Clonal Chromosomal Abnormalities Research Articles

Genomic Landscape of Ccus Compared to MDS Indicates a Potential Applicability of the IPSS-M

Background: The 5 th edition of the WHO classification newly included myeloid precursor lesions introducing CCUS as an entity. CCUS is defined as clonal hematopoiesis (CH) in the presence of unexplained, persistent cytopenia requiring detection of either somatic mutation in certain CH associated genes or clonal chromosomal abnormalities. Aim: Characterize a large CCUS cohort with respect to cytogenetics and molecular genetics and compare the findings to an MDS cohort to evaluate differences in the genomic landscape. Methods: The CCUS cohort comprised 222 cases (median age: 76 y; female: 33%), the MDS cohort 698 cases (median age: 73 y; female: 43%). The diagnoses were established following WHO 2022. All samples were analyzed by cytomorphology, cytogenetics, targeted NGS panel (median coverage 1500x) and whole genome (median coverage 100x) sequencing. Mutation (MUT) status of 59 genes associated with myeloid malignancies were analyzed in detail (54 CH associated; 5 relevant for IPSS-M). Results: Significant differences in age, gender, BM blasts, blood parameters and number and types of cytopenias were detected. CCUS patients (pts) were older, had more WBC, less PLT, harbored a higher HB, less BM blasts and were even more predominantly male compared to MDS pts (all p<0.05). Within the CCUS cohort 29 pts (13%) showed cytogenetic abnormalities (CA) only (thereof 69% loss of chromosome Y (Y-loss)), while the remaining 193 pts (87%) showed at least one MUT (VAF ≥2%) in any of the 54 CH associated genes (thereof 84% MUT only; 16% MUT+CA). Regarding cytogenetics 162 CCUS pts (73%) harbored normal karyotypes while CA were detected in 27% (MDS: 43%; p<0.001) with Y-loss being most frequent (39/60; 65%). Y-loss was more frequently found in CCUS than in MDS (17% vs. 5%; p<0.001), while complex karyotypes and del(5q) were more frequent in MDS (11% and 16% vs. 1% and 0%; both p<0.001). Based on WGS data additional pts with Y-loss (n=7; confirmed by FISH) and recurrent CN-LOH (4q: n=4; 7q: n=5) were observed. With respect to somatic MUTs, 356 MUTs were found within the entire CCUS cohort (in 28 CH genes, 3 non-CH genes). Overall, CCUS pts showed less MUTs (median: 1 [0-6]) compared to MDS (median: 2 [0-12]; p<0.001). CCUS pts most frequently harbored MUTs in DNMT3A (32%), TET2 (28%) and ASXL1 (14%) followed by MUTs in splicing genes and TP53 (Fig. 1A). MUTs in DNMT3A and PPM1D were more frequent in CCUS than MDS while MUTs in ASXL1, TP53, SF3B1, STAG2, RUNX1, NRAS, CUX1 were less frequent in CCUS (each p<0.05). Notably, biallelic TP53 MUTs were less frequent in CCUS compared to MDS ( TP53bi of mutated: 14% vs. 55% p=0.008). Within the 15 most frequently mutated genes in CCUS, MUTs in 6 genes ( DNMT3A, SF3B1, TP53, CBL, STAG2, PPM1D) showed a median VAF <10%. Nine of the top 15 genes showed significantly lower median VAFs than in MDS ( DNMT3A, TET2, ASXL1, SRSF2, U2AF1, SF3B1, TP53, PPM1D, IDH2; each p<0.05). Thus, the frequency and VAF of ASXL1, TP53, SF3B1 MUTs were significantly lower in CCUS than in MDS, while DNMT3A and PPM1D MUTs were significantly more often detected in CCUS but at a lower VAF than in MDS. Combining cytogenetics and mutational analysis we calculated the IPSS-M, a patient-specific risk score for MDS resulting in six risk categories, and detected a clear skewing towards low risk categories in CCUS (Fig. 1B). The frequency of categories very low (VL) and low (L) were significantly higher in CCUS than in MDS (30% vs. 15%; 50% vs. 40%; both p<0.013), while high and very high categories were more rare (1% vs. 13%; 1% vs. 14%; both p<0.001). Although CCUS follow-up data were short (median: 1.5 y), no differences in overall survival were observed between CCUS VL+L and MDS VL+L, while within CCUS survival differed between VL+L and the remaining cases ( p=0.05). Conclusions: Our data confirm the comparable mutational spectrum between CCUS and MDS but clearly show major differences in the frequency and VAF of distinct gene mutations. These biological differences hint towards different subgroups within CCUS with cases closer to MDS than others. A combined risk score for MDS and CCUS would reflect this continuous spectrum and has the potential to derive an objective risk assessment irrespective of observer-dependent grading of dysplasia. Our study indicates plausible short term results for CCUS patients stratified according to the IPSS-M. However, the definite applicability of the IPSS-M needs to be confirmed in larger CCUS studies with longer follow-up.

CASE REPORT: CML PROGRESSION TO AML-MR WITH CLONAL CHROMOSOMAL ABNORMALITIES

CASE REPORT: CML PROGRESSION TO AML-MR WITH CLONAL CHROMOSOMAL ABNORMALITIES

Hematological Response to Avatrombopag Switch in Thrombopoietin Receptor Agonist-Refractory Aplastic Anemia

