly due to focal deletions and low tumor purity. Because the TP53 gene is included in the custom capture panel, we increased the sensitivity by determining six separate regions of coverage using on-target probes, which enabled the detection of focal del(17p), increasing the sensitivity to 78%. The NGS approach also allowed the characterization of 16 (6%) samples with unavailable FISH. Of the 27 samples with del(17p) detected by FISH, NGS was able to identify TP53 mutations (i.e., biallelic inactivation) in 14 samples (52%). Due to the existence of multiple and promiscuous chromosomal partners involved in IgH translocation, the identification of the chromosomal partners is limited to the clinical FISH probe sets; in fact, 19 study samples presented abnormal IgH without a known partner by FISH. Our NGS assay was able to identify these partners, confirming MYC-IGH translocations in seven (37%) of these cases. Additionally, we detected MYC translocations with immunoglobulin light chains: 14 (5%) cases with IGK, and eight (3%) with IGL. Although IgL translocations, particularly IgL-MYC translocations, have been recently associated to poor prognosis, they are not routinely evaluated in FISH assays. The risk stratification and clinical utility of the NGS assay was demonstrated by the shorter overall survival observed in samples classified as high risk (mSMART 3.0; hazard ratio [HR]= 2.81; CI:1.89-4.17; p<0.0001), which was also observed with risk assessment by FISH (HR = 2.48; CI:1.72-3.58; p<0.0001). Therefore, in addition to the detection of clinically relevant somatic mutations, the targeted NGS approach showed robust detection of recurrent MM specific IgH translocations and CNV. Furthermore, the detection of relevant abnormalities not routinely assessed in most clinical labs, such as TP53 bi-allelic inactivation, IgL and MYC rearrangements, supports the use of this small, targeted panel as a clinically relevant, single molecular assay, for the comprehensive profiling of MM.

Duplication of 8q24 in Chronic Lymphocytic Leukemia: Cytogenetic and Molecular Biologic Analysis of MYC Aberrations

Chronic lymphocytic leukemia (CLL) with cytogenetics findings, such as complex karyotype and deletions of TP53 or ATM, is associated with adverse clinical outcomes. Additional chromosomal abnormalities further stratify patients into groups with diverse prognoses. Gain of 8q24 is one of the abnormalities considered as prognostically unfavorable. In our study, we performed a FISH analysis in an initial cohort of 303 consecutive CLL patients and determined the frequency of +8q to be 6.3 %. Our analysis confirmed the association with TP53/ATM aberrations and CK, as the frequency of +8q reached 26.7 % in an extended delTP53/ATM+CK cohort. M-FISH analysis enabled the identification of partner chromosomes where the segment of the duplicated 8q arm was localized. More detailed mapping of the gained 8q region using the M-BAND method determined the smallest amplified region 8q23-8qter. We observed significantly shorter overall survival (OS; 9.0 years in +8q-positive vs. 10.6 years in +8q-negative; p=0.02) and detected slightly higher MYC mRNA/protein levels in +8q-positive vs. +8q-negative patients.

Abstract 3412: Liquid biopsy for t(11;14)/blc2 identification in multiple myeloma

Abstract Background: Multiple myeloma (MM) is a spatially heterogeneous plasma cell malignancy of the bone marrow (BM). Venetoclax is a potent inhibitor of the anti-apoptotic protein BCL2 with evidence in MM that t(11;14) and high BCL2 expression confers greater sensitivity to venetoclax. BM biopsy is required for fluorescent in-situ hybridisation (FISH) detection of t(11;14) and the evaluation of MM cell BCL2 expression, with success being entirely dependent on the quality of the BM sample procured. We hypothesised that a liquid biopsy strategy to enable interrogation of cell-free circulating tumour (ct)DNA and extra-cellular (ex)RNA would represent a more practical and less invasive alternative strategy to BM biopsy for the detection of t(11;14) and the determination of MM cell BCL2 expression, respectively. Methods: Blood and BM were obtained from 24 FISH characterised MM patients - normal karyotype (n=9), t(11;14) (n=11) and t(4;14) (n=4). Plasma derived ctDNA and exRNA was extracted utilising the QIAamp circulating nucleic acid kit and matched PBMC DNA was obtained for germ line controls. BM CD138+ MM cells for DNA and RNA extraction were selected with Miltenyi Biotec CD138 microbeads. A low density custom panel of unique capture probes (SSEL XT HS2, SureSelect Custom Tier 2, Agilent Technology), spanning approximately 10% of the CCND1 and IGH loci involved in t(11;14), was used for sequencing with approximately 5 million reads per sample with adapter sequences trimmed from the reads and aligned to the hg38 using bwa mem algorithm. Structural variant caller, Lumpy was used for identifying translocations, with low confidence reads excluded with a defined Z-score threshold. Droplet digital PCR (ddPCR) for BCL2 was performed on exRNA. Results: The low-density capture seq identified t(11;14) in 6 of 11 (55%) of the CD138+ BM samples, but not in paired germ-line samples nor in the non-t(11;14) cohort BM samples. Interrogation of ctDNA identified t(11;14) in 1 of 11 samples but also in ctDNA from a patient labelled as being t(4;14) positive raising questions about the validity of the FISH attribution. We are now utilising a modification of the assay using ultra-high density capture probes with coverage approaching 100% of the IGH region and higher ctDNA input to detect translocations involving the IGH region on chromosome 14 with any partner chromosome, including translocations undetectable by FISH. BCL2 exRNA transcripts were detected in all patients with a range of 2-120 copies/ml of plasma with 79% of patients classified as ‘high’ BCL2 expression (&amp;gt;40 copies/ml of plasma) compared to 59% of the matched CD138+ BM samples. Conclusion: We provide proof-of-concept of the potential of ctDNA and exRNA for t(11;14) detection and ddPCR quantitation of BCL2 expression, respectively, providing a rationale for further exploration of ctDNA and exRNA liquid biopsy testing as a strategy for identifying patients with t(11;14) and/or high BCL2. Citation Format: Andrew Spencer, Sridurga Mithraprabhu, Jaynish Shah, Kawa Choi, Ashley George, Carlos Hader, Tiffany Khong. Liquid biopsy for t(11;14)/blc2 identification in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3412.

