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. 2023 Apr;9(2):a006241. doi: 10.1101/mcs.a006241

Clonal cytopenia of undetermined significance (CCUS)-associated reversion of donor-derived, transient αβ T-cell large granular clonal lymphocytosis, emerging post-transplant in a patient with a history of γδ T-cell large granular lymphocytic leukemia

Siba El Hussein 1,✉, Andrew G Evans 1, John M Fitzsimmons 1, Nufatt Leong 1, Meghan Buldo 1, Jeremy P Segal 2, Audrey N Jajosky 1, Paul G Rothberg 1, Jane L Liesveld 3,4, Zoltán N Oltvai 1,✉
PMCID: PMC10240839  PMID: 37160316

Abstract

Autologous and allogeneic hematopoietic stem cell transplantation (HSCT) has revolutionized the therapy of hematolymphoid malignancies. Yet, how to best detect or predict the emergence of HSCT-related complications remain unresolved. Here, we describe a case of donor-derived, transient Alpha Beta (αβ) T-cell large granular clonal lymphocytosis and cytopenia that emerged post-HSCT in a patient with a history of gamma delta (γδ) T-cell large granular lymphocytic leukemia (T-LGLL). Clonal unrelatedness of post-transplant T-LGL lymphocytosis to the patient's pretransplant T-LGLL was first identified by T-cell receptor (TCR) PCR showing different sized fragments of rearranged gamma chains, in addition to shift from γδ to αβ TCR expression by flow cytometry analyses. Donor-derivation of the patient's post-transplant clonal lymphocytosis was confirmed by serial chimerism analyses of recipient's blood specimens demonstrating 100% donor DNA. Moreover, oncogenic DNMT3A and RUNX1 mutations were detected by next-generation sequencing (NGS) only in post-transplant specimens. Intriguingly, despite continued increase in DNMT3A and RUNX1 mutation load, the patient's clonal lymphocytosis and anemia eventually largely resolved; yet, the observed mutation profile with persistent thrombocytopenia indicated secondary clonal cytopenia of undetermined significance (CCUS) in the absence of overt morphologic evidence of myeloid neoplasm in the marrow. This case illustrates the utility of longitudinal chimerism analysis and NGS testing combined with flow cytometric immunophenotyping to evaluate emerging donor-derived hematolymphoid processes and to properly interpret partial functional engraftment. It may also support the notion that driver mutation-induced microenvironmental changes may paradoxically contribute to reestablishing tissue homeostasis.

Keywords: leukemia, T-cell chronic lymphocytic lymphoma/leukemia, vacuolated lymphocytes

INTRODUCTION

The development of autologous and allogeneic hematopoietic stem cell transplantation (HSCT) has revolutionized the treatment of hematolymphoid malignancies. However, HSCT is not without complications, including graft versus host disease, lack of engraftment, partial functional engraftment only, and, rarely, the development of donor-derived leukemia (Narumi et al. 2004; Gill et al. 2012; Hidalgo Lopez et al. 2016). Regarding the latter, the increasing use of older donors and the more likely presence of gene mutations associated with clonal hematopoiesis of indeterminate significance (CHIP) in older populations is an emerging concern (Nawas et al. 2021). It is also increasingly recognized that CHIP clones at a very low level can appear at a young age (Fabre et al. 2022; Mitchell et al. 2022) and perhaps even in utero (Williams et al. 2022).

T-cell large granular lymphocytic leukemia (T-LGLL) is mostly an indolent neoplasm characterized by a persistent (>6 mo) increase in peripheral blood large granular lymphocytes (LGLs) between 2 and 20 × 109/L (Swerdlow et al. 2017). Patients often present with neutropenia, splenomegaly, and autoimmune disorders (Swerdlow et al. 2017). The LGLs are typically clonally expanded cytotoxic T cells, expressing CD3, CD8, and TCR αβ (most commonly) (Swerdlow et al. 2017) harboring a heterogeneous mutation profile, with STAT3 mutations being the most common (Cheon et al. 2022).