ATG combined with CsA is the first-line treatment for patients with SAA who cannot choose HLA-matched HSCT because of various conditions. The HR rate is only approximately 60-80%, and the CR is not high. The lack of residual HSC in patients is an important reason for this unsatisfactory effect. RhTPO/TPO-RA combine with HSC membrane receptors to promote their proliferation and differentiation, expand multipotent hematopoietic progenitor cells in vivo. Studies have found that TPO-RA eltrombopag (EPAG) combined with IST can significantly increase the HR rate and improve the quality of the HR. From 40-50% hematologic improvement can also be obtained with refractory SAA. However, TPO-RA treatment is ineffective in some patients. Avatrombopag (APAG) acts on the transmembrane region of the TPO receptor, has a stronger effect than EPAG, and has no obvious liver toxicity. We analyzed the efficacy and safety of IST combined with rhTPO and/or EPAG in patients with SAA of switching to APAG. We retrospectively analyzed the data of 10 patients who were hospitalized between January and December 2021. All patients have been diagnosed with V/SAA. They received IST combined with rhTPO/TPO-RA treatment, failed to obtain a HR, and were converted to APAG. The patients were treated with IST for at least 6 months, combined with rhTPO and/or EPAG for at least 3 months, and did not get HR. None of the patients included in this study was suitable for undergo allogeneic HSCT. APAG was administered orally at a dose of 40 mg/day once a day for at least 6 months.The efficacy criteria for refractory SAA were defined as follows: one-line response included platelet response (HI-P), neutrophil response (HI-N), and erythroid response (HI-E). The 2-line HR could be a combination of any two of the aforementioned one-line responses. A trilineage response was defined as a patient with simultaneous platelet, erythroid, and neutrophil responses, and the quality of the trilineage response was the same as that of the initial treatment with SAA/TDNSAA. Ten patients were included in the study, with a median age of 54 (14-68) years, including five males and five females. All patients received IST for a median of 8.5 (6-40) months. Four patients received rhTPO for 1-2 months followed by EPAG for 2-8 months. Six patients received only EPAG for 3-4 months. All the patients received rhTPO/TP-RA for a median of 3.5 (3-10) months. Five patients with PNH clones were positive by flow cytometry, and there was no hemolytic or biochemical evidence of hemolysis. Two patients had clonal chromosomal abnormalities: one with 47,XY; +8 [2/20] and the other with 45,XY; -Y [10/20]. Ten patients were converted to APAG treatment for a median of 6 (6-10) months. After 3 months of follow-up , seven patients obtained a HR (7/10, 70%), of which two patients obtained a HI-P and HI-N, and five patients (5/10, 50%) obtained a three-line HR (1-CR, 3-GPR, 1-PR). Seven patients (7/10, 70%) obtained HR after 6 months of follow-up, of which one patient obtained HI-P/N and six patients (6/10, 60%) obtained three-line HR (3-CR, t2-GPR, 1-PR). The patient with two-line HR further improved after 8 months of APAG, reaching a three-line blood cell response to PR. At the end of the last follow-up, the HR rate of all patients with APAG conversion therapy was 70%, and all of them achieved three-line blood cell responses (4-CR, 1-GPR, 2-PR). During the follow-up period, no APAG-related adverse events of grade 2 or above were found in any patient, no thrombotic events occurred, and no fibrous tissue hyperplasia was found in the bone marrow pathology review after 6 months of treatment. Among the five patients with PNH clones before APAG conversion therapy, three patients did not change significantly. Otherwise one patient's granulocyte PNH clone decreased from 16.2% at baseline to 6.3%, and the other patient's PNH clone increased from 9.8% at baseline to 19.6%, whereas there was no symptomatic hemolysis in all patient. One patient (1/10, 10%) with normal chromosomal before therapy get a new clonal chromosomal abnormality after 3 months. It was 46XX, t(X;4 )(p11.4;q21) [4/20]; the HR was assessed as GPR. In conclusion, patients with SAA refractory to TPO-RA, drug conversion therapy can be based on the safety, pharmacological, and pharmacodynamic characteristics of TPO-RA drugs. Therefore, APAG is a better choice for switch therapy in these patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Cytogenetics in Fanconi Anemia: The Importance of Follow-Up and the Search for New Biomarkers of Genomic Instability.

Fanconi Anemia (FA) is a disease characterized by genomic instability, increased sensitivity to DNA cross-linking agents, and the presence of clonal chromosomal abnormalities. This genomic instability can compromise the bone marrow (BM) and confer a high cancer risk to the patients, particularly in the development of Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemia (AML). The diagnosis of FA patients is complex and cannot be based only on clinical features at presentation. The gold standard diagnostic assay for these patients is cytogenetic analysis, revealing chromosomal breaks induced by DNA cross-linking agents. Clonal chromosome abnormalities, such as the ones involving chromosomes 1q, 3q, and 7, are also common features in FA patients and are associated with progressive BM failure and/or a pre-leukemia condition. In this review, we discuss the cytogenetic methods and their application in diagnosis, stratification of the patients into distinct prognostic groups, and the clinical follow-up of FA patients. These methods have been invaluable for the understanding of FA pathogenesis and identifying novel disease biomarkers. Additional evidence is required to determine the association of these biomarkers with prognosis and cancer risk, and their potential as druggable targets for FA therapy.