EP060: Characterizing the complex chromosomal rearrangements in a Philadelphia negative BCR/ABL1 positive case

The Philadelphia chromosome (Ph), produced by the reciprocal translocation t(9;22)(q34;q11.2), is a derivative chromosome 22 with a chimeric BCR/ABL1 fusion gene encoding constitutively active tyrosine kinase protein. The Ph occurs in 95% of chronic myeloid leukemia (CML) cases and the BCR/ABL1 fusion is a diagnostic hallmark for CML. A small subset of patients (around 5-10%), displaying typical features of CML, lack a Ph by chromosome analysis, but are shown to carry BCR/ABL1 fusion either by fluorescence in situ hybridization (FISH) or molecular analysis. Two major mechanisms have been proposed to explain how BCR/ABL1 rearrangements arise in these Ph-negative BCR/ABL1 positive cases: a cryptic insertion between chromosomes 9 and 22; or via two sequential translocations where a standard t(9;22) translocation is followed by an additional reverse translocation resulting in the reconstitution of the normal morphology of the partner chromosomes. The bone marrow specimen of a 66 year old female patient, presented with persistent leukocytosis with myelocytes and metamyelocytes, along with thrombocytosis, was sent to our lab for both BCR/ABL1 RT-PCR analysis and chromosome analysis, due to suspicious myeloproliferative neoplasm. The RT-PCR analysis showed the presence of the BCR/ABL1 major (p210) breakpoint fusion transcript b2a2 (e13a2). Initial chromosome analysis did not identify a typical t(9;22)(q34;q11.2) reciprocal translocation; instead, structural abnormalities were observed on the short arm of chromosome 12 and the long arm of chromosome 22. Overall, the abnormal chromosome 22 resembles the morphology of a “Philadelphia chromosome” while both homologs of chromosome 9 show normal morphology. This leads us to hypothesize that a complex rearrangement involving chromosomes 9, 12, and 22 might be contributing to this case. Metaphase fluorescence in situ hybridization (FISH) analysis was performed to test our hypothesis using BCR/ABL1 fusion probes, where the only cryptic BCR/ABL1 fusion signal was mapped to the long arm of chromosome 9 that has normal morphology at band q34, while the long arm of the derivative chromosome 22 is absent for BCR/ABL1 fusion signals, suggesting that the Ph-like derivative chromosome 22 is not a real Philadelphia chromosome, making this case a true Ph negative case. The final ISCN for the case is designated as: 46,XX,der(9)(9pter->9q34::22q11.2->22q11.2::9q34->9qter),der(12)(22qter->22q11.2::12p13->12qter),der(22)(22pter->22q11.2::12p13->12pter), and this can be explained by an initial three way translocation between chromosomes 9, 12, and 22 followed by an additional sequential reverse translocation between the derivative chromosomes 9 and 22, leading the cryptic BCR/ABL1 fusion being inserted into the long arm of chromosome 9 and bringing chromosome 9 back to a normal morphology. Cryptic BCR/ABL1 fusion has been characterized as a variant rearrangement in CML patients lacking Ph, and up to 50% of these cases with variant BCR/ABL1 fusion exhibit a del(9q) of the derivative chromosome 9, which has been considered to be a marker for poor prognosis in CML patients. Thus, chromosomal microarray analysis (CMA) was also performed for this case, and no deletions in 9q were identified. Here, we presented a case where FISH mapping was utilized to further characterize the complex chromosomal rearrangements between chromosomes 9, 12, and 22 in a Ph negative BCR/ABL1 positive case, which reveals that the cryptic BCR/ABL1 is inserted into the long arm of chromosome 9 with normal morphology at band q34, while the Ph-like derivative chromosome 22 is not a true Ph chromosome. Our study illustrates the challenge of recognizing the complex pattern of cytogenetic aberrations in these Ph negative BCR/ABL1 positive CML cases with variant BCR/ABL1 rearrangements, and highlights the importance of utilizing comprehensive cytogenomic and molecular techniques (including metaphase FISH, RT-PCR and CMA) in characterizing these complex chromosomal abnormalities for proper diagnosis and prognosis prediction.

