Recurrent Cytogenetic Abnormalities Research Articles

Cytogenetic and Molecular Characteristics in Adult Hispanic Acute Myeloid Leukemia Patients From Puerto Rico.

The cytogenetic and molecular heterogeneity of acute myeloid leukemia (AML) is characterized as a contributing factor in the disparity of treatment outcomes and clinical outcomes seen among ethnic and racial groups. In this study, we have retrospectively evaluated the karyotypes of 800 adult Hispanic AML patients from Puerto Rico (PR). Acute promyelocytic leukemia with PML-RARA is the most common recurrent cytogenetic abnormality, compatible with previously published results. Among these AML patients, 163 patients had 21 gene panels performed. Twenty-six (15.95%) patients showed no detectable mutations, and 137 patients (84.05%) showed at least one mutation. Compared with previously published data from other examined Hispanic AML populations in the United States, mutational frequencies of these 21 genes, except for ASXL1, WT1, and KRAS,show no significant difference. This is the largest study to date about the landscape of cytogenetic and molecular abnormalities in Hispanic AML patients and a first report regarding the frequencies of these abnormalities in Puerto Rican Hispanic AML patients.

Clinicopathologic features and outcomes of acute leukemia harboring PICALM::MLLT10 fusion

The PICALM::MLLT10 fusion is a rare but recurrent cytogenetic abnormality in acute leukemia, with limited clinicopathologic and outcome data available. Herein, we analyzed 156 acute leukemia patients with PICALM::MLLT10 fusion, including 12 patients from our institutions and 144 patients from the literature. The PICALM::MLLT10 fusion preferentially manifested in pediatric and young adult patients, with a median age of 24 years. T-lymphoblastic leukemia/lymphoma (T-ALL) constituted 65% of cases, acute myeloid leukemia (AML) 27%, and acute leukemia of ambiguous lineage (ALAL) 8%. About half of T-ALL were classified as an early T-precursor (ETP)-ALL. In our institutions’ cohort, mediastinum was the most common extramedullary site of involvement. Eight of 12 patients were diagnosed with T-ALL exhibiting a pro-/pre-T stage phenotype (CD4/CD8-double negative, CD7-positive), and frequent CD79a expression. NGS revealed pathogenic mutations in 5 of 6 tested cases, including NOTCH1, and genes in RAS and JAK-STAT pathways and epigenetic modifiers. Of 138 cases with follow-up, pediatric patients (<18 years) had 5-year overall survival (OS) of 71%, significantly better than adults at 33%. The 5-year OS for AML patients was 25%, notably shorter than T-ALL patients at 54%; this distinction was observed in both pediatric and adult populations. Furthermore, adult but not pediatric ETP-ALL patients demonstrated inferior survival compared to non-ETP-ALL patients. Neither karyotype complexity nor transplant status had a discernible impact on OS. In conclusion, PICALM::MLLT10 fusion is most commonly seen in T-ALL patients, particularly those with an ETP phenotype. AML and adult ETP-ALL patients had adverse prognosis. PICALM::MLTT10 fusion testing should be considered in T-ALL, AML, and ALAL patients.

The numerous facets of 1q21+ in multiple myeloma: Pathogenesis, clinicopathological features, prognosis and clinical progress (Review).

Multiple myeloma (MM) is a malignant neoplasm characterized by the clonal proliferation of abnormal plasma cells (PCs) in the bone marrow and recurrent cytogenetic abnormalities. The incidence of MM worldwide is on the rise. 1q21+ has been found in ~30-40% of newly diagnosed MM (NDMM) patients.1q21+ is associated with the pathophysiological mechanisms of disease progression and drug resistance in MM. In the present review, the pathogenesis and clinicopathological features of MM patients with 1q21+ were studied, the key data of 1q21+ on the prognosis of MM patients were summarized, and the clinical treatment significance of MM patients with 1q21+ was clarified, in order to provide reference for clinicians to develop treatment strategies targeting 1q21+.

Immunophenotypic portrait of leukemia-associated-phenotype markers in B acute lymphoblastic leukemia.