T-LGL expansions post-HSCT have also been well-described, most of which are reactive in nature, with a self-limiting clinical course in asymptomatic patients (Dolstra et al. 1995; Mohty et al. 2002; Nann-Rutti et al. 2012; Kim et al. 2013). Rare cases of T-LGL leukemia after solid organ transplant (Feher et al. 1995; Gentile et al. 1998) and HSCT have been also reported, the latter in single case reports (Au et al. 2003; Wong et al. 2003; Narumi et al. 2004; Chang et al. 2005; Kusumoto et al. 2007; Hidalgo Lopez et al. 2016) or small case series, with a frequency of 0.5% in allogeneic and autologous HSCT (Gill et al. 2012). The time interval from transplant to the appearance of post-transplant T-LGLL in previously described cases varied from 3 mo to 11 yr (Feher et al. 1995; Deeg and Socie 1998; Gentile et al. 1998; Narumi et al. 2004; Gill et al. 2012; Hidalgo Lopez et al. 2016).

Here, we describe a case of donor-derived, transient αβ T-cell large granular clonal lymphocytosis emerging and then resolving in a patient with a history of γδ T-LGL leukemia, with acquired donor-derived clonal cytopenia of undetermined significance (CCUS) and gradually expanding oncogenic DNMT3A and RUNX1 mutation load, without overt morphologic evidence of myeloid neoplasm in the marrow. This case illustrates that aberrant T-LGL expansions occurring post-autologous or allogeneic HSCT seem to have a different pathogenesis and clinical course when compared to de novo T-LGLL, as it has been previously suggested (Gill et al. 2012). It also demonstrates the diagnostic utility of combined flow cytometric immunophenotyping, chimerism, and next-generation sequencing (NGS) testing to detect and properly assess emerging donor-derived proliferations in the post-HSCT setting. It also supports the notion that pathogenic driver mutations may contribute to tissue allostasis in the post-transplant setting.

RESULTS

Case Presentation

A 54-yr-old female with a history of collagen vascular disease was diagnosed with T-LGLL of the rare gamma delta (γδ) variant in 2017 (Fig. 1A) with associated red cell aplasia, which was positive both then (not shown) and in spring 2020 for a monoclonal T-cell population by T-cell receptor gamma (TCRG) gene rearrangement testing by polymerase chain reaction (TCRG-PCR) assay (Fig. 1B, lower panel). Flow cytometry showed 76% lymphocytes, of which 99% were CD3+ T cells with a CD4/CD8 ratio of 1:10. Ninety percent of lymphocytes were CD3/CD5/TCR γδ-positive T cells. This T-cell population coexpressed CD2, CD7 (variable), CD16 (partial), and CD8 (partial, 30%). A subset of these T cells (65%) was double negative for CD4 and CD8.

Figure 1.

Figure 1.

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Clinical timeline since initial diagnosis of T-cell large granular lymphocytic (T-LGL) leukemia and post-allogeneic stem cell transplant. (A) Representative flow cytometry plots from winter 2017–2018, illustrating patient's gamma delta (γδ) T-LGL leukemia in the blood (purple cell population); (B) pretransplant T-cell receptor gene rearrangement analysis by polymerase chain reaction (TCRG-PCR) of donor's blood specimen showing a polyclonal pattern (upper panel), and patient's blood specimen, showing a prominent clonal population (red arrow) (lower panel); (C) allogeneic stem cell transplant took place in summer 2020; (D) flow cytometry analysis performed post-transplant on the recipient's blood specimens in winter 2020–2021, showing T-LGL regeneration (purple population), with a reversed CD4:CD8 ratio and polyclonal TCRG-PCR; (E,F) follow-up chimerism analyses had demonstrated 100% donor DNA; however, flow cytometry immunophenotyping identified aberrant αβ T-LGL populations in the bone marrow in summer 2021, and in the blood in autumn 2021 (purple populations), in addition to clonal T-cell population in these two specimens, showing rearranged fragments that were of different size from patient's pretransplant LGL leukemia clone (red arrows). The constellation of these findings corroborates the diagnosis of donor-derived αβ T-cell large granular clonal lymphocytosis.

The patient showed no clinical response to oral cyclophosphamide, prednisone, and intravenous cyclosporine treatment. She then received fludarabine/cyclophosphamide/total body irradiation conditioning and a subsequent haploidentical allogenic HSCT in summer 2020, with her son serving as donor (Fig. 1C) with polyclonal T-cell population in his peripheral blood (Fig. 1B, upper panel). The donor and recipient (patient) ABO/Rh and CMV serostatus were O+/O+ and −/+, respectively. Graft versus host disease prophylaxis consisted of post-transfusion cyclophosphamide, mycophenolate mofetil, and tacrolimus therapy. Mycophenolate was stopped at day 30 and tacrolimus at approximately day +180 after allografting.