Frequency of Chromosomal Translocations Are Significantly Higher in Therapy Related Myeloid Neoplasm As Compared to Primary MDS

Background: Myelodysplastic syndromes (MDS) are a heterogeneous group of disorders with variable outcomes. Acquired chromosome abnormalities in patients with myelodysplastic syndromes (MDS) are among the most valuable determinants of prognosis. However, chromosomal translocations are rare in MDS, and their prognostic implication is not well known. To elucidate factors driving chromosomal translocations in MDS, we compared the frequency of MDS patients with and without prior exposure to DNA damaging cytotoxic therapies.Methods: We evaluated diagnostic bone marrow metaphase cytogenetic data of MDS patients enrolled in South Australian MDS/AML registry data. Appropriate Institutional Review Board approval was obtained. Diagnosis and subclassification of MDS were done according to World Health Organization (WHO) 2016 criteria and risk stratified according to the revised International Prognostic Scoring System (R-IPSS) (Greenberg et al., 2012). Standard G-banding method was used for cytogenetic analysis.To decipher changes induced by cytotoxic therapy (CT) from that of myeloid neoplasm in general, we compared cytogenetic profile of three carefully selected cohorts: (i) tMN, in which a myeloid neoplasm occurred in a cancer survivor following exposure to cytotoxic (n = 206, 15.5%); (ii) myeloid neoplasm developing in unrelated cancer survivors with NO prior exposure to CT (a diagnosis of primary MN and another cancer; pMN+Ca) (n =148, 12%). This would include myelodysplastic syndrome or acute myeloid leukemia diagnosed in patients managed with surgical resection of breast cancer and NO exposure to CT; (iii) primary MDS without preceding independent cancer and/or exposure to CT (pMDS) (n = 893,72.5%)Results: The median age of 1247 patients analyzed was 72.50 (IQR 64.9 to 79) and 64% patients were male. Majority of patients were diagnosed with pMDS (n=893; 72.5%), while 206 (15.5%) and 148 (12%) were classified as t-MN and pMN+Ca respectively.Ten percent (114/1247) patients harbored translocations at the time of diagnosis. The most frequent chromosomes involved in translocation are chromosome 1 (37/114, 34.5%), 3 (15/114, 13.8%), 5 (6/114= 5.3%) (Fig.1A). Most common chromosome partners for translocation with chromosome 1 were chromosome 3, 7 and 21.The frequency of chromosomal translocations in patients with monsomal (55/114, 48%) and complex (70/114, 61%) karyotype were higher than patients without. There is a clear relationship in incidence of chromosomal translocations with increasing IPSS-R scores. The frequency of translocations in the Very high risk IPSS-R risk category was significantly higher compared to patients with Very low risk category (27% vs.1.1%).Importantly, frequency of chromosomal translocations was significantly higher in t-MN as compared pMN+Ca and P-MN patients who were not exposed to DNA damaging cytotoxic therapies prior to their diagnosis (17% vs. 4% vs. 8%, P=0.0004; Fig 1B). Notably, the survival of patients with chromosomal translocation was significantly poorer as compared to patients without translocation (Fig.1C). Poor OS of patients with translocation is most probably due to association with complex karyotype (Fig.1D).Conclusion: This comprehensive analysis demonstrates that chromosomal translocations are observed in 10% of MDS patients. Chromosomal translocations are significantly higher in patients who had prior exposure to cytotoxic therapy as compared to patients without. [Display omitted] DisclosuresHiwase: Novartis: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees.