Multiparametric flow cytometry (MFC) is an essential diagnostic tool in B acute lymphoblastic leukemia (B ALL) to determine the B-lineage affiliation of the blast population and to define their complete immunophenotypic profile. Most MFC strategies used in routine laboratories include leukemia-associated phenotype (LAP) markers, whose expression profiles can be difficult to interpret. The aim of our study was to reach a better understanding of 7 LAP markers' landscape in B ALL: CD9, CD21, CD66c, CD58, CD81, CD123, and NG2. Using a 10-color MFC approach, we evaluated the level of expression of 7 LAP markers including CD9, CD21, CD66c, CD58, CD81, CD123, and NG2, at the surface of normal peripheral blood leukocytes (n = 10 healthy donors), of normal precursor B regenerative cells (n = 40 uninvolved bone marrow samples) and of lymphoblasts (n = 100 peripheral blood samples or bone marrow samples from B ALL patients at diagnosis). The expression profile of B lymphoblasts was analyzed according the presence or absence of recurrent cytogenetic aberrations. The prognostic value of the 7 LAP markers was examined using Maxstat R algorithm. In order to help the interpretation of the MFC data in routine laboratories, we first determined internal positive and negative populations among normal leukocytes for each of the seven evaluated LAP markers. Second, their profile of expression was evaluated in normal B cell differentiation in comparison with B lymphoblasts to establish a synopsis of their expression in normal hematogones. We then evaluated the frequency of expression of these LAP markers at the surface of B lymphoblasts at diagnosis of B ALL. CD9 was expressed in 60% of the cases, CD21 in only 3% of the cases, CD58 in 96% of the cases, CD66c in 45% of the cases, CD81 in 97% of the cases, CD123 in 72% of the cases, and NG2 in only 2% of the cases. We confirmed the interest of the CD81/CD58 MFI expression ratio as a way to discriminate hematogones from lymphoblasts. We observed a significant lower expression of CD9 and of CD81 at the surface of B lymphoblasts with a t(9;22)(BCR-ABL) in comparison with B lymphoblasts without any recurrent cytogenetic alteration (p = 0.0317 and p = 0.0011, respectively) and with B lymphoblasts harboring other cytogenetic recurrent abnormalities (p = 0.0032 and p < 0.0001, respectively). B lymphoblasts with t(1;19) at diagnosis significantly overexpressed CD81 when compared with B lymphoblasts with other recurrent cytogenetic abnormalities or without any recurrent alteration (p = 0.0001). An overexpression of CD58 was also observed in the cases harboring this abnormal cytogenetic event, when compared with B lymphoblasts with other recurrent cytogenetic abnormalities (p = 0.030), or without any recurrent alteration (p = 0.0002). In addition, a high expression of CD123, of CD58 and of CD81 was associated with a favorable prognosis in our cohort of pediatric and young adult B ALL patients. We finally built a risk score based on the expression of these 3 LAP markers, this scoring approach being able to split these patients into a high-risk group (17%) and a better outcome group (83%, p < 0.0001). The complexity of the phenotypic signature of lymphoblasts at diagnosis of B ALL is illustrated by the variability in the expression of LAP antigens. Knowledge of the expression levels of these markers in normal leukocytes and during normal B differentiation is crucial for an optimal interpretation of diagnostic cytometry results and serves as a basis for the biological follow-up of B ALL.

A Retrospective Review Assessing the Development of Myelodysplastic Syndromes in Patients with Plasma Cell Dyscrasias

Introduction Myelodysplastic syndrome (MDS) are hematopoietic disorders defined by dysplasia of the myeloid lineage accompanied by persistent cytopenias, and recurrent cytogenetic abnormalities (MDS). Whilst therapy related myeloid disorders in patients with Multiple Myeloma have been well described, there is lack of information regarding co-occurrence of these disease states in early plasma cell disorders as well as multiple myeloma at diagnosis which may also then allude to whether there is a founder effect to the clones. Recent advances in commercial sequencing assays of genomic data have allowed for identification of relevant gene mutations in patients with hematologic malignancies, such that the presence of these gene mutations have been incorporated into classification systems. In our study, we aimed to assess the occurrence of reported MDS in patients with plasma cell disorders i.e. Monoclonal Gammopathy of unknown significance (MGUS), Smoldering myeloma (SM) and Multiple myeloma (MM) as well as relevant gene mutations that would be implicated in the pathogenesis of MDS even in those patients who do not meet the full criteria for MDS (clonal hematopoiesis of indeterminate potential; CHIP mutations). Methods In this retrospective review, clinical and bone marrow based genomic data (Tempus xT ® 684 gene DNA-based NGS platform) was gathered on 57 patients with a diagnosis of MGUS, SM, and/or MM. Pathogenic gene mutations that are frequently associated with MDS were reported in our patient population. Bone marrow morphologic assessments for MDS related changes were also collected at the time of genomic data abstraction. Those patients who had defined MDS were further subclassified using the World Health Organization 2022 fifth edition classification system. Those observed as having subtle MDS findings on bone marrow biopsy, but with identified CHIP mutations, not meeting criteria for MDS, were also identified. Results Among 57 patients with MGUS (28), SM(9), and MM(20), 2 had met criteria for defined MDS at the time of their assessment. An additional 5 had bone marrow morphologic findings concerning for MDS, but did not meet criteria to be fully defined. A total of 14 myeloid disorder related pathogenic mutations were identified in our patient population (6 in MM at disease diagnosis, 7 in MGUS, and 1 in SM). Overall, the most frequently observed recurring mutations in the cohort were that of DNMT3A (57.1%, mean VAF 4.45%), TET2 (14.3%, mean VAF 4.0%), and SF3B1 (14.3%, mean VAF 39.2%). Patients with defined MDS had recurring pathogenic mutations in genes SF3B1, ASXL1 and DNMT3A. Discussion In our cohort, we identified several patients with features both diagnostic and suggestive of myeloid disorders (MDS) during work up for a plasma cell disorder, in particular MGUS, SM, and/or MM prior to therapeutic intervention. Molecular work up also identified gene mutation subsets in plasma cell disorder patients that have otherwise been implicated in the pathogenesis of myeloid disorders. The identification of both morphologic and molecular abnormalities in our cohort may inform therapeutic considerations as well as vigilance for the discovery or evolution of myeloid disorders during follow-up , in particular when unexpected cytopenias or new clinical concerns emerge. We expect to design future prospective studies, based on our observations, to further inform of the risk of myeloid disorders in our plasma cell disorder patient cohorts, as well as understand its impact on therapies such as immunomodulatory agents, alkylators and/or autologous stem cell transplant.