Post-Transplantation Disease Course

Post-HSCT, the patient developed transfusion-dependent anemia, with hemoglobin averaging 9 g/dL (normal range [NR], 11.2–15.7 g/dL) along with persistent profound thrombocytopenia of 9 × 109/L (NR 160–370 × 109/L) without associated lymphadenopathy or splenomegaly. Chimerism analyses of the bone marrow from summer 2020 and blood specimens from 3 mo (autumn 2020) and 6 mo (winter 2020–2021) post-transplant revealed 11%, 48%, and 73% donor DNA, respectively, indicating gradual ongoing engraftment.

From summer 2020 until autumn 2020, several flow cytometry analyses performed on blood specimens were negative for aberrant T-LGL populations (not shown). However, in winter 2020–2021, flow cytometry analyses performed on blood specimens showed T-LGL regeneration, with a reversed CD4:CD8 ratio, and negative TCRG-PCR (Fig. 1D; Supplemental Figs. 1 and 2). In spring 2021, the patient started having a steady increase in blood lymphocytosis reaching up to 6.6 × 109/L (NR 1.2–3.7 × 109/L) in the absence of lymphadenopathy and without any clinical symptoms, in addition to persistent transfusion-dependent anemia and thrombocytopenia. Peripheral blood smears were notable for increased intermediate-sized lymphocytes with moderate to abundant cytoplasm, irregular nuclear contours and prominent azurophilic granules (Supplemental Fig. 3).

This prompted follow-up flow cytometry analysis on a bone marrow specimen in summer 2021, which revealed a reversed CD4:CD8 ratio with an aberrant T-LGL population expressing CD8 (strong uniform) with decreased CD5 and CD7 expression, negativity for CD4, CD56, and γδ TCR expression, and a monoclonal T-cell population by TCRG-PCR (Fig. 1E, top and middle panels). The pattern of rearranged fragments in this clone (Fig 1E, bottom panel) was different from that detected in the patient's pretransplant LGL leukemia (Fig. 1B, bottom panel), suggesting that these two clones were likely not directly-related. Follow-up flow cytometry and TCRG-PCR analyses on blood specimens in autumn 2021, showed similar but more pronounced findings (Fig. 1F). Chimerism analyses performed on four consecutive blood specimens in summer/autumn 2021, and winter/spring 2022 showed 100% engraftment (i.e., pretransplant donor DNA pattern), supporting the diagnosis of a donor-derived T-LGL clone.

Next-Generation Sequencing

In house NGS of the patient's post-HSCT bone marrow and blood samples revealed pathogenic mutations in DNMT3A NM_022552.4: c.2645G > A (p.Arg882His) and RUNX1 NM_001754.4: c.502G > T (p.Gly168Ter) at comparable variant allele frequencies (VAFs) of ∼22% (summer 2021, bone marrow) and 11% (autumn 2021, blood), respectively, likely denoting their presence in the same clone.

Subsequent NGS testing at an outside laboratory performed on a blood specimen from winter 2021 revealed the following pathogenic mutations: DNMT3A R882H NM_022552.4: c.2645G > A with a VAF of 30.1%, and RUNX1 G168* NM_001754.4: c.502G > T with a VAF of 23.6%; in addition to the following variants with unknown clinical significance: ARID1A P887L NM_006015.6: c.2660C > T with a VAF of 22.3%, and PLCG2 N639S NM_002661.5: c.1916A > G with a VAF of 53.3% (Table 1) (note, ARID1A and PLCG2 are not covered on the myeloid NGS panel we used for in-house NGS; see Methods). In contrast, expanded panel retrospective NGS did not detect any pathogenic mutation, including variants in STAT3 or STAT5B, in the patient's pre-HSCT bone marrow specimen (from autumn, 2018). Retrospective NGS testing of DNA from the donor's pretransplant blood was also negative for any clinically significant variants using our in-house NGS panel. This suggested that if the donor harbored any variants associated with clonal hematopoiesis of undetermined significance (i.e., CHIP mutations), their VAFs were too low to be detectable by standard NGS testing.

Table 1.