MYC Rearrangements in Myeloid Neoplasms

Background:8q24 /MYC rearrangements are an important mechanism of MYC protein dysregulation and most frequently occur in lymphoid neoplasms. Induced MYC expression in murine bone marrowrapidly induces leukemia and preferentially stimulates myeloid leukemogenesis in the absence of anti-apoptotic mutations (Luo et al, Blood, 2006). However, clinical data on patients with myeloid neoplasms and MYC rearrangement is very limited.Methods:We searched out databases for all cases with 8q24/ MYC rearrangements in the past 11 years and identified eight cases of myeloid neoplasms with MYC rearrangements which were confirmed by FISH analysis with MYC break apart rearrangement probe. The clinical and laboratory data, response to treatment and patient outcome were obtained through medical records. All samples were collected following institutional guidelines with informed consent in accordance with the Declaration of Helsinki.Results:Patients' clinical, pathological, and cytogenetic features are summarized in Table 1. There were 5 males and 3 females, with a median age of 60 years. Three patients were diagnosed with acute myeloid leukemia (AML), 1 with myelodysplastic syndrome (MDS) with excess blasts-2, and 4 with blastic plasmacytoid dendritic cell neoplasm (BPDCN).Flow cytometry immunophenotyping was performed in all 8 patients. Of the 3 patients with AML, case #1 had aberrant expression of CD2 and cCD3, cases #2-3 had an aberrant expression of CD7. Case #4 had a myeloid phenotype. The immunophenotype of cases #5-8 were consistent with BPDCN with expression of CD2, CD4, and bright 123. MYC immunohistochemistry stain was performed in 5 patients, the percentage of cells with MYC expression was variable, but largely correlated with the percentage of blasts in the bone marrow.Case #1 showed a normal diploid karyotype, but FISH revealed a cryptic der(14)ins(8;14)(q24;q32q32); Cases #4 and #8 had t(8;14)(q24;q32) as the sole chromosomal abnormality; the other 5 cases had a complex karyotype. The partner chromosomes for 8q24 were 14q32 (cases # 1, 4 and 8), 6p21 (case #5), Xq24 (case #6), 3p25 (case #7), and unknown in cases #2 and #3. FISH showed MYC rearrangement in a median of 80% cells (ranges, 4.5% -97%).The treatments were summarized in Table 1. Three AML patients did not response to chemotherapies or had a quick relapse after a short remission, all 3 patients died within 1 year. Case #4 (MDS) developed AML at 1 month and died at 2.2 months after the diagnosis of MDS. Of the four BPDCN patients, case #4 had an early death due to infection, the other 3 patients responded well to ALL-based regimens (Hyper-CVAD, n=2; COG ALL L0434, n=1), two were alive and in complete remission, one had been in remission but died of pulmonary hemorrhage at 26 months.Conclusions:8q24/ MYC rearrangement occurs rarely in myeloid neoplasms, often with partner genes other than the ones encoding immunoglobulin. Patients with AML/MDS were often resistant to chemotherapies, while patients with BPDCN responded well to ALL-based regimens. [Display omitted] DisclosuresPemmaraju:cellectis: Research Funding; stemline: Consultancy, Honoraria, Research Funding; LFB: Consultancy, Honoraria; affymetrix: Research Funding; roche diagnostics: Consultancy, Honoraria; novartis: Consultancy, Honoraria, Research Funding; Incyte Corporation: Consultancy, Honoraria; abbvie: Research Funding.

Evaluation of cytogenetic response in CML patients with variant Philadelphia translocation.

Molecular mechanism of translocation and outcome in variant chronic myeloid leukaemia (vCML) has been a topic of debate. While several cytogenetic studies suggest a low response to Imatinib Mesylate, others demonstrate a similar disease course in both classical and vCML. Besides, many studies comprehensively also link tyrosine kinase domain (TKD) mutations with aggressive clinical outcome. Thus, we aim to study the molecular mechanism of translocation, identify the third partner chromosomes and comment on the disease course and clinical outcome. We cytogenetically characterised 25 vCML cases to determine the third partner chromosome, mechanism of translocation and prognostic outcome. We also compared vCML cases with and without TKD mutation to most appropriately outline the clinical consequence and ascertain the potent cause of unresponsiveness to treatment. Third partner chromosome in variant translocation was defined by conventional and molecular cytogenetics. Although in our study most cases showed inadequate clinical response attributable to TKD mutation rather than variant translocation, we observed an inferior outcome in cases involving chromosome 5 as the third partner. Thus, we conclude that characterising and reporting new cases of variant translocations, involving various different chromosomes as third partner (with different breakpoints) by cytogenetics, will lead to a better understanding of the disease. To the best of our knowledge, this kind of delineate study has not been applied to precisely comment on the prospects of cytogenetically characterised vCML.

11q23/MLL rearrangements in adult acute leukemia.