Characterization of MDS and CMML Patients Harboring Trisomy 8 Abnormality: Clinical, Autoimmune, and Mutational Features

Introduction Trisomy 8, seen in 5-7% of MDS and CMML patients, is regarded as an intermediate-risk cytogenetic abnormality in IPSS-R but not an MDS-defining cytogenetic abnormality. Recent studies have demonstrated an association between systemic autoimmune disease and trisomy 8, attributed to CD8 positive T-cell expansion and WT1 over-expression resulting in inflammation and suppression of hematopoiesis. Further, MDS and CMML with trisomy 8 respond favorably to immunosuppressive therapy. In this study, we investigated the clinical (including autoimmune manifestations), molecular and outcome characteristics of MDS/CMML with trisomy 8 in comparison to MDS with normal karyotype. Methods We retrieved all patients with trisomy 8 by conventional cytogenetic studies from our cytogenetic files between 2015 and 2023 who underwent bone marrow examination and met the WHO/ICC criteria for MDS/CMML diagnosis. In some cases, FISH testing was performed for confirmation. Majority of patients underwent amplicon-based targeted next-generation sequencing with a panel of 81 genes associated with myeloid malignancies (2500X coverage; LOD: 2% VAF). Clinicopathologic features were collected from medical records. For comparison, we retrieved a control cohort of all MDS patients diagnosed within the same period with normal karyotype (MDS-NK) at our institution. Results From a total of 362 patients with trisomy 8 (215 treatment naïve) detected at any time point during the disease course, we identified 165 MDS/CMML patients with trisomy 8 at baseline. These included 57 (35%) with isolated trisomy 8, 21 (13%) with 1 additional and 78 (53%) with ≥2 additional cytogenetic abnormalities (meeting the criteria for complex karyotype). Recurrent additional cytogenetic abnormalities included del(13)(q11q22) (n=3) and trisomy 21, del(5q), del(11q) involving KMT2A, and loss of Y, all seen in 2 patients each. We focused our subsequent analysis on 57 treatment naïve MDS/CMML patients with isolated trisomy 8 and compared them to 162 treatment naïve MDS/CMML patients with diploid karyotype (MDS-NK) at baseline. The median age was 74 years (range, 30-90) and 34 (60%) were men, with baseline characteristics shown in Table 1. There were no significant differences in age, gender distribution, median hemoglobin, ANC, MCV or platelet counts when comparing the two populations. Trisomy 8 MDS/CMML had a higher median baseline BM blast percentage [5% (1-18%) vs. 3% (0-17%); p=0.034] compared to MDS-NK. Distribution of IPSS-R subgroups in trisomy 8 MDS/CMML revealed a significantly lower representation of “Very Low” [0% vs. 13%; p=0.0027] and “High” [18% vs. 49%; p=0.0001], and higher representation of “Very High” [16% vs. 3%; p =0.0004] categories. NGS data was available in 36 (63%) of treatment naïve patients with isolated trisomy 8. The most frequent mutation was seen in ASXL1 (n=20, 56%), followed by TET2 (n=12, 33%), RUNX1 (n=12, 33%), SRSF2 (n=8, 25), KRAS/NRAS (n=8, 22%), and EZH2 (n=7, 19%). Compared to MDS-NK, the following mutations were significantly enriched in trisomy 8: ASXL1, EZH2, STAG2, RUNX1, KRAS/NRAS, IDH2 and PTPN11. Over a median follow-up time of 19 months, 28 (49%) with trisomy 8 and 19 (12%) MDS-NK patients died. MDS/CMML with trisomy 8 had a significantly lower median overall survival (OS) compared to MDS-NK [23.7% vs. not reached; p-value: &amp;lt;0.0001]. A total of 27 (7.6%) MDS/CMML patients with trisomy 8 (21 with trisomy 8 at baseline) were found to have a bona fide autoimmune disease, including autoimmune uveitis/scleritis (n=3), (rheumatoid and psoriatic), polyarteritis nodosa, giant cell arteritis with polymyalgia rheumatica, autoimmune sclerosing pancreatitis, immune hemolytic anemia, synovitis, and Grave's disease. Conclusions MDS/CMML with trisomy 8 showed unique clinicopathologic characteristics compared to MDS-NK: higher baseline BM blasts, higher representation of “Very High” IPSS-R risk categories with characteristic mutational signatures and a variety of underlying autoimmune diseases.

Intra-nucleus mosaic pattern (InMop) and whole-cell Haralick combined-descriptor for identifying and characterizing acute leukemia blasts on single cell peripheral blood images.