Summary of all detected gene variants observed in patient's blood specimen in winter 2021 (21 mo post-transplant)

Gene Chromosome Variant VAF Classification
DNMT3A 2 NM_022552.4:c.2645G > A, p.R882H 30% Oncogenic
RUNX1 21 NM_001754.4:c.502G > T, p.G168* 24% Oncogenic
ARID1A 1 NM_006015.6:c.2660C > T, p.P887L 22% VUS
PLCG2 16 NM_002661.5:c.1916A > G, p.N639S 53% Likely benign
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(VUS) Variant of unknown significance, (VAF) variant allele frequency.

Post-Secondary T-LGL Clonal Lymphocytosis Course and Mutation Load Changes

The patient did not receive any specific treatment for her post-HSCT clonal lymphocytosis. However, she was started on steroids and the thrombopoietin receptor agonist eltrombopag to manage her persistent profound thrombocytopenia, in addition to weekly platelet and packed red blood cells transfusions. In early 2022, the patient's lymphocyte counts started normalizing to 2.5 × 109/L (NR 1.2–3.7 × 109/L), her anemia has also resolved in summer 2022 with an average hemoglobin of 12.5 g/dL (NR, 11.2–15.7 g/dL). The patient continued receiving treatment with eltrombopag and later with the thrombopoietin analog romiplostim, and her thrombocytopenia by summer 2022 improved to up to 92 × 109/L (NR 160–370 × 109/L).

Finally, we performed retrospective NGS on serial post-transplant blood samples within the span of 1 yr (Table 2). Despite the normalization of the lymphocyte count and resolution of the patient's anemia, the VAF of the DNMT3A R882H and RUNX1 G168* mutations continued to increase from 10% to nearly 40% within this time frame (Table 2). TCRG PCR testing on the same specimens showed a gradual reduction of the monoclonal T-cell population detected in autumn 2021 (Supplemental Fig. 4), while other T-cell clones appeared to become more prominent within the polyclonal background (Supplemental Fig. 4). A bone marrow biopsy performed in early 2023 showed a normocellular marrow without overt morphologic evidence of myeloid neoplasm, with presence of DNMT3A R882H and RUNX1 G168* at VAF of 43% and 64%, respectively, and persistent thrombocytopenia at 98 × 109/L (NR 160–370 × 109/L). These findings were supportive of the diagnosis of CCUS.

Table 2.

Mutational burden (variant allele frequency [VAF]) observed over time in the peripheral blood, post-hematopoietic stem cell transplant

Autumn 2021 (18 mo post-transplant) Winter 2022 (21 mo post-transplant) Spring 2022 (25 mo post-transplant) Summer 2022 (29 mo post-transplant)
DNMT3A p.R882H 10% 29% 36% 31%
RUNX1 p.G168* 11% 31% 36% 40%
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DISCUSSION

Post-transplant T-LGL clonal lymphocytosis was distinguished from reactive conditions in our case based on persistent large lymphocytosis with aberrant phenotype by flow cytometry analyses and demonstration of a clonal T-cell population by TCRG-PCR. Our patient developed a donor-derived T-LGL clonal lymphocytosis ∼1-yr post-transplant. This case is unusual to the extent that the patient's initial T-LGL leukemia was of the very rare γδ variant (Teramo et al. 2022), whereas the donor-derived T-LGL clone was of the common αβ TCR-bearing variant. This immunophenotype shifting of LGLs (from γδ to αβ TCR) provided a hint that the newly emerging T-LGL clone was likely clonally unrelated to the patient's initial leukemia. In comparison with other γδ TCR-bearing lymphoproliferative disorders, γδ T-LGL leukemia has an excellent prognosis and is generally considered an indolent disease (Saito et al. 2002; Makishima et al. 2003; Shichishima et al. 2004; Ahmad et al. 2005). However, studies directly comparing it with emergent, post-transplant αβ T-LGL clonal lymphocytosis have not been reported.