To detect the frequency, diagnostic and prognostic significance of 11q23/MLL rearrangements and to determine the chromosomes that are most frequently involved in 11q23/MLL abnormalities in adult acute leukemia (AL). Cytogenetic investigations of bone marrow and/or peripheral blood cells from 140patients with acute myeloid leukemia (AML) and 57patients with acute lymphoblastic leukemia (ALL) were performed. The methods of conventional cytogenetics (GTG-banding) and fluorescence in situ hybridization were used. Chromosomal abnormalities in leukemia cells were found by conventional cytogenetic methods in 80 (57%) and 37 (65%) adult patients with AML and ALL, respectively. 11q23/MLL rearrangements were found in 7 (5%) and 8 (14%) patients with AML and ALL, respectively. Among them, 8 (53.4%) patients had translocations, 2 (13.3%)- had deletions and 5 (33.3%) patients had trisomies or tetrasomies of chromosome 11. With respect to the distribution of partner chromosomes involved in 11q23/MLL translocations chromosome 4was found to participate in 3 (37.5%) cases of 11q23/MLL translocations, 9- in 2 (25%) cases and chromosomes 10, 14and non-identified chromosome were involved in 1 (12.5%) case each. Nine patients (60%), besides abnormal ones, had 9-86% normal metaphases in their karyotypes. Of 15patients with 11q23/MLL rearrangements, 5 (33%) patients had only 11q23/MLL rearrangements, whereas other 10 (67%)- had additional cytogenetic abnormalities, besides 11q23/MLL rearrangements. Chromosomal abnormalities of various kinds were found in 57% and 65% adult patients with AML and ALL, respectively. The frequency of 11q23/MLL rearrangements in patients with AML and ALL was 5% and 14%, respectively. Since AL patients with 11q23/MLL rearrangements are attributed to cytogenetic categories of AL with a poor or intermediate risk prognosis, cytogenetic methods should be included in the standard examination of AL patients for diagnosis, prognosis and selection of the optimal treatment strategy.

Multicolor Spectral Karyotyping Identifies New Recurring Breakpoints and Translocations in Multiple Myeloma

Karyotypic information on multiple myeloma (MM) is less extensive than that on other myeloid or lymphoid malignancies due to low mitotic activity of plasma cells. An add(14)(q32) marker chromosome has been reported to be the most frequent recurring abnormality in clonally abnormal cases; in approximately one third of the latter cases, this marker has been identified as a der(14)t(11;14)(q13;q32) chromosome. To map chromosomal breakpoints, characterize the add(14)(q32) marker chromosomes, and to identify other recurring translocations in MM, we used spectral karyotyping (SKY) to analyze a panel of nine bone marrow (BM) biopsy samples from eight patients and 10 tumor cell lines derived from MM patients. SKY involves hybridization of 24 fluorescently labeled chromosome painting probes to metaphase spreads in such a manner that simultaneous visualization of each of the chromosomes in a different color is accomplished. By this method, it was possible to define all chromosomal rearrangements and identify all of the clonal marker chromosomes in tumor cells. By detailed mapping of breakpoints of rearrangement, it was also possible to identify several novel recurring sites of breakage that map to the chromosomal bands 3q27, 17q24-25, and 20q11. The partner chromosomes in translocations that generated the add (14)(q32) marker chromosomes were identified in all cases in which they were detected by G-banding (one biopsy and six cell lines). In addition, two new translocations involving band 14q32, ie, t(12;14)(q24;q32) and t(14;20)(q32;q11) have also been identified. These studies demonstrate the power of SKY in resolving the full spectrum of chromosome abnormalities in tumors.© 1998 by The American Society of Hematology.

Abstract 6559: Novel anti-myeloma potential of CAPE analogs: Downregulation of IKZF1-IRF4-MYC axis