Acute leukemia is usually diagnosed when a test of peripheral blood shows at least 20% of abnormal immature cells (blasts), a figure even lower in case of recurrent cytogenetic abnormalities. Blast identification is crucial for white blood cell (WBC) counting, which depends on both identifying the cell type and characterizing the cellular morphology, processes susceptible of inter- and intraobserver variability. The present work introduces an image combined-descriptor to detect blasts and determine their probable lineage. This strategy uses an intra-nucleus mosaic pattern (InMop) descriptor that captures subtle nuclei differences within WBCs, and Haralick's statistics which quantify the local structure of both nucleus and cytoplasm. The InMop captures WBC inner-nucleus structure by applying a multiscale Shearlet decomposition over a repetitive pattern (mosaic) of automatically-segmented nuclei. As a complement, Haralick's statistics characterize the local structure of the whole cell from an intensity co-occurrence matrix representation. Both InMoP and Haralick-based descriptors are calculated using the b-channel from Lab color-space. The combined-descriptor is assessed by differentiating blasts from nonleukemic cells with support vector machine (SVM) classifiers and different transformation kernels, in two public and independent databases. The first database-D1 (n = 260) is composed of healthy and acute lymphoid leukemia (ALL) single cell images, and second database-D2 contains acute myeloid leukemia (AML) blasts (n = 3294) and nonblast (n = 15,071) cell images. In a first experiment, blasts versus nonblast differentiation is performed by training with a subset of D2 (n = 6588) and testing in D1 (n = 260), obtaining a training AUC of 0.991 ± 0.002 and AUC = 0.782 for the independent validation. A second experiment automatically differentiates AML blasts (260 images from D2) from ALL blasts (260 images from D1), with an AUC of 0.93. In a third experiment, state-of-the-art strategies, VGG16 and RESNEXT convolutional neural networks (CNN), separate blast from nonblast cells in both databases. The VGG16 showed an AUC of 0.673 and the RESNEXT of 0.75. Reported metrics for all the experiments are area under the ROC curve (AUC), accuracy and F1-score.

Childhood B-lymphoblastic leukemia: Integrating cytogenetics with other prognostic parameters and correlation with post-induction remission

BackgroundCytogenetics is irrefutably one of the most significant prognostic markers which have imparted ease and feasibility in the classification of acute leukemia. It is an essential prognostic tool in major treatment protocols for all hematological malignancies. This study aimed to note the frequency of different cytogenetic aberrations in children with B-ALL, their association with risk factors, and impact on post-induction remission status. MethodsA retrospective study was conducted in the Hematology department of The Indus Hospital, Karachi. All newly diagnosed patients of B-ALL (aged 1–17 years) from October 2, 2020 to October 13, 2021 were included. They were treated with modified Children's Oncology Group (COG) protocol. Cytogenetic studies including conventional karyotyping and fluorescence in situ hybridization (FISH) for BCR::ABL1, ETV6::RUNX1, and MLL gene rearrangement were performed on semi-automated cell imaging. ResultsAmong 238 patients, cytogenetic abnormalities were identified in 146 (61.3%) cases. A normal karyotype was observed in 92 (38.7%), high hyperdiploidy in 56 (24%), random structural abnormalities in 22 (9.2%), t (1:19) in 21 (8.8%), t (9; 22) in 12 (5.0%), complex karyotype in 10 (4.2%), 11q abnormalities in 5 (2.1%) and monosomy 7 in 1 (0.4%) case(s). FISH positivity for MLL, BCR::ABL1 and ETV6::RUNX1 was observed in 6 (2.5%), 18 (7.5%), and 20 (8.4%), cases, respectively. Post induction morphological remission was observed in 98.3% of cases. ConclusionThe overall frequency of cytogenetic abnormalities in the current cohort is comparable to previously reported studies. Our data also suggest the value of combining the karyotype and FISH techniques to increase the likelihood of detection of recurrent cytogenetic abnormalities. Even though there was a lack of correlation between genetic abnormalities and early outcome, a follow-up may be useful to ascertain the correlation with long-term survival.

Ganglioneuroblastoma intermixed: Clinicopathological implications of diagnosis at presentation and genomic correlations.

Ganglioneuroblastoma intermixed (GNBI) is classified as "favorable" histology by International Neuroblastoma Pathology Classification system. However, the International Neuroblastoma Risk Group (INRG) stratifies patients using wider clinicopathological and cytogenetic/molecular parameters. While the diagnosis of GNBI is typically made on resected tumor, it may sometimes be rendered on initial biopsy. We studied GNBI noted at diagnosis to evaluate its correlation with INRG staging and other clinicopathological and molecular features. In this retrospective study, clinical, radiological, pathological, cytogenetic, and molecular information from patients with GNBI at diagnosis seen between 1995 and 2021 was analyzed. INRG staging was performed. Of the 15,827 neuroblastoma specimens, GNBI was noted in 237 patients. Of these, 53 had the initial pathological diagnosis of GNBI; median follow-up 3.5 (range: 0.2-14) years. Disease was locoregional in 41 (77%, 16 stage L1 and 25 L2); none relapsed. Twelve (23%) had metastatic disease at presentation; six (50%) relapsed, and two died of disease. MYCN was amplified in two metastatic tumors. Six of 31 (19%) tumors tested had recurrent cytogenetic abnormalities and nonrecurrent somatic gene mutations in 10/23 (43%). The presence of any adverse molecular/cytogenetic findings was associated with metastatic disease (p<.05). For patients with localized GNBI undergoing both biopsy and resection, GNBI was diagnosed in both in 17/19 (90%). Localized GNBI at diagnosis has excellent long-term clinical outcome even without cytotoxic therapy. For localized GNBI, a biopsy sample is adequate to make the diagnosis. When associated with metastasis at diagnosis, prognosis is poorer, possibly due to associated adverse biological features.