The pathogenesis of T-LGL leukemia remains undefined. Chronic antigenic stimulation has been postulated to lead to polyclonal and subsequent clonal escape due to emerging oncogenic mutations (most commonly in STAT3 and STAT5B) and proliferation of a neoplastic population of cytotoxic CD8+ T-large granular lymphocytes (Sokol and Loughran 2006; Semenzato and Zambello 2022). The occurrence of post-transplant T-LGL proliferations in both autologous and allogeneic cells suggests that neoplastic transformation may be related to post-HSCT predisposition rather than an inherent dysfunction in T-large granular lymphocytes. No solid links implying relationships to underlying CMV and EBV infections have been made so far in the setting of post-transplant T-LGL expansions. Earlier studies of polyclonal T-LGL proliferations suggested that “reactive” T-LGL expansions might mediate a graft versus leukemia effect, with a favorable effect on the pretransplant hematologic neoplasm (i.e., decreased recurrences) (Dearden 2011; Dolstra et al. 1995). Whether reported donor-derived T-LGL leukemia cases were similarly directly related to favorable outcomes described in these reports remains to be defined (Narumi et al. 2004; Gill et al. 2012; Hidalgo Lopez et al. 2016). Regardless, T-LGL leukemia occurring post-HSCT seems to have a different pathogenesis and clinical course when compared with de novo T-LGL leukemia, as patients often remain stable for years and do not require specific treatment (Narumi et al. 2004; Gill et al. 2012; Hidalgo Lopez et al. 2016). Indeed, in one series, patients were made only aware of the lymphocytosis, but not of the pathological diagnosis of donor-derived T-LGL leukemia, made on the basis of meeting all the diagnostic criteria of de novo T-LGL leukemia as proposed by the World Health Organization (WHO) classification of hematologic diseases (Gill et al. 2012). Prudence in disclosing the pathological diagnosis was, in part, with respect to the psychological effects on patients who have already undergone an HSCT for another hematological malignancy, and in part because of the lack of characterization of emerging aberrant T-LGL populations in the post-transplant setting.

Our patient underwent slow engraftment, as her post-transplant chimerism analysis of bone marrow and blood specimens between summer 2020 and autumn 2020 demonstrated gradual increase of donor DNA, reaching up to 73% in winter 2020–2021. During this phase, she was managed for pure red cell aplasia/graft failure. On follow-up in early 2022, the patient's anemia and thrombocytopenia persisted despite gradual normalization of lymphocytic count and supportive therapy including transfusions. These findings suggest that the patient's cytopenias may be not only due to possible hematopoietic suppression mediated by expanding recipient's reactive T-LGL and natural killer (NK) cells during early stages of HSCT (residual host immunity), leading to instability of the hematopoietic engraftment (Imamura 2021); instead, it might have been caused later on by gradually expanding CCUS-related mutations in DNMT3A and RUNX1, contributing to poor graft function in a 100% donor-derived marrow, and translating into long-duration cytopenias clinically, despite normalization of neutrophil and lymphocyte levels. It is notable that this mutation profile is highly unusual, both for STAT-mutated and STAT wild-type T-LGLLs (Coppe et al. 2017; Teramo et al. 2020; Cheon et al. 2022). However, related TET2 mutations are common in NK-LGL (Olson et al. 2021). Alternatively, occurrence of LGL clones with associated bone marrow failure syndromes such as (hypoplastic) myelodysplastic syndrome (MDS) were described (Kochenderfer et al. 2002), perhaps through T-lymphocyte-mediated inhibition of hematopoiesis. In contrast to classic T-LGLL, in hypoplastic MDS, mutations in DNMT3A, TET2, and RUNX1 are not unusual (Fattizzo et al. 2021) and are common in CCUS (Vobugari et al. 2022).

There are two mechanisms by which clonal hematopoiesis can arise in allogenic-HSCT recipients post-transplant with near-complete engraftment (as in our case): The clone may be transferred from the donor and engraft in the recipient, or the clone may arise de novo from donor cells post-transplant (Nawas et al. 2021). Furthermore, donor T- and NK-cell rapid expansion has been shown to facilitate donor hematopoietic stem cell engraftment through inhibition of residual host immunity, with a collateral suppression of donor-derived hematopoiesis (Muller et al. 2010), which may explain, in the current case, both the aberrant donor-derived αβ T-LGL expansion with positive TCRG-PCR and long-duration cytopenias, while background chimerism is 100% donor. Of note, LGLL (in nontransplant settings) has been shown by one group to be associated with the presence of CHIP-like mutations (Durrani et al. 2020): The authors hypothesized that LGLL may evolve as a consequence of immune surveillance reaction to CHIP mutations; alternatively, emergence of CHIP mutations may be the result of recurrent DNA damage provoked by LGL proliferation, immune inhibition of myelopoietic evolution and persistence of a myeloid escape clone (Sloand et al. 2011; Durrani et al. 2020). The latter scenario may explain the emergence of CHIP mutations post-transplant, in the setting of clonal LGLL expansion in our patient.