Abstract Background: Multiple myeloma (MM) is the second most common hematological malignancy and an incurable bone marrow cancer so far. The hallmark of myeloma is chromosomal translocations that transform plasma cells into malignant myeloma cells. 50-75% of myeloma patients exhibit translocations at the immunoglobulin heavy chain (IgH) locus juxtaposing oncogenes from partner chromosome under the control of strong 3' IgH enhancers. Overexpression of oncogenes such as Cyclin D1, Cyclin D3, interferon regulatory factor (IRF4) etc. occurs, depending on the translocating partner locus. We have earlier reported that elevated expression of octameric binding protein-2 (OCT2), a key transcription factor in IgH translocations as a poor prognostic factor and shown its association with reduced survival in MM patients. IRF4 is another indispensable transcription factor for plasma cell differentiation, and its juxtaposition with IgH locus leads to IRF4 overexpression mediated tumorigenesis. Both OCT2 and IRF4 are regulated by the transcription factor, nuclear factor kappa B (NF-κB). Current chemotherapeutics exhibit several side effects, and face the challenge of chemoresistance, thus warranting the need for novel therapeutics for myeloma. Caffeic acid phenethyl ester (CAPE) is an active principle of propolis from honeybee hives, with potent NF-κB inhibitory activity. Hence, we synthesized and evaluated the structure-activity relationship of twenty-one CAPE analogs for their anti-myeloma potential. Methods: Human myeloma cell lines, lenalidomide, CAPE and its analogs were used for the study. Myeloma cell growth inhibition by CAPE analogs was determined by PrestoBlue cell viability assays. Effect of CAPE analogs on IRF4, OCT2, CyclinD3, caspase-3, Ikaros (IKZF1), MYC levels were evaluated by qRT-PCR and/or immunoblotting methods; apoptosis induction detected by Annexin V-PI flow cytometry assays. Results: Cyclohexylethyl and two phenpropyl ester analogs exhibited high myeloma cell growth inhibition in comparison with CAPE and lenalidomide. IRF4 was significantly downregulated both at mRNA and protein levels by the phenpropyl ester analog that demonstrated a remarkable increase in cleaved caspase-3 levels and apoptotic cell number. For the first time, we have demonstrated the ability of CAPE analogs to decrease OCT2, IKZF1, IRF4 and MYC levels in myeloma cells. Downregulation of the IKZF1-IRF4-MYC axis by CAPE and its key analogs revealed their novel anti-myeloma mechanism of action. Furthermore, the lead analogs showed no adverse cytotoxic effect on normal human mononuclear or naïve B-cells, also exhibited appropriate in silico pharmacokinetic properties and drug-likeness. Conclusion: Thus, our findings suggest the promising application of CAPE analogs to target Ikaros/IRF4 addiction, the so-called Achilles heel of myeloma, thereby leading to better therapeutic outcomes. Citation Format: Alli Murugesan, Gregoire Lasalle-Claux, Lauren Hogan, Elise Vaillancourt, Ayyoub Selka, Katie Luiker, Andy Hong, MinJi Kim, Mohamed Touaibia, Tony Reiman. Novel anti-myeloma potential of CAPE analogs: Downregulation of IKZF1-IRF4-MYC axis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6559.

The role of phosphorylation in the elasticity of the tethers that connect telomeres of separating anaphase chromosomes

ABSTRACT Elastic tethers, connecting telomeres of all separating anaphase chromosome pairs, lose elasticity when they lengthen during anaphase. Treatment with phosphatase inhibitor CalyculinA causes anaphase chromosomes to move backwards after they reach the poles, suggesting that dephosphorylation causes loss of tether elasticity. We added 50nM CalyculinA to living anaphase crane-fly spermatocytes with different length tethers. When tethers were short, almost all partner chromosomes moved backwards after nearing the poles. When tethers were longer, fewer chromosomes moved backwards. With yet longer tethers none moved backward. This is consistent with tether elasticity being lost by dephosphorylation. 50nM CalyculinA blocks both PP1 and PP2A. To distinguish between PP1 and PP2A we treated cells with short tethers with 50nM okadaic acid which blocks solely PP2A, or with 1µM okadaic acid which blocks both PP1 and PP2A. Only 1µM okadaic acid caused chromosomes to move backward. Thus, tether elasticity is lost because of dephosphorylation by PP1.

Prenatal identification of partial 3q duplication syndrome

BackgroundThe 3q duplication syndrome is a result of duplication of a large fragment of the long arm of chromosome 3, mainly 3q21-qter, and in most cases it is diagnosed only after birth. The phenotypic consequences resulting from genetic imbalance are an important source of information for genetic counselling, especially in prenatal diagnostics. However, in most cases it is impossible to define them precisely because the final clinical presentation is a result of an overlap, usually due to different sizes of deletions and/or duplications not only chromosome 3 but also of translocation partner chromosome. In this article, we present a prenatal diagnosis of the 3q duplication syndrome in a foetus, arising from a balanced insertion ins (7,3)(q21.2;q12.3q29) carried by the mother.Case presentationThe article presents a case of a 29-year-old woman referred to the Genetic Outpatient Clinic for consultation in the 12th week of her fifth pregnancy with a diagnosis of generalised hydrops foetalis. The analysis of karyotype using GTG technique and FISH allowed diagnosis of a balanced aberration in the mother, and determined the type of chromosomal rearrangement, which allowed the identification of the origin of the additional genetic material in the foetus and the previous malformed child of the same couple. The use of molecular karyotyping techniques (FISH and aCGH) allowed a precise determination of the size of the imbalanced fragments in the affected siblings.ConclusionsThe aCGH technique is particularly valuable for the diagnostics of submicroscopic deletions and duplications, if no imbalanced chromosomal aberrations are detected by routine cytogenetic tests. It is also a valuable technique for identifying and fully characterizing genetic material of unknown origin, which can’t be identified using routine cytogenetic techniqes. However, it does not allow identification of balanced aberrations in carriers.

Cytogenetic analysis of 130 renal oncocytomas identify three distinct and mutually exclusive diagnostic classes of chromosome aberrations.

