Incorporating flow cytometry and next-generation sequencing in the diagnosis of CMML. Are we ready for prime?

Dr. Faiman considers the use of qualitative interviews to understand the patient perspective on the clinical benefits and tolerability of belamaf. She also highlights the safety profile and noninferiority of subcutaneous daratumumab compared with IV daratumumab, as described in the APOLLO trial. Finally, Dr. Faiman emphasizes the importance of achieving MRD negativity measured by multiparameter flow cytometry and next-generation sequencing.

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Clonal Hierarchy of Piga Mutant Cells Assessed By Next Generation Sequencing Is More Complex Than Previously Recognized in Paroxysmal Noctural Hemoglobinuria

Blood ◽

10.1182/blood.v128.22.1506.1506 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1506-1506

Author(s):

Michael J. Clemente ◽

Bartlomiej P Przychodzen ◽

Cassandra M. Hirsch ◽

Jaroslaw P Maciejewski

Keyword(s):

Flow Cytometry ◽

Next Generation Sequencing ◽

The Body ◽

Next Generation ◽

Gpi Anchor ◽

Clone Size ◽

Hematopoietic Stem ◽

Positive Fraction ◽

Generation Sequencing ◽

Pnh Clone

Abstract Since the pivotal revelation of the PIGA gene mutations responsible for glycosylphosphatidylinositol (GPI) anchor deficiency over 20 years ago, molecular and clinical research into the evolution of Paroxysmal Nocturnal Hemoglobinuria (PNH) has significantly advanced the current understanding of the disease, expanding upon the foundational work by numerous investigators over the previous two centuries. The discovery of multiple PIGA mutations in normal individuals using the bacterial toxin aerolysin as well as with florescent activated cell sorting (FACS) clearly demonstrated that PIGA mutations are common in normal hematopoiesis. A strong association of PNH with Aplastic Anemia (AA) and the failure of PIGA clones to expand in animal models argued for the necessity of permissive conditions, largely understood to be immune mediated bone marrow failure. While GPI anchor deficiency may lead to escape of the PNH clone from autoimmunity, recent research has added to the body of knowledge by demonstrating that the PNH clone may acquire additional mutations in other genes that promote clonal expansion in the absence of competition from normal hematopoiesis as found in AA. Sequencing studies of PIGA in PNH patients suggested that one, two, or at most three hematopoietic stem cells were sufficient to supply the necessary blood cells for survival. Furthermore, specific monoclonal antibodies combined with the fluorescently labeled inactive proaerolysin variant (FLAER) currently used to perform PNH diagnostic assays visualized the relatively frequent occurrence of both Type II and Type III PNH cells, suggesting the presence of two PIGA mutations in a significant number of patients. Using a combination of multiparameter FACS and a custom designed multiamplicon next generation sequencing (NGS) assay targeting PIGA, our results suggest that this may be an underestimate. 17 sequential patients with PNH (N=7, 3 Male, 4 Female) or AA/PNH (N=10, 5 Male, 5 Female) were enrolled in this study. A flow cytometry panel consisting of CD235a/CD59 for RBCs and FLAER/CD24/CD15/CD45 for granulocytes was used to assess PNH clone size and to sort WBCs into PNH positive and negative fractions. Mean RBC clone size was 31.6% (range 1.1-63.5%); mean WBC clone size was 53.2% (range 0.17-99.7%). Sort purity was confirmed at >98% in both fractions, DNA was extracted and subjected to analysis using an NGS assay and a stringent bioanalytic pipeline with an average depth of 18,000 reads. At least one PIGA mutation was detected in the PNH positive fraction of every patient. A total of 68 PIGA mutations were observed, consisting of 31 nonsynonymous SNVs, 16 frameshift insertion/deletions, 12 stopgains, 7 splice site, and 2 nonframeshift deletions. 13/17 (76%) had more than one mutation, and 12/17 (70%) had 3 or more mutations (range 3-14). Analysis of variant allelic frequency (VAF) indicated that multiple clones with distinct PIGA mutations greater than 5% VAF of the PNH positive fraction were found in 9/17 (53%) patients with a median VAF of 11% (range 5-86%) and 5/9 demonstrating 3 mutations >5%. Repeat identical experiments from three patients were performed on samples obtained roughly one month apart with concordant results. Overall, our results suggest that a complex clonal hierarchy with multiple dominant and/or subdominant yet expanded clones is relatively common in PNH. The clonal hierarchy in PNH patients can include up to 14 PNH clones with distinct frequencies and mutations. In addition, it is a widely held notion that PIGA mutations occur only in a hematopoietic stem cell, thus affecting all lineages, yet anomalous cases of PNH have been reported where the RBC PNH clone size is markedly higher than that of the granulocyte clone, and comparison between monocyte and granulocyte clone size is significantly different. We have identified two such cases in this cohort, with flow cytometry revealing RBC PNH clones of 18.63% and 35.61%, while granulocyte clones were 0.17% and 8.11%, and monocyte clones were 53.12% and 50.35%, respectively. Current experiments isolating and sequencing both sorted PNH positive cell fractions as well as hematopoietic precursors for PIGA and other commonly mutated genes found in hematologic malignancies are underway to confirm and elucidate the complex clonal hierarchy these results suggest. Disclosures No relevant conflicts of interest to declare.