FISH Analysis of CDKN2A/B Deletion in Chronic Lymphocytic Leukemia

Introduction: The course of chronic lymphocytic leukemia (CLL) ranges from indolent to rapid disease progression, including transformation to an aggressive lymphoma (Richter transformation, RT). A subset of CLL pts have been shown to acquire deletions of the tumor suppressor CDKN2A/B. RT has been associated with deletion of CDKN2A/B, however, prior studies’ sample populations were limited or not primarily focused on those with this deletion. Due to the rarity of this genetic abnormality in CLL and severity of RT, additional studies concerning this subgroup should be investigated. Here, we examine a larger cohort of CLL pts with CDKN2A/B deletion by fluorescence in situ hybridization (FISH) to provide further clarity on this genetic lesion. Methods: We conducted a retrospective case study for all pts for whom cytogenetic clinical samples were submitted for extended CLL FISH panel analysis over a 3.5 year (yrs) period, between 2015 and 2018 at The Ohio State University. Pts were screened for CDKN2A/B deletion with a new or follow up diagnosis of CLL or small lymphocytic lymphoma (SLL). Cytogenetic testing was performed on cells stimulated with pokeweed mitogen, phorbol myristate acetate (PMA), and CpG oligonucleotides. FISH for D13S319, D12Z3, ATM, TP53, BCL6, MYC, REL, SEC63, MYB, and CDKN2A/B was performed via manufacturer's recommendations. IGHV mutational status was determined using PCR. A 50-gene hematologic sequencing panel was performed via Ion Torrent. Overall survival (OS) from first detection of abnormal CDKN2A/B results were examined. Cox regression model was used to evaluate OS post CDKN2A/B deletion. The Fine and Gray Model was used to examine correlation of variables with regard to RT, treating death without RT as a competing risk. Results: 636 pts had sample(s) submitted for extended CLL FISH panel analysis. Of these, 43 (6.8%) pts with confirmed CLL or SLL were found to have loss of CDKN2A/B by FISH, including 6 pts within one year of diagnosis. Median age at diagnosis was 54.9 yrs (range 41.9-77.7). Loss of CDKN2A/B was detected at a median of 7.8 yrs (range diagnosis-26.1) from diagnosis. Of those with known IGHV status (n=33), 91% were unmutated. A complex karyotype (>3 clonal chromosomal abnormalities) was found in 90% percent of pts. In regard to CDKN2A/B deletion, 77%, 16%, and 7% of pts had heterozygous, homozygous, or subclonal populations of heterozygous as well as homozygous loss, respectively. Additional FISH testing showed 23% had gain of REL, 16% had BCL6 gain/rearrangement, 16% had deletion 6q, 37% had gain/rearrangement of MYC, 16% had loss of ATM, 14% had trisomy 12, and 58% had deletion 13q. Deletion of TP53 was seen in 63%. Sequencing data was available for 15 pts (35%), of note, 10 pts had mutations in TP53, with 4 also having mutations in SF3B1. In terms of OS, there were 30 deaths in the cohort, with median OS of 10.8 yrs from diagnosis (95% CI 8.0-14.4). With a median follow-up of 4.9 yrs among survivors, the median OS from time of detection of CDKN2A/B deletion was 1.7 yrs (95% CI 0.3-4.7). Multivariable analysis of OS post CDKN2A/B detection was significant for increasing beta-2-microglobulin measured at first visit at our institution (p=0.0044, hazard ratio (HR) 1.32 (95% CI 1.09-1.60)) and CDKN2A/B subclonal heterozygous and homozygous loss (p=0.0024, HR 9.81 (95% CI 2.25-42.73)). Of the 43 pts with loss of CDKN2A/B, 21 (49%) progressed via RT (n=20) or prolymphocytic leukemia (PLL) (n=1). The Cumulative Incidence Rate of RT development 12-months post CDKN2A/B deletion was 23.5% (95% CI 10.9-38.9), at 24-months, it was 29.4% (15.1-45.3). Deletion of TP53 was seen in 85.7%. Albeit there was limited pts with detection of CDKN2A/B deletion prior to RT, the multivariable model for RT found loss of TP53 (p=0.02, HR 4.53 (95% CI 1.33-15.43)) and homozygous and heterozygous loss of CDKN2A/B (p=0.0008, HR 6.08 (95% CI 2.13-17.39)) were independently significant. Conclusion: Here we show that deletion of CDKN2A/B in CLL is a very rare occurrence and was seen predominantly in pts with highly aggressive disease characteristics. Close to half of our cohort progressed with RT or PLL. Additional investigation of serial samples is needed to establish when CDKN2A/B deletion occurs relative to RT. Our results indicate that prospective FISH analysis for CDKN2A/B deletion, in conjunction with other high-risk genetic alterations, to establish RT prognostication in high-risk CLL is warranted.

Labial Mucosa Stem Cells: Isolation, Characterization, and Their Potential for Corneal Epithelial Reconstruction.

PurposeThe purpose of this study was to characterize labial mucosa stem cells (LMSCs) and to investigate their potential for corneal epithelial reconstruction in a rabbit model of total limbal stem cell deficiency (LSCD).MethodsRabbit LMSCs (rLMSCs) and human (hLMSCs) LMSCs were derived from labial mucosa and characterized in terms of their proliferation activity by the evaluation of proliferation index (PI) and colony forming efficiency (CFE), cell senescence, and differentiation abilities. The expression of various limbus-specific, stem cell-specific, and epithelial markers was assessed via immunocytochemistry. Flow cytometry was used to evaluate mesenchymal and hematopoietic cell surface markers expression. Chromosomal stability of the derived cells was examined using the conventional GTG-banding technique. To assess the impact of LMSCs on corneal epithelial reconstruction, rLMSCs were seeded onto a decellularized human amniotic membrane (dHAM), thereafter their regeneration potential was examined in the rabbit model of total LSCD.ResultsBoth rLMSCs and hLMSCs showed high proliferation and differentiation abilities, entered senescence at later passages, and expressed different stem cell-specific (ABCB5, ALDH3A1, ABCG2, and p63α), mesenchymal (vimentin), and epithelial (CK3/12, CK15) markers. Cell surface antigen expression was similar to other described mesenchymal stem cells. No clonal structural chromosome abnormalities (CSCAs) and the low percentage of non-clonal structural chromosome abnormalities (NSCAs) were observed. Transplantation of rLMSCs promoted corneal epithelial reconstruction and enhanced corneal transparency.ConclusionsLMSCs have significant proliferation and differentiation abilities, display no detrimental chromosome aberrations, and demonstrate considerable potential for corneal repair.

POLQ suppresses genome instability and alterations inDNA repeat tract lengths.

DNA polymerase theta (POLQ) is a principal component of the alternative non-homologous end-joining (ANHEJ) DNA repair pathway that ligates DNA double-strand breaks. Utilizing independent models of POLQ insufficiency during telomere-driven crisis, we found that POLQ–/– cells are resistant to crisis-induced growth deceleration despite sustaining inter-chromosomal telomere fusion frequencies equivalent to wild-type (WT) cells. We recorded longer telomeres in POLQ–/– than WT cells pre- and post-crisis, notwithstanding elevated total telomere erosion and fusion rates. POLQ–/– cells emerging from crisis exhibited reduced incidence of clonal gross chromosomal abnormalities in accordance with increased genetic heterogeneity. High-throughput sequencing of telomere fusion amplicons from POLQ-deficient cells revealed significantly raised frequencies of inter-chromosomal fusions with correspondingly depreciated intra-chromosomal recombinations. Long-range interactions culminating in telomere fusions with centromere alpha-satellite repeats, as well as expansions in HSAT2 and HSAT3 satellite and contractions in ribosomal DNA repeats, were detected in POLQ–/– cells. In conjunction with the expanded telomere lengths of POLQ–/– cells, these results indicate a hitherto unrealized capacity of POLQ for regulation of repeat arrays within the genome. Our findings uncover novel considerations for the efficacy of POLQ inhibitors in clinical cancer interventions, where potential genome destabilizing consequences could drive clonal evolution and resistant disease.