The cytogenetic alterations in renal oncocytoma (RO) are poorly understood. We analyzed 130 consecutive RO for karyotypic alterations. Clonal chromosome abnormalities were identified in 63 (49%) cases, which could be categorized into three classes of mutually exclusive cytogenetic categories. Class 1 (N = 20) RO had diploid karyotypes with characteristic 11q13 rearrangement in balanced translocations with 10 or more different chromosome partners in all cases. We identified recurrent translocation partners at 5q35, 6p21, 9p24, 11p13-14, and 11q23, and confirmed that CCND1 gene rearrangement at 11q13 utilizing fluorescence in situ hybridization (FISH). Class 2 RO (N = 25) exhibited hypodiploid karyotypes with loss of chromosome 1 and/or losses of Y in males and X in females in all cases. The class 3 tumors comprising of 18 cases showed diverse types of abnormalities with the involvement of two or more chromosomes exclusive of abnormalities seen in classes 1 and 2 tumors. Furthermore, karyotypically uninformative cases were subjected to FISH analysis to identify classes 1 and 2 abnormalities. In this group, we found similar frequencies of CCND1 rearrangement, loss of chromosome 1 or Y as with karyotypically abnormal cases. We validated our results against 91 tumors from the Mitelman database. Correlation of clinical data with all the three classes of ROs showed no clear evidence of overall patient survival. Our findings support the hypothesis that RO exhibit three principal cytogenetic categories, which may have different roles in initiation and/or progression. These cytogenetic markers provide a key tool in the diagnostic evaluation of RO.

The frequency of chromosomal abnormalities in AL amyloidosis and their impact on treatment response.

e19539 Background: Chromosomal abnormalities (CA) found in AL amyloidosis (AL) are similar to those found in multiple myeloma (MM.) However, t(11;14) has been reported more frequently in AL, anywhere from 30% - 50% in studies. It has been reported that the presence of t(11;14) confers a lower hematologic response rate with bortezomib-based therapy (BBT) and a better hematologic response rate with high dose melphalan and autologous stem cell transplantation (HDM/SCT). Methods: We performed a retrospective review of 307 newly diagnosed patients seen at our center between January 2013 and December 2017, for whom interphase fluorescent in-situ hybridization (iFISH) data, performed on bone marrow aspirate using standard MM FISH probes on enriched plasma cells after magnetic separation, were available. Patients who already had iFISH performed prior to their visit with us were also included. We collected data on the incidence of CA, first line treatment, and hematologic response to this treatment. Results: Of the 307 patients, CA were not detected in 37%. 21% had t(11;14), 25% had 13q deletion, 21% had IgH rearrangements with an undetermined partner chromosome, and 11% had 1q21 gain. In those for whom initial treatment and hematologic response data were available, in patients with t(11;14), hematologic complete response (CR) was achieved by 18% following HDM/SCT and 27% following BBT. For those with 1q21 gain, CR was achieved by 33% following HDM/SCT and 19% following BBT. In contrast, hematologic CR was achieved by 47% following HDM/SCT and 40% following BBT for those who had no detectable CA with iFISH. Conclusions: Our study shows a lower incidence of t(11;14) in AL compared to other studies. This may be partly explained by the high number of patients with IgH rearrangements with undetermined partner chromosomes, some of whom may indeed harbor t(11;14). Our study also did not show a better hematologic response rate with HDM/SCT in t(11;14) patients compared with BBT. Hematologic response rates were much better both with HDM/SCT and BBT for those with no detected CA compared to patients who had t(11;14) or 1q21 gain.

Shugoshin protects centromere pairing and promotes segregation of nonexchange partner chromosomes in meiosis

Faithful chromosome segregation during meiosis I depends upon the formation of connections between homologous chromosomes. Crossovers between homologs connect the partners, allowing them to attach to the meiotic spindle as a unit, such that they migrate away from one another at anaphase I. Homologous partners also become connected by pairing of their centromeres in meiotic prophase. This centromere pairing can promote proper segregation at anaphase I of partners that have failed to become joined by a crossover. Centromere pairing is mediated by synaptonemal complex (SC) proteins that persist at the centromere when the SC disassembles. Here, using mouse spermatocyte and yeast model systems, we tested the role of shugoshin in promoting meiotic centromere pairing by protecting centromeric synaptonemal components from disassembly. The results show that shugoshin protects the centromeric SC in meiotic prophase and, in anaphase, promotes the proper segregation of partner chromosomes that are not linked by a crossover.

WGS and RNA-Seq Is Superior to Conventional Diagnostic Tests in Multiple Myeloma: Ready for Prime Time?