The gradual decline of the original post-transplant monoclonal T-cell clone with subsequent emergence of two other clonal T-cell populations concomitant with increasing DNMT3A and RUNX1 mutation load, yet simultaneous normalization of the patient's hematologic parameters, is an intriguing finding. The fact that the effect of various oncogenic mutations can be efficiently buffered to maintain normal tissue homeostasis (i.e., allostasis) has been well-established (Martincorena et al. 2015, 2018; Yokoyama et al. 2019). Moreover, the mutation profiles of clones bearing oncogenic variants in normal tissues are different from those observed in true malignancies (Kakiuchi and Ogawa 2021). This highlights the need to evaluate a larger number of patients with hematologic neoplasms displaying transient post-transplant lymphocytosis to clarify if the emergence of mutations is consistently observed, and, if yes, whether they are associated with distinct mutation profiles.

In summary, although reactive T-LGL expansions may occur in the post-transplant settings, this report illustrates a CCUS-associated, transient donor-derived T-LGL clonal lymphocytosis of the αβ variant, arising ∼1 yr post-HSCT, in a patient with history of T-LGL leukemia of the γδ variant. It also emphasizes that aberrant T-LGL expansions occurring post-autologous or allogeneic HSCT seem to have different pathogenesis and clinical course when compared to de novo T-LGLL, as has been previously suggested (Gill et al. 2012), urging the need to provide an updated clinical characterization of clonal T-LGL populations emerging in the post-transplant setting in future versions of the WHO classification of hematologic diseases. Furthermore, donor-derived clonal hematopoiesis may contribute to persistent cytopenias post–full engraftment, supporting the potential utility of molecular screening of donors’ specimens for CHIP mutations pretransplant. However, although myeloid neoplasms are much more likely to develop in patients with CCUS than in patients without clonality (Vobugari et al. 2022), paradoxically these driver mutations may also contribute to tissue allostasis in the post-transplant setting.

METHODS

Clinical Specimens

The patient was monitored and treated at the University of Rochester Medical Center (URMC) between May 2017 and February 2022. Bone marrow aspirate/biopsy and/or peripheral blood samples were collected at various time points after initial URMC assessment.

Specimen Processing and Morphologic Assessment

Bone marrow aspirate and biopsy specimens were processed using the automated tissue processors Leica ASP300S and Leica Peloris II (Leica Biosystems Division of Leica Microsystems Inc.). Three micron-section tissue slides were cut from the processed paraffin blocks and stained with hematoxylin and eosin (H&E) using H&E automated strainers (Sakura Finetek USA; Leica Biosystems, Division of Leica Microsystems). Morphologic assessment of the H&E-stained specimens was performed by a board-certified hematopathologist. Wright–Giemsa stain was performed on peripheral blood sample with the Midas III Stainer (Fisher Scientific, Part of Thermo Fisher Scientific; Sysmex America, Inc.; Sigma-Aldrich). Complete blood count (CBC) was performed on a Sysmex XN-10 Automated Hematology Analyzer (Sysmex America) and followed with a manual differential.

Flow Cytometric Immunophenotyping

Immunophenotyping assays were performed by URMC Clinical Flow Cytometry Laboratory for standard clinical care, using a Beckman Coulter Navios Flow Cytometer, FDA approved 10-Color ClearLLab lyophilized immunophenotyping tubes and Kaluza C analysis software (Beckman Coulter Life Sciences). Peripheral blood samples were processed using a stain/lyse/wash protocol. Cell concentrations were adjusted to 3–20 × 106/mL to ensure optimal antibody staining and the cells were washed three times before acquisition. Viability was assessed using 7AAD and CD45. Following morphological review of peripheral slide, 10-color analyses were performed for the following surface and cytoplasmic antigens: Kappa, Lambda, CD10, CD5, CD200, CD34, CD38, CD20, CD19, CD45, TCR alpha/beta, TCR delta/gamma, CD4, CD2, CD56, CD7, CD8, CD3, CD45, CD16, CD7, CD13, CD64, CD34, CD14, HLA-DR, CD11b, CD15, CD123, CD117, CD33, CD45, 6AC1: cy-TdT, cy-79a, CD22, 6AC2: cy-MPO, CD1a, cy-CD3. Cells were gated to exclude debris (forward scatter vs. side scatter and time of flight), to exclude cell doublets (forward scatter height vs. forward scatter width) and to isolate leukocyte populations (CD45 vs. side scatter). Primary analysis and quality control was performed by the flow cytometry supervisor. Final gating and reporting were performed by a board-certified hematopathologist.