PB1914: CYTOGENETIC FINDINGS IN 18 CHILDREN WITH MYELODYSPLASTIC SYNDROMES BY KARYOTYPIC AND FISH ANALYSIS

Background: Pediatric myelodysplastic syndromes (MDS) are rare disorders characterized by hematopoietic cell dysfunction, an increased risk of AML and a poor prognosis. Although it is known that cytogenetic abnormalities are seen in approximately half of children diagnosed with MDS, their detailed cytogenetic profile has not been elucidated yet due to the rarity of the disease. Aims: We tried to detect chromosomal abnormalities in pediatric patients with MDS, including submicroscopic aberrations such as gene alterations associated with the disease and possible malignant transformation by karyotypic and molecular cytogenetic analysis (fluorescence in situ hybridization, FISH) to contribute to the elucidation of the cytogenetic profile of pediatric MDS and the investigation of the pathogenesis of the disease. Methods: In this study 18 children with MDS are included. Karyotypic analysis was performed on 24h, 48h and 72h bone marrow (BM) cultures using GTG-banding. Karyotypic analysis was carried out on 25 at least metaphases. FISH analysis was performed on cultured BM cells using IVD probes for centromere (CEP) of chromosomes 7 and 8 and the chromosomal regions 7q22, 7q31, 8q24(MYC), 11q22.3(ATM), 11q23(MLL), and 17p13(TP53). For each sample at least 200 interphase cells (iFISH) and 5 metaphases (mFISH) were analyzed to increase sensitivity. Normal cut-off was estimated at 3%. Results: The sex ratio was 1,25 (10 M/8 F) and the mean age was 9,42 years (range 0,4y - 19y). Eleven patients had RCC, 5 MDS-EB and 2 t-MDS. Normal karyotypes were detected in 10 patients (55,6%), 6 patients with RCC, 3 with MDS-EB and 1 with t-MDS. Abnormal karyotypes were found in 8 patients (44,4%), 4 with RCC, 2 with MDS-EB and 1 with t-MDS. Monosomy 7 was observed in 2/18 patients (>70% of metaphases), +8 in 2/18 patients (5-6.6% of metaphases), inv(17)(p11.2q21) in 1/18 patients (18.75% of metaphases), complex karyotypes in 2/18 patients (31.3-73.2% of metaphases including abnormalities of 1q, 2q, 4q, 5q, 16q, 18 and Xp), in one patient after SCT in the transformation of MDS into s-AML and in the other at diagnosis with t-MDS) and chromatid and chromosome breaks in 1/18 patients, (8,6-9,6% of metaphases) in 2 different BM samples taken with an interval of 3 months, which were also found in his brother’s BM karyotype. FISH revealed -7 in 2/18 patients confirming their karyotypes, loss of one p53 allele in 3/18 patients at low frequencies (6,5%, 4,5%, 3,5%) all with hypocellular RCC and transfusion-dependency that underwent SCT, +8 in 2/18 (in 8% and 2,7% of cells) confirming trisomy 8 in their karyotypes and loss of one MLL allele and MLL rearrangement in a patient that was transformed to s-AML in 4,4% of cells. A normal pattern of hybridization was observed for the above probes in the rest cases and for MYC and ATM genes in all the examined cases. Summary/Conclusion: Karyotypic aberrations were detected in 44,4% of children including -7, +8, inv(17), complex karyotypes and chromatid and chromosome breaks, revealing that other chromosome aberrations except -7/del(7q) and trisomy 8 are characterized this disease. Clonal chromosome abnormalities can be detected at low frequencies in the karyotypes of pediatric MDS patients so except from iFISH, mFISH is also needed especially in cases with small clones or submicroscopic translocations. The low frequency of submicroscopic aberrations such as p53 deletions observed in 3 cases of hypocellular RCC must be confirmed in larger studies by molecular analysis as they may play a potential role in MDS pathogenic evolution.

A Multicenter, Prospective Study for Pediatric Chronic Myeloid Leukemia in Chronic Phase: The JPLSG CML-08 Study