Background: The diagnosis and risk stratification of multiple myeloma (MM) is based on clinical and cytogenetic tests. Magnetic CD138 enrichment followed by interphase FISH is the gold standard to identify prognostic translocations and copy number alterations. Gene expression (GEP) studies show that MM might consist of various subgroups with distinct clinical outcomes (Zhan et al., Blood 2006). Whole genome sequencing (WGS) and targeted sequencing studies have further shed light onto recurrent mutations in MM and clinical implications are being derived (Lohr et al., Cancer Cell 2014). We set up a single workflow to analyze MM by WGS and RNA-Seq to evaluate whether this diagnostic workup is superior to conventional diagnostic testing.Methods: The cohort comprised 211 patients (pts) diagnosed with MM at our institution from 2011 to 2017. For all pts FISH and WGS was performed on CD138 enriched cells. For WGS 150bp paired-end sequences where generated on Illumina HiseqX and NovaSeq 6000 (Illumina, San Diego, CA). A mixture genomic DNA from multiple anonymous donors was used as normal controls. To remove potential germline variants, each variant was queried against the gnomAD database, variants with global population frequencies >1% where excluded. 47 genes recurrently mutated in MM were selected for evaluation (Kortüm et. al., Blood 2016). Copy number alterations (CNAs) were called using GATK4 and structural variations (SVs) were called using MANTA accounting for missing matched-normal samples. For transcriptome analysis total RNA was sequenced and the resulting estimated gene counts were pre-processed and normalized, applying trimmed mean of M-values normalization method.Results: WGS allowed us to detect 98/102 (96%) translocations that had previously been identified by FISH. Specifically we confirmed 24/24 of t(4;14), 6/7 of t(6;14), 11/12 of t(8;14), 51/53 of t(11;14) and 6/6 of t(14;16) cases by WGS. Moreover, by conventional FISH 12 pts had an IGH (n=4) or MYC (n=8) translocation with an unknown partner chromosome. We identified all 12 translocations by WGS. By WGS we also identified 679/740 (92 %) copy number alterations (CNA) detected by FISH. In detail these were 100/103 del(13q), 17/21 del(17p), 10/10 del(1p) and 79/87 +1q. Concordance rates for trisomies 3, 5, 9, 11, 15 and 19 were 80/91, 75/87, 92/97, 87/97, 53/55 and 86/92, respectively.Zhan et al. defined 7 MM subgroups (CD-1, CD-2, HY, MF, MS, LB, PR) based on GEP. 4 groups (CD-1/CD-2, MF, MS) were genetically defined by recurrent translocations and 1 by hyperploidy (HY). They used 700 probes to separate the groups of which 400 transcripts were recovered in our RNA-Seq analysis (GEPSeq). Supervised clustering grouped all 211 pts at the following frequencies: CD-1 (5%), CD-2 (25%), HY (30%), MF (5%), MS (11%), LB (14%), (PR 10%). 56/62 (90%) of pts that were allocated to CD-1/CD-2 had the characteristic translocation (t(11;14) or t(6;14)), while 23/24 (92%) of pts and 5/10 (50%) pts in GEPSeq group MS and MF had the respective t(4;14) or t(14;16). GEP allocates pts with hyperdiploidy to group HY and 51/63 (81%) pts in HY had a hyperdiploid karyotype by FISH. We specifically queried the WGS data for patients with discrepant FISH and GEPSeq results: WGS identified hyperdiploidy in 10/12 patients that were allocated to HY and could not be assigned by FISH due to insufficient material for complete testing, resulting in a 97% final concordance of GEPSeq and karyotype. One patient in group MS without FISH data for t(4;14) could be confirmed by WGS as harbouring the translocation (concordance 100%). Interestingly in 5/5 patients that were allocated to the MF group by GEPSeq 2 had an IGH-MYC or IGH-MYCN rearrangement respectively and 2 had another IGH rearrangement involving chromosome 8q. By WGS the most frequently mutated (mut) genes were KRAS (26%), NRAS (23 %), TP53 (8%), BRAF (4%) and ATM (2%), which is in line with published data (Lohr 2014). NRASmut was significantly associated with GEPSeq groups CD-2 and HY (p=0.001) and ATMmut with MF, MS and PR (p=0.047).Conclusion: RNA-Seq and WGS prove highly valuable in differentiating genetically distinct MM subgroups. The simultaneous analysis of gene mutations might have future implications for study design and selecting treatment options. A single workflow based on WGS and RNA-Seq provides a comprehensive genetic analysis in MM, is feasible and might substitute conventional diagnostic testing in the near future. DisclosuresHöllein:MLL Munich Leukemia Laboratory: Employment. Twardziok:MLL Munich Leukemia Laboratory: Employment. Walter:MLL Munich Leukemia Laboratory: Employment. Hutter:MLL Munich Leukemia Laboratory: Employment. Hernández:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.