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Multicolor Flow Cytometry and Multigene Next-Generation Sequencing Are Complementary and Highly Predictive for Relapse in Acute Myeloid Leukemia after Allogeneic Transplantation

Biology of Blood and Marrow Transplantation ◽

10.1016/j.bbmt.2017.03.017 ◽

2017 ◽

Vol 23 (7) ◽

pp. 1064-1071 ◽

Cited By ~ 59

Author(s):

Bartlomiej M. Getta ◽

Sean M. Devlin ◽

Ross L. Levine ◽

Maria E. Arcila ◽

Abhinita S. Mohanty ◽

...

Keyword(s):

Flow Cytometry ◽

Acute Myeloid Leukemia ◽

Next Generation Sequencing ◽

Myeloid Leukemia ◽

Allogeneic Transplantation ◽

Next Generation ◽

Multicolor Flow Cytometry ◽

Generation Sequencing ◽

Acute Myeloid

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Recurrent and Refractory Hemophagocytic Lymphohistiocytosis in an Elderly. Role of Immune Aberration Due to Myeloid Gene Mutation

Blood ◽

10.1182/blood-2020-133776 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 24-26

Author(s):

Suthanthira kannan Ramamoorthy ◽

Tina Noutsos ◽

David Wei ◽

Alexandra Yasmin Laidman ◽

Ferenc Szabo

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Next Generation Sequencing ◽