Background: Tyrosine kinase inhibitor (TKI) has been used in pediatric chronic myeloid leukemia (CML) for more than 10 years, but only a few prospective clinical studies have been conducted in pediatric patients with CML due to their rarity. We conducted the JPLSG CML-08 study to determine the efficacy and tolerability of TKIs in children and adolescents with newly diagnosed CML in chronic phase (CML-CP).Methods: The JPLSG CML-08 study was a prospective multicenter observational study (UMIN000002581). Patients under 18 years of age with untreated BCR-ABL1-positive CML-CP were eligible and treated according to the modified ELN-2009 recommendation, and the efficacy and safety of TKIs were evaluated.Results: From October 2009 until September 2014, 79 patients were enrolled in 46 hospitals in Japan. A total of 78 patients (49 males and 29 females) were eligible for inclusion. Median age at diagnosis was 11 years (range, 1-17). Median observational period for survivors was 82 months (range, 48-118). Median WBC, Hb and platelet counts were 275x10 9/L (range, 8-765), 9.6g/dL (range, 5.8-14.6) and 560x10 9/L (range, 110-2875), respectively. Splenomegaly was found in 76%. High risk of Sokal, Hasford, EUTOS, and ELTS scores were observed in 21, 13, 27, and 9%, respectively. Clonal chromosome abnormalities in Ph-positive cells occurred in 1 patient at diagnosis. Imatinib, dasatinib, and, nilotinib were used as a first-line treatment in 69 (88%), 7 (9%), and 2 (3%) patients, respectively. The median initial dose of imatinib, dasatinib, and nilotinib was 276, 63, and 262mg/m2, respectively. 5y-PFS and OS was 96.2% (95%CI, 88.6 to 98.7%) and 97.4% (95%CI, 90.1 to 99.4%), respectively. Deaths were observed in 2 patients due to transplant complications. Hematopoietic cell transplantation was conducted in 14 patients (18%). Nine patients (12%) discontinued TKI with the aim of treatment-free remission (TFR), and five of them achieved TFR. In 69 patients with first-line imatinib, complete hematologic response was achieved in 95.7% at 3 months, complete cytogenetic response in 75.4% at 12 months, major molecular response (MMR) in 40.1% at 18 months, and MR4.0 in 52.8% at 60 months; If a transplant was performed, the follow-up period was censored at the date of transplant. Of the 69 patients, 52% changed treatment from imatinib to another TKI or transplant due to poor response, and 20% did due to intolerance. The most common cause of intolerance to imatinib was musculoskeletal events. BCR-ABL1 (IS) <10% at 3 months strongly correlated with higher achievement of MMR, MR4.0, and MR4.5. The EUTOS score was significantly associated with achievement of IS <10% at 3 months. Patients with a first-line second-generation TKI had a higher cumulative incidence of MR4.5 (P = 0.0191) than patients with a first-line imatinib. Second-generation TKI was used as first-line therapy only in patients older than 9 years, but other clinical characteristics, including risk scores, did not differ significantly between the two groups. The incidence of grade 3/4 adverse events (≥ 10%) included neutropenia (47%), anemia (39%), leukopenia (13%), arthralgia (13%), and myalgia (11%) for imatinib, neutropenia (21%), anemia (13%), and thrombocytopenia (11%) for dasatinib, and neutropenia (14%), elevated ALT (14%), hyperbilirubinemia (14%), skin rash (14%), and high CPK (14%) for nilotinib. Gastrointestinal bleeding was an adverse event specific to dasatinib (11% in all grades).Conclusion: This clinical study extends and confirms previous data showing that first-line treatment with imatinib is effective in children and adolescents, with response rates similar to those seen in adults. Although longer follow-up is needed to fully assess the long-term toxic effects, adverse events with imatinib, dasatinib, and nilotinib have been acceptable. As reported in adults, there was an advantage in selecting second-generation TKI over imatinib as first-line therapy to achieve deep molecular remission (DMR). Since discontinuation of TKI after achieving DMR is the preferred strategy, second-generation TKI is expected to become the standard therapy for children and adolescents. DisclosuresNo relevant conflicts of interest to declare.

Outcomes in Patients with Poor-Risk Cytogenetics with or without TP53 Mutations Treated with Venetoclax Combined with Hypomethylating Agents

Outcomes in Patients with Poor-Risk Cytogenetics with or without TP53 Mutations Treated with Venetoclax Combined with Hypomethylating Agents

Cytogenetic findings of ectopic endometriotic tissue in women with endometriosis and review of the literature

ObjectiveTo determine chromosome and gene alterations in ectopic endometrial (EM) tissue which may be implicated in the clinical course or the progression of endometriosis and to review the literature concerning the cytogenetic findings of women with endometriosis. Study design15 women who underwent laparoscopic endometriosis surgery at the Athens Genesis Clinic were enrolled in the study. Ectopic endometrial tissue was surgically removed and further analyzed by conventional and molecular cytogenetic techniques. Fluoresent in situ hibridization (FISH) with probes for p53, ATM, MYC, MLL1 and IGH genes, the centromeres of chromosomes 7 and 8 and 7q22/7q31 chromosomal regions was carried out. ResultsKaryotypic analysis revealed no clonal chromosomal abnormalities. However, an increased frequency of polyploidy (55.6%) and sporadic chromosomal abnormalities (40.0%) concerning chromosomes 9, 11, 17 and X were noticed involving mainly deletions, trisomies or monosomies. FISH analysis showed IGH gene rearrangements in 54% of the EM cases and MLL gene rearrangements in 73% of the examined samples. Normal hybridization patterns were observed for p53, ATM and MYC. The increased frequency of polyploidy shown by conventional karyotyping was also confirmed by FISH. ConclusionPolyploidy, sporadic chromosomal abnormalities, as well as IGH and MLL gene rearrangements, may provoke genetic instability and play a potential role in the development of endometriosis. IGH and MLL gene rearrangements indicate a genetic relation between endometriosis and carcinogenesis. Confirmation of the above gene rearrangements in a large series of women may allow the determination of their possible involvement in the pathogenesis of this complex disease and their possible contribution in the early identification of women in danger for malignant transformation.

Tetraploidy in Acute Myeloid Leukemia Is Associated with Poor Outcomes Regardless of the Presence of Favorable Risk Features, and Requires an Allogeneic Stem Cell Transplant in First Remission