TRCG-PCR

Genomic DNAs were extracted from bone marrow and/or peripheral blood samples using the QIAGEN DNeasy blood kit, as per the manufacturer's instructions. In routine clinical testing TCRG gene rearrangement testing by PCR was performed using an in-house laboratory developed assay using primers from Invivoscribe that are the BioMed II set (van Dongen et al. 2003) as described previously (Rothberg et al. 2012). In the research setting we retested all specimens using the Invivoscribe's T-cell receptor gamma gene rearrangement assay as per the manufacturer's instructions using an ABI 3500× capillary electrophoresis system for result readout.

Chimerism Testing

Genomic DNA were extracted from bone marrow and/or peripheral blood samples using the QIAGEN DNeasy blood kit, as per the manufacturer's instructions. Chimerism testing was done using the short tandem repeat polymorphic markers D11S554 and ACTBP8 (SE33), resolved by capillary electrophoresis on the ThermoFisher 3500XL genetic analyzer and analyzed using GeneMapper software.

NGS Testing by Illumina TruSight Panel

Genomic DNA were extracted from bone marrow or peripheral blood samples using the QIAGEN DNeasy blood and tissue kit per the manufacturer's instructions (QIAGEN). Sequencing libraries were prepared for sequencing on the Illumina TruSightTM Myeloid sequencing panel per the manufacturers’ protocols. The enriched DNA libraries were sequenced on an Illumina MiSeq instruments (version 3 chemistry, 300 base pair [bp] paired-end reads; Illumina, Inc.) (TruSight Myeloid panel). FASTQ files were processed through vendor-provided bioinformatics pipelines. Variant call files (vcfs) were filtered to remove subthreshold calls with <500× coverage and/or VAF less than defined, validated thresholds ranging from 1%–5%, depending on the type of mutation, as follows: 5% for single-nucleotide variants (SNVs); 1% for insertion-deletion mutations <3 bp; and 5% for insertion-deletion mutations 3 bp or larger. Clinically relevant mutations from this VAF were annotated by a board-certified molecular genetic pathologist manually and reported. Sequenced regions (i.e., mutational hotspot regions, consisting of indicated exons) of the clinically ordered gene set for this patient on the TruSight panel were as follows: ASXL1 (NM_015338.5): 12; BCOR (NM_001123385.1): all; BRAF (NM_004333.4): 15; CBL (NM_005188.3): 8,9; CSF3R (NM_156039.3): 14–17; DNMT3A (NM_022552.4): all; ETV6 (NM_001987.4): all; EZH2 (NM_004456.4): all; FBXW7 (NM_033632.3): 9–11; FLT3 (NM_004119.2): 14,15,20; GATA1 (NM_002049.3): 2; GATA2 (NM_032638.4): 2–6; IDH1 (NM_005896.2): 4; IDH2 (NM_002168.2): 4; JAK2 (NM_004972.3): 12,14; KIT (NM_000222.2): 2,8–11,13,17; KRAS (NM_033360.2): 2,3; MPL (NM_005373.2): 10; MYD88 (NM_002468.4): 3–5; NOTCH1 (NM_017617.3): 26–28,34; NPM1 (NM_002520.6): 12; NRAS (NM_002524.4): 2,3; PHF6 (NM_032458.2): all; PTPN11 (NM_002834.3): 3,13; RUNX1 (NM_001754.4): all; SETBP1 (NM_015559.2): 4; SF3B1 (NM_012433.2): 13–16; SRSF2 (NM_001195427.1): 1; STAG2 (NM_001042749.1): all; TET2 (NM_001127208.2): 3–11; TP53 (NM_000546.5): 2–11; U2AF1 (NM_001025203.1): 2,6; WT1 (NM_024426.4): 7,9; ZRSR2 (NM_005089.3): all.