T Cell ◽

Myelodysplastic Syndrome ◽

Hemophagocytic Lymphohistiocytosis ◽

Bone Marrow Examination ◽

Next Generation ◽

Tet2 Mutation ◽

Generation Sequencing

Hemophagocytic lymphohistiocytosis (HLH) is a potentially fatal hyper-inflammatory disease induced by aberrant immune activation and subsequent proliferation of macrophages, histiocytes and T-helper cells. In this abstract we present a case of HLH, which relapsed twice despite ongoing treatment, and we hypothesize on possible causes and mechanisms. A 77 year old female presented to our hospital with ongoing fevers and worsening cytopenia. Blood counts from three years before the current presentation showed Hb 120g/L, WBC 4.0 x 10^9/L, Neutrophil count 1.8 x 10^9/L, Lymphocytes count 1.8 x 10^9/L and Platelet count 104 x 10^9/L. A bone marrow examination at that time revealed a normocellular marrow with 28% lymphocytes of which70% were CD 4+, CD 3+, CD5+ and CD 7-. Molecular studies confirmed T cell receptor (TCR) gamma gene rearrangement. The karyotype on the bone marrow was normal. In the absence of clinical symptoms, the patient was regularly followed up without specific therapy. During the current admission, however, the patient was febrile, had progressive pancytopenia and biochemistry suggestive of HLH (Fig 1). She was extensively evaluated which ruled out infective and malignant causes. A bone marrow aspirate and biopsy was performed and treatment initiated as per HLH-94 protocol. The bone marrow examination showed marked features of haemophagocytosis on a normocellular background. A small clone of T-lymphocytes was again noted with similar features as in the first biopsy. In addition, a prominent population (10%) of promonocytes was apparent with an uncertain significance. Karyotype was normal. Next Generation Sequencing showed TET2 frame shift mutation at low variant allele frequency (5%). Patient responded well to treatment. While on tapering dose of steroids, the disease flared up (Fig 1) and the patient was restarted on high dose steroids with etoposide. After a quick initial response, while still on active treatment, she again relapsed within 3 weeks, coupled with sepsis and acute myocardial ischemia, followed by sudden death. We were unable to identify a cause for HLH. There were 10% promonocytes in bone marrow and evidence of aberrant T-cells on flow cytometry. Although there was no obvious evidence of dysplasia on microscopy, the flow cytometry showed up- regulation of CD 64 and CD 14, down regulation of CD13 and 11b, and CD 34 expression in granulocytes possible indicating dysplasia as per the Wells criteria. (Wells et al., Blood 2003; 102(1):393) The Next generation sequencing showed TET2 mutation as mentioned above. Mutations in TET2 have been found to have overrepresented in chronic myelomonocytic leukemia in as much as 50% of patients and around 20-35% of patients with myelodysplastic syndrome (MDS). These patients with TET2 mutation have been found to have altered methylation. Recently TET2 has been implicated in immune regulation with evidence of abnormal CD 4 T cell proliferation (present in our patient) and disruption of T cell homeostasis. In addition, patients with TET2 mutation associated myelodysplastic syndrome are known to have auto-immune manifestations (Yimei Feng et al., Frontiers in Oncology, 2019 (9):1). Alyssa H et al have shown that TET2 mutant in patients with MDS (Myelodysplasia) leads to alteration of immune environment in the macrophage differentiation (Alyssa et al., Experimental Hematology, 2017:55; 56). Whether these immune aberration caused recurrent florid relapse of hemophagocytosis in our patient within a span of 2-3 months remained unclear and it could be considered in future research. Even though the occurrence of hemophagocytic syndrome has been described in acute leukemias, the association of the same with myeloid gene aberrations with or without overt myelodysplastic features is unknown. However since hemophagocytosis itself is not common, a careful evaluation to look for uncommon associations which may be a triggering factor may pave the way for identifying their possible role in the pathogenesis. And therapeutic options such as hypo methylating agents can evolve when such associations are confirmed in future studies. Figure 1 Disclosures No relevant conflicts of interest to declare.

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Highly Sensitive Detection of Minimal Residual Disease in Acute Lymphoblastic Leukemia Using Next-Generation Sequencing of Immunoglobulin Heavy Chain Variable Region

Blood ◽

10.1182/blood.v118.21.2540.2540 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2540-2540

Author(s):

Malek Faham ◽

Tom Willis ◽

Martin Moorhead ◽

Victoria Carlton ◽

Jianbiao Zheng ◽

...

Keyword(s):