Clinical characteristics of 11 patients with chronic lymphocytic leukemia with t (14;19) (q32;q13)

Objective: To analyze the clinicopathological characteristics of 11 cases of chronic lymphocytic leukemia (CLL) with t (14;19) (q32;q13) . Methods: The case data of 11 patients with CLL with t (14;19) (q32;q13) in the chromosome karyotype analysis results of the Blood Diseases Hospital, Chinese Academy of Medical Sciences from January 1, 2018, to July 30, 2022, were retrospectively analyzed. Results: In all 11 patients, t (14;19) (q32;q13) involved IGH::BCL3 gene rearrangement, and most of them were accompanied by +12 or complex karyotype. An immunophenotypic score of 4-5 was found in 7 patients and 3 in 4 cases. We demonstrated that CLLs with t (14;19) (q32;q13) had a mutational pattern with recurrent mutations in NOTCH1 (3/7), FBXW7 (3/7), and KMT2D (2/7). The very-high-risk, high-risk, intermediate-risk, and low-risk groups consisted of 1, 1, 6, and 3 cases, respectively. Two patients died, 8 survived, and 2 were lost in follow-up. Four patients had disease progression or relapse during treatment. The median time to the first therapy was 1 month. Conclusion: t (14;19) (q32;q13), involving IGH::BCL3 gene rearrangement, is a rare recurrent cytogenetic abnormality in CLL, which is associated with a poor prognosis.

Association between Immunophenotypic Parameters and Molecular Alterations in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is a hematologic malignancy that occurs due to alterations such as genetic mutations, chromosomal translocations, or changes in molecular levels. These alterations can accumulate in stem cells and hematopoietic progenitors, leading to the development of AML, which has a prevalence of 80% of acute leukemias in the adult population. Recurrent cytogenetic abnormalities, in addition to mediating leukemogenesis onset, participate in its evolution and can be used as established diagnostic and prognostic markers. Most of these mutations confer resistance to the traditionally used treatments and, therefore, the aberrant protein products are also considered therapeutic targets. The surface antigens of a cell are characterized through immunophenotyping, which has the ability to identify and differentiate the degrees of maturation and the lineage of the target cell, whether benign or malignant. With this, we seek to establish a relationship according to the molecular aberrations and immunophenotypic alterations that cells with AML present.

Single-cell analysis highlights a population of Th17-polarized CD4+ naïve T cells showing IL6/JAK3/STAT3 activation in pediatric severe aplastic anemia

Acquired aplastic anemia (AA) is recognized as an immune-mediated disorder resulting from active destruction of hematopoietic cells in bone marrow (BM) by effector T lymphocytes. Bulk genomic landscape analysis and transcriptomic profiling have contributed to a better understanding of the recurrent cytogenetic abnormalities and immunologic cues associated with the onset of hematopoietic destruction. However, the functional mechanistic determinants underlying the complexity of heterogeneous T lymphocyte populations as well as their correlation with clinical outcomes remain to be elucidated. To uncover dysfunctional mechanisms acting within the heterogeneous marrow-infiltrating immune environment and examine their pathogenic interplay with the hematopoietic stem/progenitor pool, we exploited single-cell mass cytometry for BM mononuclear cells of severe AA (SAA) patients pre- and post-immunosuppressive therapy, in contrast to those of healthy donors. Alignment of BM cellular composition with hematopoietic developmental trajectories revealed potential functional roles for non-canonically activated CD4+ naïve T cells in newly-diagnosed pediatric cases of SAA. Furthermore, single-cell transcriptomic profiling highlighted a population of Th17-polarized CD4+CAMK4+ naïve T cells showing activation of the IL-6/JAK3/STAT3 pathway, while gene signature dissection indicated a predisposition to proinflammatory pathogenesis. Retrospective validation from our SAA cohort of 231 patients revealed high plasma levels of IL-6 as an independent risk factor of delayed hematopoietic response to antithymocyte globulin-based immunosuppressive therapy. Thus, IL-6 warrants further investigation as a putative therapeutic target in SAA.

Presentation and outcomes of patients with multiple myeloma harboring gain or amplification of 1q21 and receiving novel agent therapies: results from a single-center study

ABSTRACTObjectiveGain or amplification 1q21 (1q21+) is one of the most common recurrent cytogenetic abnormalities in multiple myeloma (MM). Our aim was to explore the presentation and outcomes of patients with MM harboring 1q21 + .MethodsWe retrospectively analyzed the clinical features and survival outcomes in 474 consecutive patients with MM receiving immunomodulatory drugs or proteasome inhibitor-based regimens as first-line therapies.Results1q21 + was detected in 249 (52.5%) patients. Patients with 1q21 + had a higher proportion of subtypes of IgA, IgD, and λ-light chain than non-1q21 + . 1q21 + was associated with more advanced ISS stage and was more frequently accompanied by del(13q), elevated lactate dehydrogenase and lower levels of hemoglobin and platelets. Patients with 1q21 + had shorter PFS (21 months vs. 31 months, P = 0.001) and OS (43 months vs. 72 months, P < 0.001) than those without 1q21 + . Multivariate Cox regression analysis confirmed that 1q21 + was an independent prognostic factor for both PFS (HR 1.277, P = 0.031) and OS (HR 1.547, P = 0.003). Patients with 1q21 + del(13q) double-abnormality had shorter PFS (P < 0.001) and OS (P = 0.001) than those with no FISH abnormalities, and they also had shorter PFS (P = 0.018) and OS (P = 0.026) than those with del(13q) single abnormality. No significant difference in PFS (P = 0.525) or OS (P = 0.245) was found between patients with 1q21 + del(13q) double-abnormality and 1q21 + del(13q) multiple-abnormality.ConclusionsPatients with 1q21 + were more likely to have coexisting negative clinical features and del(13q). 1q21 + was an independent prognostic factor associated with poor outcomes. Concurrence with such unfavorable features may account for poor outcomes given 1q21 + .