Introduction: While tetraploidy (92 chromosomes) and near-tetraploidy (>80 chromosomes) confer favorable prognosis in B-cell lineage pediatric acute lymphoblastic leukemia, their prognostic significance in acute myelogenous leukemia (AML) remains ill-defined. Moreover, the mechanisms underlying their pathobiology and impact on known prognostic factors in AML are unknown. Combining data collected at our institution along with a review of the literature, we provide the most complete analysis of tetraploidy and near-tetraploidy in AML including data on biology, treatment outcomes, impact on other prognostic markers, and survival to assist in clinical management.Patients and methods: A systematic literature search was performed and included studies published from June 1st, 1994 to June 1st, 2017 in PubMed, Embase, and Web of Science. We used the key words tetraploidy, near-tetraploidy, acute myeloid leukemia, acute myelogenous leukemia, and AML. Data was tabulated regarding the following seven variables: Fluorescence in situ hybridization (FISH) analysis, molecular analysis, karyotype, risk status, overall survival (OS), history of allogeneic stem cell transplantation, and chemotherapy. Data was reported as percent and total number of cases with available data of each variable.Results and discussion: We identified 104 AML cases with confirmed tetraploidy or near-tetraploidy (102 cases in 24 articles, and 2 cases from our institution). Incidence was nearly equal between tetraploidy (55/104) and near-tetraploidy (49/104). The median age at diagnosis was 54. A notable male predominance was observed with a male-to-female ratio of 2.2:1 (71 males and 33 females). While insufficient molecular data were available to include in risk stratification, cytogenetics including FISH and karyotype were available in all cases. We stratified our cases irrespective of tetraploidy or near-tetraploidy into favorable risk (defined by the presence of the following genetic alterations in core-binding factors: inv(16) or t(16;16) or t(8;21), or t(15;17)), unfavorable risk (defined by complex karyotype [≥3 clonal chromosomal abnormalities], monosomal karyotype, 5q-, 7q-, -5,-7, t(6;9), inv(3), t(3;3), 11q23 - non t(9;11), t(9;22)) and intermediate risk (the rest). Consistent with the literature, cases with other cytogenetic abnormalities in addition to those defined in the favorable-risk group, were included within the favorable-risk category (Byrd Blood 2002). Interestingly, t(8;21) was the only favorable risk feature represented, seen in 13 patients (12.5%), thus grouped under favorable-risk AML. 51 patients (49.0%) were grouped under unfavorable-risk AML and included 24 patients with complex karyotype, 9 patients with -7, 7 patients with 5q-, 4 patients with -5, 1 patient with 7q-, 1 patient with t(9;22), 3 patients with both -5 and -7, 1 patient with both 5q- and -7, and 1 patient with both -5 and 7q-. Intermediate risk was observed in 40 patients (38.5%) who were found to have normal or non-defined cytogenetics. We used the Kaplan-Meier method to stratify survival data, and the log-rank test to compare the survival between subgroups. The overall outcomes were poor, the median OS (mOS) of the whole cohort was 6 months (M) (Figure 1A). The prognosis was equally poor for both tetraploidy (mOS 6.2M) and near-tetraploidy (mOS 6M) (P=0.81; Figure 1B). Surprisingly, risk stratification showed no impact on overall survival (P=0.57; Figure 1C), which suggest that the unfavorable risk imposed by tetraploidy or near-tetraploidy may overcome favorable-risk features, such as t(8;21), challenging current data on AML risk stratification. Twenty-one patients (20.2%) had an allogeneic stem cell transplantation. Interestingly, we noticed a significant improvement in outcomes (mOS 17M) in patients who received an allogeneic stem cell transplantation compared to those who did not (mOS 4M) (P=0.0085; Figure 1D).Conclusion: Regardless of the risk status, the prognosis of tetraploid or near-tetraploid AML is dismal and should be incorporated within the unfavorable risk group. Prompt evaluation for allogeneic stem cell transplant should be initiated at diagnosis. Indeed, longer survival can be achieved with chemotherapy followed by allogeneic stem cell transplantation at first complete remission. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Clinical and Molecular Characterization of Clonal Chromosomal Abnormalities Appearing in Philadelphia-Negative Metaphases of Chronic Phase CML

▪BackgroundClonal chromosomal abnormalities in Philadelphia chromosome-negative metaphases (CCA/Ph-) emerge as patients (pts) with chronic phase chronic myeloid leukemia (CP-CML) are treated with tyrosine kinase inhibitors (TKI). The clinical significance of CCA/Ph- remains unclear specifically among pts treated with second generation TKI. Very little is known about the molecular abnormalities associated with them.MethodsWe conducted a retrospective analysis of pts enrolled on prospective, single-institution frontline trials for CML-CP. Of the 598 pts enrolled, 279 pts (46%) were treated with imatinib, 126 (21%) with nilotinib, 143 (23%) with dasatinib and 50 (8%) with ponatinib. CCA/Ph- were defined as abnormalities present in ≥2/20 Philadelphia-negative (Ph-) metaphases or if present in one metaphase in ≥2 cytogenetic assessments. The clinical outcomes of pts with CCA/Ph- were compared to a control group without additional chromosomal abnormalities (ACAs). We performed targeted deep sequencing (median depth 490x) on a pilot study from 10 pts with various CCA/Ph- (panel of 295 genes recurrently mutated in hematological malignancies). Single nucleotide variants (SNV) and small insertions and deletions (indels) were called against virtual normal sequence developed in-house. An automatic pipeline was used to filter and annotate raw variants, and then select non-polymorphism, protein-coding changing mutation, thus identifying high confidence driver mutations.ResultsCCA/Ph- occurred in 58 pts (10%) and the most common were - Y in 25 (43%) and trisomy 8 in 7 pts (12%). Most CCA/Ph- appeared within the first year of the start of the TKI (median 6 months, range 3-78 months). CCA/Ph- were transient and were detected in 2 or less time points in 36/58 cases (62%). The median number of Ph-negative metaphases containing CCA/Ph- per analysis was 5/20 (range 1-20). We further categorized CCA/Ph- into those where - Y was the only clonal abnormality, and all others. Pts who developed CCA/Ph- were significantly older than those without ACAs (median age 59 vs 47; P<0.001). Specifically, pts with -Y CCA/Ph- were the oldest (median age 64; P<0.001), followed by those with non-Y CCA/Ph- (median age 52; P=0.003) when compared to those without ACAs (median age 47). Four pts with ACAs in Ph+ metaphases who subsequently developed CCA/Ph- were excluded from response and survival analyses. Response to TKI therapy was similar for pts with CCA/Ph- and those without ACAs. With a median follow-up for survivors of 7.6 years (range 0.2-14.7 years), we found that pts with non -Y CCA/Ph- had worse failure-free survival (FFS), event-free survival (EFS), transformation-free survival (TFS) and overall survival (OS) compared to those without ACAs with the following 5-year rates: FFS (52% vs 70%, P = 0.02), EFS (68% vs 86%, P = 0.02), TFS (76% vs 94%, P < 0.01) and OS (79% vs 94%, P = 0.03). In a multivariate analysis, non -Y CCA/Ph- increased the risk of transformation or death when baseline characteristics were considered with a hazard ratio (HR) of 2.81 (95% CI 1.15-6.89, P = 0.02). However, this prognostic impact was not statistically significant when achieving BCR-ABL < 10% at 3 months was included in the analysis (Table 1).A total of 46 high-confidence driver mutations in 18 genes in 10/10 (100%) pts with CCA/Ph- were identified (Figure 1). Median number of driver mutations was 2.5 (range 1-9). ASXL1 frameshift mutation was detected in all examined pts (10/10 pts) mostly at low variant allelic frequency (VAF) with a median VAF of 1.6% (range 0.7% - 4.2%). Mutations were also commonly detected in PTPN11 (7/10 pts). Mutations in PTPN11 have been reported in about 4% of pts with acute myeloid leukemia (Papaemmanuil et al. NEJM 2016). Mutations in DNMT3A (R635Q) and ABL (Y253H, associated with resistance to imatinib) were observed in the same case in addition to ASXL1. In this cohort, the observed mutation rate is higher than what is expected in CP-CML when comparing to published literature (Kim et al. Blood 2017). Sequencing of a control cohort from pts with deep molecular response without ACAs is ongoing.ConclusionIn conclusion, non -Y CCA/Ph- are associated with decreased survival when emerging in pts with chronic phase CML across various TKIs. Preliminary results from deep sequencing indicate a higher than expected rate of mutations in CP-CML pts with CCA/Ph- compared to those without ACAs, mostly detected at low VAF. [Display omitted] DisclosuresKantarjian:Pfizer: Research Funding; Bristol-Meyers Squibb: Research Funding; ARIAD: Research Funding; Novartis: Research Funding; Amgen: Research Funding; Delta-Fly Pharma: Research Funding. Takahashi:Symbio Pharmaceuticals: Consultancy. Jabbour:Bristol-Myers Squibb: Consultancy. Cortes:ARIAD: Consultancy, Research Funding; Sun Pharma: Research Funding; BMS: Consultancy, Research Funding; Novartis Pharmaceuticals Corporation: Consultancy, Research Funding; Teva: Research Funding; ImmunoGen: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding.