Flow Cytometry ◽

Next Generation Sequencing ◽

Lymphoblastic Leukemia ◽

Patient Specific ◽

Next Generation ◽

Conventional Pcr ◽

Employment Equity ◽

Equity Ownership ◽

Generation Sequencing

Abstract Abstract 2540 Background: Minimal Residual Disease (MRD) assessment is increasingly used for treatment stratification since it is a strong predictor of outcome in Acute lymphoblastic leukemia (ALL). The most widely used MRD assays include flow cytometric detection of aberrant immunophenotypes and PCR amplification of patient-specific antigen-receptor sequences. The latter approach has proven to provide reliable clinical information but requires the development of patient-specific reagents which is laborious, time-consuming, and generates assays with variable sensitivities. In addition, this methodology may miss clonal changes that can occur during the course of the disease, such as the emergence of subclones as well as genetic evolution. To overcome these limitations, we developed a universal amplification assay with a sequencing readout that eliminates the need for patient-specific reagents, allows the assay to detect leukemic cells that have genetically evolved, and has a higher sensitivity than conventional tests. Methods and Results: To amplify all the IgH sequences, we developed a PCR assay to amplify all alleles of all the V and J segments with very low amplfication bias. Amplified molecules were then subjected to clonal sequencing to obtain >1 million reads to measure the frequency of the different IgH clonotypes in the sample. It should be noted that current next generation sequencing costs of this deep sequencing are similar to those of an MRD test conducted by flow cytometry. We tested the sensitivity of the method by in serial dilutions of genomic DNA from a leukemia sample known to carry two IgH clonotypes with into genomic DNA obtained from peripheral blood sample from a healthy donor. The material from the dilution series was then sequenced and analyzed to measure the level of these clones. The leukemic clonotypes could be readily detected even when diluted 1 million fold. To directly compare the our method to established MRD assays in ALL, we studied diagnostic and follow-up samples from 10 ALL patients whose MRD levels have been previously assessed by both real-time PCR amplification of IgH genes and flow cytometry. The results of these tests were not disclosed until completion of the deep sequencing analysis. The follow up samples were collected during (n = 3) or at the end of remission induction therapy (n = 4), or during continuation therapy (n = 3). Samples were processed similarly to identify the leukemia-specific sequence in the diagnostic samples and determine the level of these sequences in the follow up samples. The sequencing-based method identified all 5 samples that were MRD-positive according to flow cytometry and PCR (Figure 2), with highly concordant estimates of MRD levels. Notably, among the remaining 5 samples, scored as MRD-negative by both flow cytometry and conventional PCR, the sequencing method detected residual leukemic sequences at a very low level (∼10−6) in one of the samples. The other 4 samples were MRD-negative by all three methods. Studies with a larger cohort of ALL samples are ongoing. Contrary to conventional PCR-based MRD testing, the sequencing technology allows for the detection of leukemic clones that evolve by V replacement or other mechanisms. In this study, we identified clonotypes in several of the diagnostic samples that appeared to be the result of V replacement. These and other newly appearing related clones can be monitored in subsequent samples using the generic amplification and sequencing assay. Conclusions: We developed a highly sensitive and specific MRD detection method based on next-generation sequencing of IgH genes. This method has substantial advantages over conventional PCR MRD in that it eliminates the need for patient-specific reagents, can follow genetic evolution, and has potential for higher sensitivity. Disclosures: Faham: Sequenta Inc: Employment, Equity Ownership. Willis:Sequenta Inc: Employment, Equity Ownership. Moorhead:Sequenta Inc: Employment, Equity Ownership. Carlton:Sequenta Inc: Employment, Equity Ownership. Zheng:Sequenta Inc: Employment, Equity Ownership.

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Clinical impact of panel-based error-corrected next generation sequencing versus flow cytometry to detect measurable residual disease (MRD) in acute myeloid leukemia (AML)

Leukemia ◽

10.1038/s41375-021-01131-6 ◽

2021 ◽

Author(s):

Nikhil Patkar ◽

Chinmayee Kakirde ◽

Anam Fatima Shaikh ◽

Rakhi Salve ◽

Prasanna Bhanshe ◽

...

Keyword(s):

Flow Cytometry ◽

Next Generation Sequencing ◽

Residual Disease ◽

Multiparameter Flow Cytometry ◽

Next Generation ◽

Free Survival ◽

Additional Information ◽

Next Generation Sequencing Ngs ◽

Kinetics Of ◽

Generation Sequencing

AbstractWe accrued 201 patients of adult AML treated with conventional therapy, in morphological remission, and evaluated MRD using sensitive error-corrected next generation sequencing (NGS-MRD) and multiparameter flow cytometry (FCM-MRD) at the end of induction (PI) and consolidation (PC). Nearly 71% of patients were PI NGS-MRD+ and 40.9% PC NGS-MRD+ (median VAF 0.76%). NGS-MRD+ patients had a significantly higher cumulative incidence of relapse (p = 0.003), inferior overall survival (p = 0.001) and relapse free survival (p < 0.001) as compared to NGS-MRD− patients. NGS-MRD was predictive of inferior outcome in intermediate cytogenetic risk and demonstrated potential in favorable cytogenetic risk AML. PI NGS-MRD− patients had a significantly improved survival as compared to patients who became NGS-MRD− subsequently indicating that kinetics of NGS-MRD clearance was of paramount importance. NGS-MRD identified over 80% of cases identified by flow cytometry at PI time point whereas FCM identified 49.3% identified by NGS. Only a fraction of cases were NGS-MRD− but FCM-MRD+. NGS-MRD provided additional information of the risk of relapse when compared to FCM-MRD. We demonstrate a widely applicable, scalable NGS-MRD approach that is clinically informative and synergistic to FCM-MRD in AML treated with conventional therapies. Maximum clinical utility may be leveraged by combining FCM and NGS-MRD modalities.

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Intrapatient Functional Clonality Deconvoluted By Coupling Intracellular Flow Cytometry and Next Generation Sequencing in Human Leukemia

Blood ◽

10.1182/blood.v128.22.3161.3161 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3161-3161

Author(s):

Qing Zhang ◽

Markus Ball ◽

Zhao Yulong ◽

Maria E. Balasis ◽

Alan F List ◽

...