CD20 expression: A risk stratification factor for newly diagnosed multiple myeloma with t(11;14).

Translocation (11;14) is one of the most frequent recurrent cytogenetic abnormalities in multiple myeloma (MM), while its clinical prognostic value remains controversial. CD20 expression is uncommon in MM while strongly associated with t(11;14). This study aimed to investigate whether CD20 could provide further prognostic value in MM patients harboring t(11;14). CD20 expression detected by flow cytometry was retrospectively analyzed in 211 newly diagnosed MM patients with t(11;14). The clinical characteristics and outcomes were analyzed between CD20 positive and negative patients. CD20 expression was found in 34.6% (73/211) newly diagnosed MM (NDMM) patients with t(11;14), associated with lower serum creatine levels and lower incidence of plasmacytoma. Based on similar treatment regimens, CD20 positive patients had a comparable overall response rate to CD20 negative patients, whereas had a lower CR/sCR (complete response/stringent complete response) rate than the latter (31.4% vs. 46.4%, P =0.045). Nevertheless, CD20 positive patients had a longer tendency of progression-free survival (PFS) (59.0 vs. 29.0 months, P =0.163) and significantly longer overall survival (OS) (99.0 vs. 56.0 months, P=0.003) than CD20 negative patients. Further investigation among CD20 expression proportion showed that strong expression of CD20 (>80% of bone marrow plasma cells) exhibited the longest OS (median not reached, P =0.011). However, the favorable impact of CD20 expression on survival was eliminated with the contaminant presence of cytogenetic abnormalities besides t(11;14). Autologous stem cell transplantation (ASCT) could improve the prognosis of CD20 negative t(11;14) patients. Multivariate analysis confirmed that CD20 expression was an independent favorable indicator for longer OS in t(11;14) MM patients. CD20 expression is a favorable prognostic factor in NDMM with t(11;14) and could provide further risk-stratification value in this heterogeneous disease subgroup.

Single Cell Analysis Highlights Th17-Polarized CD4+ Naive T Cells with JAK3/STAT3 Pathway Activation in Pediatric Severe Aplastic Anemia

Background: The pathogenesis of acquired aplastic anemia has been recognized as a multifaceted autoimmune bone marrow (BM) failure mainly attributed to the offsetting of functional T lymphocyte homeostasis. Although bulk genomic landscape or transcriptomic profiling have contributed a better understanding of recurrent cytogenetic abnormalities and immunologic cues associated with the onsets of hematopoietic destruction, the functional determinants underlying the complexity of heterogeneous T lymphocytes as well as their clinical significances remained to be exquisitely elucidated. Methods: We rationally adopted single cell mass cytometry (also known as 'cytometry by time-of-flight' or CyTOF) from severe aplastic anemia (SAA) bone marrow mononuclear cells (BMMCs) at pre- or post-IST stages and healthy BMMCs to functionally define critical BM cellular compartments correlated with diseases onsets and therapeutic responses. Building on the functionally characterized composition in T-lineage compartment, we deeply validated the potential regulatory mechanisms underlying immune-based hematopoietic sabotage in aplastic anemia by single cell RNA sequencing, pinpointing indicative biomarkers correlated with clinical outcomes as well as feasible therapeutic targets in autoimmune diseases of similar mechanisms. Results: Comprehensive profiling of BMMCs from 9 SAA samples at diagnosis (Dx-SAA), 3 SAA samples after immunosuppressive therapy (postIST-SAA), and 4 BMMCs from pediatric healthy donors (HD) with a predefined marker panel including critical surface antibodies (n = 31) and phosphorylated antibodies against key protein kinases (n = 11) partitioned the 27 clusters into 8 majorly functional categories, including stem/progenitor cells, granulocytes, erythrocytes, and immune subsets (monocytes(Mos), dendritic cells(DCs), B cells, T cells and NK cells). Of note, the proportion of all the non-immune subsets, and Mos and DCs in immune subsets were extremely decreased in Dx-SAA, whereas the fraction of T cells was significantly elevated in Dx-SAA, in contrast to both postIST-SAA and HD. Based on CyTOF signatures as well as flow cytometric validation on the potentially active T lineage subsets in SAA onsets, a subgroup of non-canonical CD4+ Naïve T cells with elevated phosphorylation of NFkB (pNFkBS529) and STAT3 (pSTAT3Y705) was speculated to be putatively associated with pathogenesis of SAA (Fig.1A-B). To further clarify the precise roles of the distinct CD4+ naïve T cells in the occurrences of pediatric AA, we carried out high-throughput droplet-based 5'-single-cell RNA-seq (scRNA-seq) and paired T cell receptor sequencing (scTCR-seq) of isolated CD3+ BMMCs from the aforementioned five Dx-SAAs and two HDs. Among the 14 sub-clustered T-lineage cell types, the CD4+CAMK4+ naïve T cells with functional pathway such as Th17 differentiation (JAK3 andSTAT3), TCR pathway activation (ITK and ZAP70), and the epigenetic regulators (KDM6A and XIST) caught our attention, corresponding to the features from aforementioned CD4+ naïve T cells by CyTOF analysis (Fig. 1C-D). Concordant with this discovery, the phospho-flow cytometric analysis for STAT3pY705 and STAT3pY727 from CD4+ cells in BMMCs of SAA patients and HDs strongly implied the aberrant activation of JAK3/STAT3 pathway in Th17-polarized CD4+ naïve T cells among SAA onsets. Conclusions: To our knowledge, our findings from a combination of single cell mass cytometry and single cell transcriptomic profiling have shed light on the functional roles of separated compartments of BM-infiltrating T cells in immune-mediated hematopoietic devastation in severe aplastic anemia. By tailoring scRNA-seq and CyTOF analytic tools to decipher the molecular determinants of autoimmunity, we innovatively identified the potential Th17-polarization with JAK3/STAT3 activation underlying the pathogenesis of CD4+CAMK4+ naïve T cells in SAA at single-cell transcriptomic and proteomic levels, warranting further investigation for targeted therapy with activated JAK3/STAT3 signaling in SAA. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