High Frequency of P53 Mutations Detected by a Microarray Resequencing Assay, the AmpliChip P53 Test, in AML with a Complex Aberrant Karyotype.

Acute myeloid leukemia (AML) is heterogeneous in morphology, cytogenetics, and prognosis. On the basis of cytogenetics it can be subdivided into three groups with favorable, intermediate, or unfavorable prognosis. Accounting for 10%–20% of all AML cases, AML with complex aberrant karyotypes is more frequent at higher ages and is the subgroup with the worst prognosis. AML with complex aberrant karyotype is generally defined by the presence of three or more clonal chromosome abnormalities. Genetically, it has a characteristic pattern of chromosomal gains and losses, resulting also in a unique gene expression pattern including upregulation of genes involved in DNA repair. One of the important characteristics of AML with complex aberrant karyotype is the high incidence of deletions and/or mutations in the tumor suppressor p53 gene (60%–80%) which occur very unfrequently in other AML subgroups. Here we present data on 119 AML cases with complex aberrant karyotypes that were analyzed with the new AmpliChip p53 Test, a microarray-based resequencing test that is designed to detect single nucleotide changes in the entire coding region of the p53 gene. The AmpliChip p53 Test queries for the presence of sequence alterations through comparative analysis of the microarray hybridization pattern of a series of probes from sample DNA to wild type (wt) reference DNA. Starting from genomic DNA, the 10-hour assay includes PCR amplification, fragmentation and labeling, hybridization to the microarray, washing and staining, and mutation data analysis. By use of this technique we have detected p53 mutations located in exons 4–10 in 89/119 (74.8%) samples. These mutations included missense mutations, frameshift mutations, splice site mutations, silent mutations, and nonsense mutations. All 119 samples had also been analyzed in parallel for p53 mutations by a combination of several alternative methods including the predecessor Affymetrix p53 GeneChip microarray, denaturing high performance liquid chromatography (DHPLC), as well as conventional sequencing. The specificity and sensitivity of the AmpliChip p53 Test were compared to the results obtained with these alternative assays. Discordant results were observed in 27 samples when comparing the AmpliChip p53 Test to the alternative assays. Among these 27 samples the AmpliChip p53 Test detected mutations in Exon 10 in 2 samples which had not been tested by DHPLC and further exclusively detected the R72P polymorphism in 9 samples. The remaining group of discordant samples contained:5 samples with a I03 dup 16bp +40 (acctggagggctgggg);3 samples containing a > 2 bp deletion;2 samples with insertion mutations; andone with a mutation in intron 3.Due to technical reasons, all of these aberrations noted above are out of the specifications of the AmpliChip p53 Test. In 5 samples the AmpliChip p53 Test detected additional mutations that were not identified by conventional methods. In conclusion, the AmpliChip p53 Test is a robust and rapid diagnostic tool that allows to identify a poor prognostic subset of AML. Unraveling the biology of this disease may provide new pathophysiologic insights applicable to therapeutic intervention, especially for the targeted therapy of AML with complex aberrant karyotype.

9. Enhanced detection of chromosomal abnormalities in Hairy Cell Leukemia by conventional cytogenetics using oligonucleotide stimulation

Hairy cell leukemia (HCL) is a mature B-cell malignancy that accounts for 2% of all adult leukemia. Few cytogenetic studies of HCL have been reported due to the rarity of the disease and the difficulties stimulating hairy-cells to divide in culture. Using a mitogen to stimulate leukemic B-cells to divide in culture can enhance detection of clonal chromosomal abnormalities. CpG oligodeoxynucleotides (CpG ODN) are a robust method for stimulating cell division in another mature B-cell malignancy, chronic lymphocytic leukemia.

Mixed phenotype acute leukemia with PML-RAR\u03b1 positive: a case report and literature review

Mixed phenotype acute leukemia (MPAL) is an uncommon type of leukemia. It is one kind of malignant clonal diseases that expresses more than one genealogical specific antigen simultaneously. Most MPAL patients are associated with clonal chromosomal abnormalities and molecular genetic changes, such as t(9;22) (q34;q11) and KMT2A (MLL) rearrangement. These specific abnormalities usually have important guiding significance in MPAL diagnosis, targeted therapy and prognosis judgment. In this paper, we reported a case of MPAL, T/myeloid (M5) with an unfrequent combination of PML-RARα positivity and t(15;17). The treatment was successful with chemotherapy for both AML and ALL with daunorubicin, cytarabine (DA) and vincristine, prednisone (VP). We reported here this suggestive MPAL case of rare disease condition and effective treatment, in order to provide experience for the early diagnosis and treatment of similar patients.