Keyword(s):

Flow Cytometry ◽

Next Generation Sequencing ◽

Somatic Mutations ◽

Mononuclear Cells ◽

Leukemic Cells ◽

Human Leukemia ◽

Next Generation ◽

Roc Curve Analysis ◽

Functional Differences ◽

Generation Sequencing

Abstract Leukemic clones within the same patient have unique sensitivities to drugs in vivo and may have unique capacities to engraft within xenograft models suggesting that there exist intra-patient functional clonal differences. However, methods to detect the functional differences assigned to genetically defined clones remain limited. Here, we describe a method to measure functional differences of clones within the same leukemia patient by coupling intracellular flow cytometry and next generation sequencing (NGS). We hypothesize that functionally distinct clones can be inferred by stimulating cells with cytokines, fluorescently staining with a relevant downstream marker of activation, and then isolating functionally distinct fractions by flow sorting. NGS can then digitally annotate somatic variants in sorted, functionally distinct samples that can be compared to the bulk leukemic cell population. To achieve this, we optimized a DNA isolation method that results in high-quality DNA despite originating from fixed/permeabilized cells. In this way, we annotate enrichment and de-enrichment of variants upon stimulation and infer the functional clonality of any given sample. To provide proof-of-concept, we explored the well-established relationship between Chronic Myelmonocytic Leukemia (CMML) and GMCSF. CMML cells are uniquely hypersensitive to GMCSF and simultaneously have a well-established mutational composition that can be annotated by sequencing only 49 genes. This allowed us to leverage an amplicon-based enrichment panel that employs emulsion PCR to achieve reproducible library preparation with 5 ng of input DNA hence bypassing the need for whole genome amplification. We tested our methodology by mixing two cell lines whose response to GMCSF was known and somatic variants had been annotated a priori. K562 (non-GMCSF responsive) and THP-1 (GMCSF responsive) leukemic cells were mixed at 1:1 and then stimulated with GMCSF. The resultant cells were stained with anti-pSTAT5 and cell sorting isolated positive and negative fractions. Unstimulated mixed cells and each positive and negative fraction was sequenced and variant allele fractions (VAF) was compared to unstimulated bulk (Figure 1A). To quantitate the change in VAF, we reasoned that summing the difference in VAF of unsorted/pSTAT5+ and unsorted/pSTAT5- populations would yield the most informative composite value as it would contain information about VAF difference for each mutation in both the GMCSF stimulated and GMCSF unstimulated fractions. Using this method, we found that 100% of THP1 and K562 variants were enriched in the pSTAT5+ and pSTAT5- fractions, respectively (Figure 1B). Receiver Operator Curves (ROC) demonstrated that the data functionally discriminated mixed THP-1 and K-562 cells with an AUC of 0.9868 (p<0.0001) (Figure1C). We additionally performed serial dilutions of THP-1 to K562 cell mixtures and demonstrated cell number dependent recovery of THP-1 variants upon stimulation with GMCSF. Next we reasoned that somatic mutations should be uniquely augmented by GMCSF stimulation making it possible for somatic variant identification. To test this, we sequenced 8 CMML bone marrow mononuclear cells evaluated in the same way as above. We additionally sorted CD3+ cells as germline controls to identify somatic mutations. Using our 49-gene panel, we identified a total of 35 somatic variants and 160 germline variants. ROC curve analysis confirmed that our assay was capable of discriminating germline from somatic mutations with an AUC of 0.9388 (p<0.001). In a representative CMML case, we counterstained with CD33 to isolate myeloid progenitors and inferred a functionally distinct clone that was hypersensitive to GMCSF and uniquely carried a JAK2 and PTPN11 mutation that was obscured by bulk leukemia sequence (Figure 2). This inference was orthogonally validated by sequencing individual colonies from the same case grown under GMCSF conditions (Figure 3). Further validation is ongoing comparing mutations identified at engraftment of cells differentially xenografted in NSG and NSGS mice that differ only in their production of human cytokines. Taken together, we describe a novel method to annotate functional differences among mutationally-defined populations and identify functionally distinct clones in primary leukemia samples. Disclosures Komrokji: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Speakers Bureau.

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