52. Novel recurrent cytogenetic abnormalities predict overall survival in tetraploid/near-tetraploid MDS/AML

<h3>Introduction</h3> Tetraploidy (4n=92 chromosomes) and near-tetraploidy (81-103 chromosomes) (T/NT) are uncommon cytogenetic events in MDS/AML (∼1%). Most patients are older adults with a male predominance and poor prognosis. Recurrent abnormalities associated with T/NT MDS/AML include -5/del(5q), -7/del(7q), -17/add(17p)/i(17q), -18, +8, and +21. However, other clinically-relevant abnormalities likely remain 'hidden' in long strings of cytogenetic nomenclature that are difficult to discern by visual inspection. To date, no studies have had the statistical power and computational methodology to identify novel recurrent abnormalities associated with T/NT karyotypes that predict overall survival (OS). <h3>Methods</h3> We used Cytogenetic Pattern Sleuth (CytoGPS), a publicly-available bioinformatic tool (http://cytogps.org) that we developed, to convert ISCN karyotypes from a combined cohort of 75 T/NT MDS/AML cases from two institutions (OSU and MDACC) into a binary Loss-Gain-Fusion model, which is analyzable using computational methods. <h3>Results</h3> We performed univariate analyses on the clinical and karyotypic data. Age (continuous variable, p=0.032), prior treatment (p=0.011), and cohort (p=0.025) were associated with OS; age ?60 years (p=0.316), gender (p=0.916), karyotypic complexity (p=0.175), time from diagnosis to identification of the T/NT karyotype (p=0.419), and T/NT clone size (p=0.316) had no effect. Univariate analyses of the CytoGPS results demonstrated that -5, -16, -18, del(11)(p15.1p15.4), del(13)(q12.11q22.3), and +8 were associated with poorer OS (unadjusted p<0.05). Next we used the results of univariate analyses to build multivariate models of OS. <h3>Conclusion</h3> The best predictor of OS in T/NT MDS/AML patients is the presence of any one of six cytogenetic abnormalities: -5, -16, -18, del(11)(p15.1p15.4), del(13)(q12.11q22.3), +8.

VERY COMPLEX KARYOTYPE IN YOUNG MYELODYSPLASTIC SYNDROME PATIENT AFTER CHRONIC EXPOSURE TO CHLOROFORM

Myelodysplastic Syndrome (MDS) is marked by biological and clinical heterogeneity, characterized by dysplasia in the bone marrow and blood cells with the presence of cytopenias. MDS is recognized as a disease in elderly individuals. About 86% of MDS cases are diagnosed at age ≥ 60 and less than 10% of patients are younger than 50. Recurrent cytogenetic abnormalities are present in 40% to 60% of cases. Cytogenetic analysis plays an important role, whether in the diagnosis or classification of the disease. Recently, the new international molecular prognostic score system (IPSS-M) reinforces the valuable prognostic contribution of cytogenetics in MDS in both survival and leukemic evolution. To report a chronic chloroform exposure as a trigger capable of inducing DNA damage. Chronic exposure to a genotoxic agent has been linked to MDS chromosomal abnormality. Among these, chloroform, and an inorganic halide derivative of hydrocarbons can induce the DNA lesion. A 24-year-old male patient with a marked history of 10-year chloroform exposure sought medical attention for anemia, fainting, and unintentional weight loss. He was diagnosed with Myelodysplastic Syndrome and refractory anemia with excess blasts (RAEB II) with initial tests: Hb = 5 g/dL; neutrophils=320/mm3; platelets= 18 x 109/L. Bone marrow aspiration showed 15% myeloblasts, immunohistochemistry profile showed CD34+, CD117+ in blasts and cytogenetic analysis showed a very complex karyotype: 45,XY,del(6)(q22),-7,add(9) (q12),del(12)(p11.2),del(17)(p13),del(20)(q12),add(21(p13)[17]/46, XY[3]. Chemotherapy with azacitidine was performed as a bridge to bone marrow transplantation, but the patient evolved into sepsis, dying eight months after diagnosis. The pathogenesis of MDS reflects the heterogeneity of the disease, thus, comprises multiple steps and includes factors that can be endogenous and/or exogenous. Therefore, this unusual case shows the extreme importance of investigating possible recurrent and/or occupational exposures in these patients combined with cytogenetics analyses.