Clinical characteristics, treatment pattern, and outcomes in newly diagnosed patients with chronic myeloid leukemia in the chronic phase by age

目的探讨初发慢性髓性白血病慢性期（CML-CP）不同年龄患者的临床特征、治疗和结局。方法回顾性分析2006年1月至2019年12月在北京大学人民医院确诊的≥14岁初诊酪氨酸激酶抑制剂（TKI）一线治疗的CML-CP连续病例。结果共收集957例患者，男性597例（62.4％），中位年龄40（14～83）岁。按年龄分为<40岁组（470例，49.1％）、40～59岁组（371例，38.8％）和≥60岁组（116例，12.1％）。随年龄增长，初诊时脾大（P<0.001）、WBC≥100×109/L（P<0.001）、贫血（P<0.001）、PLT<450×109/L（P＝0.022）、外周血原始细胞比例高（P＝0.010）和具有Ph染色体附加异常（P＝0.006）的患者比例降低，有合并症（P<0.001）、Sokal积分中/高危（P<0.001）和初始选择伊马替尼（P<0.001）的患者比例增高。而性别、ELTS危险度分布在各年龄组间差异无统计学意义（P值均>0.05）。多因素分析显示，≥60岁仅是影响患者总生存（OS）的不利因素（OR＝3.7, 95％ CI 1.5～9.2, P＝0.005），年龄与治疗反应和其他结局无显著相关性。TKI治疗中，随年龄增长，非血液学不良反应发生率显著增加（P<0.001），而血液学不良反应发生率相似。随访末期，随年龄增长，仍服用伊马替尼（P＝0.026）和服用减量TKI（P<0.001）的患者比例显著增加。结论初发CML-CP不同年龄组患者的临床特征、TKI用药选择和剂量、治疗反应、OS期和非血液学不良反应发生率存在差异。

Case Report of acute myeloid leukemia with “WT1, ATRX, CEBPA, CSMD1, IKZF1, and LRP1B mutation and translocation between chromosome 1 and 19” developing from Philadelphia-negative chronic myeloid leukemia after TKI therapy

Rationale:The success of tyrosine kinase inhibitor (TKI) therapy has greatly prolonged the survival time of patients with chronic myeloid leukemia (CML), harboring the characteristic Philadelphia (Ph) chromosome. However, a fraction of patients, achieving complete cytogenetic response after TKI therapy, develop a myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML) with additional clonal chromosomal abnormalities in Philadelphia-negative cells (CCA/Ph–).Patient concerns:A 56-year-old woman with AML, developing from Philadelphia-negative CML after TKI therapy. She showed 6 kinds of somatic variants—CEBPA, ATRX, WT1, CSMD1, IKZF1, and LRP1B mutation after diagnosed as AML.Diagnosis:The patient was diagnosed with chronic phase CML that developed to AML after achieving durable complete cytogenetic response (CCR) and major molecular response (MMR).Interventions:The patient was treated with TKI therapy at the period of CML. When diagnosed with AML, she received induction chemotherapy regimens, consolidation therapy, and allogeneic hematopoietic stem cell transplantation subsequently.Outcomes:The patient has been CCR and MMR for nearly 4 years, and has achieved complete remission after intervention related to AML. She is now preparing for allogeneic hematopoietic stem cell transplantation.Lessons:These rare occurrences highlight the importance of exploring the relevant pathogenesis of AML developing from CML after TKI therapy. In addition to monitoring molecular changes in the course of CML, cytogenetic analysis, or next-generation sequencing of CML patients should be performed.

Second-Generation Tyrosine Kinase Inhibitors for Chronic Myeloid Leukemia with Additional Chromosomal Abnormalities

Second-Generation Tyrosine Kinase Inhibitors for Chronic Myeloid Leukemia with Additional Chromosomal Abnormalities

The influence of additive clonal chromosome abnormalities in Ph negative cells on the efficacy of chronic myeloid leukemia

Objective: To investigate the influence of additional clonal chromosome abnormalities in Ph negative cells (CCA/Ph(-)) on the efficacy of chronic myeloid leukemia (CML) after tyrosine kinase inhibitors (TKI) treatment. Methods: The clinical data of 28 CML patients with CCA/Ph(-) treated in Henan Cancer Hospital from July 2014 to December 2017 were analyzed retrospectively. The univariate analysis was carried out by Kaplan-Meier method. Multivariate analysis was done by Cox proportional risk model. Results: A total of 28 CCA/Ph(-)patients were recruited including 17 males and 11 females with median age of 42.5 years old. The most common CCA/Ph(-)were trisomy 8 (60.7%), monosomy 7 (14.3%). 64.3% CCA/Ph(-)were transient and 35.7% recurrent (more than 2 times). Cytopenia in two or three lineages of peripheral blood was seen in 42.9% patients. As to the efficacy, 89.3% patients achieved major cytogenetic response (MCyR), 25% with major molecular response (MMR). The median follow-up time was 26.5 months. Treatment failure (TF) of TKI occurred in 32.1% patients with median duration of response 8 (1-41) months. Univariate analysis showed that TF rate was significantly correlated with the frequency of CCA/Ph(-)and cytopenia (all P<0.05). The MMR rate was also significantly correlated with cytopenia (P<0.05). Cytopenia of two lineages or pancytopenia was an independent risk factor related to MMR rate (RR=3.868, 95%CI 1.216-12.298, P=0.022) . Conclusions: Cytopenia in CCA/Ph(-)appears to be an independent risk factor of MMR in CML patients with TKI treatment. The recurrent CCA/Ph(-)may link to higher treatment failure rate. Drug withdrawal or alternative strategy should be considered according to response and the ABL kinase mutations.