AML-426 A Prospective Study to Evaluate the Prognostic Implications of PRDM16 Gene in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a complex hematologic malignancy distinguished by the accumulation of abnormally differentiated and uncontrolled proliferation of immature myeloid cells in the bone marrow induced due to diverse genetic alterations with recurrent cytogenetic abnormalities. PR domain-containing 16 (PRDM16) is a transcriptional coregulatory encoder for zinc finger transcription factor crucial for both normal and malignant hematopoiesis and essential for hematopoietic stem cells (HSCs) maintenance. However, to our knowledge, the molecular interaction, biological and immunological role, as well as its clinical diagnostic and prognostic value in AML is still incompletely known. This study aimed to characterize the potential role and molecular functions of PRDM16 in AML. In this study, PRDM16 expression level was studied using GEPIA2 database. Kaplan-Meier survival investigation was performed to estimate the prognostic significance of PRDM16 using UALCAN platforms. We explore the molecular mechanisms of PRDM16 in AML, its correlation, and biological function analysis by performing Gene set enrichment analysis (GSEA) and Linked Omics database. The relationship of PRDM16 expression with immune infiltration level was analyzed using TIMER 2.0 and CIBERSORT database. In our analyses, PRDM16 mRNA was significantly overexpressed in TCGA-LAML cohorts and closely associated with a worse prognosis in TCGA-LAML cohorts (P<0.05). Enrichment analysis revealed that PRDM16 was involved in tumor immunity. Notably, the PRDM16 mRNA expression was negatively linked with the infiltration of immune cells and stromal cells. In this study, we found that PRDM16 plays a significant role in AML progression and may serve as a potential prognostic biomarker and therapeutic target for the effective management of AML.

Molecular evaluation of gene mutation profiles and copy number variations in pediatric acute myeloid leukemia

BackgroundThe objectives of this study were to investigate the mutation profiles of targeted genes and copy number variations (CNVs) in normal cytogenetics (CN) pediatric acute myeloid leukemia (AML). MethodsThis prospective study was conducted from October 2018 to December 2020. The next-generation sequencing (NGS) and chromosomal microarray analyses (CMA) were performed in pediatric CN-AML patients. ResultsOut of 94 children (aged ≤18 years), 70 patients with AML (24 excluded) underwent conventional karyotyping/cytogenetic analyses. Forty-five (64.3%) of patients had abnormal/ recurrent cytogenetic abnormalities and 25 (35.7%) had normal cytogenetics. Twenty-three out of 25 CN-AML were further processed for gene mutation profile and CNVs using NGS and CMA, respectively. Twenty-two out of 23 (95.7%) patients were detected to have mutations in various genes. The common mutations were: NRAS, NPM1, CEBPA, KRAS, KIT, RUNX1, NOTCH1, WT1, GATA1, GATA2, FLT3, KMT2D, FLT3-TKD, and PHF6. Copy number variations (CNVs) were detected in nine patients (39%), and eight (34.8%) had a long contiguous stretch of homozygosity (LCSH) /loss of heterozygosity (LOH). An LCSH was detected on chromosomes 5, 7, 11, and 19. The gains were more common than losses (8 vs 2). The gains were observed on chromosomes 8, 9, 14, 19, 21, and 22, and the losses were detected on chromosomes 7 and 10. Monosomy was observed in three patients. Three patients (monosomy7, n = 2, and FLT-ITD, n = 1) were reclassified into the high-risk category. Post-induction, complete remission was achieved in all evaluable patients. ConclusionCN-AML patients have genetic abnormalities that can be detected by more advanced techniques like NGS and CMA. These genetic abnormalities play a role in risk stratification that may remain hidden in otherwise CN-AML.

Characterization of unusual iAMP21 B-lymphoblastic leukemia (iAMP21-ALL) from the Mayo Clinic and Children's Oncology Group.

Acute lymphoblastic leukemia (B-ALL) with intrachromosomal amplification of chromosome 21 (iAMP21-ALL) represents a recurrent high-risk cytogenetic abnormality and accurate identification is critical for appropriate clinical management. Identification of iAMP21-ALL has historically relied on fluorescence in situ hybridization (FISH) using a RUNX1 probe. Current classification requires ≥ five copies of RUNX1 per cell and ≥ three additional copies of RUNX1 on a single abnormal iAMP21-chromosome. We sought to evaluate the performance of the RUNX1 probe in the identification of iAMP21-ALL. This study was a retrospective evaluation of iAMP21-ALL in the Mayo Clinic and Children's Oncology Group cohorts. Of 207 cases of iAMP21-ALL, 188 (91%) were classified as "typical" iAMP21-ALL, while 19 (9%) cases were classified as "unusual" iAMP21-ALL. The "unusual" iAMP21 cases did not meet the current definition of iAMP21 by FISH but were confirmed to have iAMP21 by chromosomal microarray. Half of the "unusual" iAMP21-ALL cases had less than five RUNX1 signals, while the remainder had ≥ five RUNX1 signals with some located apart from the abnormal iAMP21-chromosome. Nine percent of iAMP21-ALL cases fail to meet the FISH definition of iAMP21-ALL demonstrating that laboratories are at risk of misidentification of iAMP21-ALL when relying only on the RUNX1 FISH probe. Incorporation of chromosomal microarray testing circumvents these risks.