NGS Testing by OncoPlus (v7.1.0) Panel

Genomic DNA was isolated as above. DNA was quantified using the Qubit fluorometric assay (Thermo Fisher Scientific). DNA was subjected to ultrasonic fragmentation and subsequent library preparation using adapter molecules containing patient-specific index sequences (HTP Library Preparation Kit, Kapa Biosystems). After library amplification, quantification and pooling, fragments originating from targeted genomic regions were enriched using a panel of biotinylated oligonucleotides (xGen Lockdown probes, IDT) supplemented with additional probes to assist with genome-wide copy-number assessment (xGen CNV Backbone Panel, IDT). After subsequent amplification and pooled library quantification, libraries were sequenced in rapid run mode on a NovaSeq 6000 system (Illumina) to produce 2 × 101-bp paired-end sequencing reads. Sequencing data was analyzed via custom-designed bioinformatics pipelines on a University of Chicago HIPAA compliant high performance computing system, using the hg19 (GRCh37) human genome reference sequence for alignment. For mutations, insertions, and deletions, limit of detection is 10% mutant alleles (roughly corresponding to 20% tumor cells). Limit of detection for fusions/translocations is 20% tumor cells. Gene fusions cannot be detected in the rare occurrence of a fusion between ALK, RET, or ROS1 and a partner gene <100,000 bp distant. Limit of detection for copy-number changes is >4× or <0.5× normal copy number, with relevant equivocal changes reported at >2× or <0.6×. For copy-number calling, sensitivity depends on the overall copy-number alteration in tumor cells as well as the proportion of malignant cells in the specimen. All copy-number results are gene-based only; the assay is not validated to report cytogenetic (larger scale) gains and losses. This test evaluates for mutations in tumor tissue only and is not capable of concretely discriminating germline from somatic events. Germline variants may be detected by the assay, but the clinical test is not intended for definitive diagnosis of germline mutations. Full coding regions of the following genes were analyzed for mutations and insertions/deletions: ABL1, AKT1, ALK, APC, ARID1A, ARID2, ASXL1, ATM, ATR, ATRX, AXL, B2M, BAP1, BCOR, BCORL1, BIRC3, BLM, BRAF, BRCA1, BRCA2, BTK, CALR, CBL, CBLB, CCND1, CCND2, CCND3, CCNE1, CD274, CDH1, CDK4, CDK6, CDK12, CDKN2A, CEBPA, CHEK1, CHEK2, CSF1R, CSF3R, CTCF, CTNNA1, CTNNB1, CUX1, CXCR4, DAXX, DDR2, DDX3X, DDX41, DICER1, DNMT3A, EGFR, ELOC, EP300, EPHA3, EPHA5, ERBB2, ERBB3, ERBB4, ERCC3, ESR1, ETV6, EZH2, FANCA, FAT3, FBXW7, FGFR1, FGFR2, FGFR3, FH, FLT3, FOXL2, GATA1, GATA2, GNA11, GNAQ, GNAS, GRIN2A, H3-3A, H3C2, H3C3, HNF1A, HRAS, IDH1, IDH2, IKZF1, ITPKB, JAK2, KDM6A, KDR, KEAP1, KIT, KMT2A, KRAS, MAP2K1, MAPK1, MDM2, MET, MEN1, MLH1, MLH3, MPL, MRE11, MSH2, MSH6, MTOR, MYC, MYCN, MYD88, NBN, NF1, NF2, NFE2L2, NOTCH1, NOTCH2, NPM1, NRAS, NTRK1, NTRK2, NTRK3, PALB2, PAX5, PBRM1, PDGFRA, PDGFRB, PHF6, PIK3CA, PIK3CB, PIK3R1, PLCG2, POLE, POT1, PPP2R1A, PTCH1, PTEN, PTPN11, RAD21, RAD50, RAD51, RB1, RET, ROS1, RUNX1, SDHB, SDHC, SDHD, SETBP1, SETD2, SF3B1, SMAD4, SMARCB1, SMC1A, SMC3, SMO, SRSF2, STAG2, STAT3, STAT5B, STK11, TERT, TET2, TP53, TRAF7, TSC1, TSC2, U2AF1, VHL, WT1, ZRSR2.

ADDITIONAL INFORMATION

Data Deposition and Access

The consent documentation signed by the patient does not expressly allow submission of full sequencing data (FASTQ, BAM/BAI, VCF) to external data repositories. The variants were deposited to ClinVar and can be found under accession numbers SCV003840279–SCV003840281.

Ethics Statement

The patient signed the institution-approved standard consent for clinical diagnostic testing by NGS, including agreement to the opt in/out clause for use of genetic and other diagnostic information for research purposes. This consent mechanism does not allow for sharing of genetic and other diagnostic information beyond that clinically relevant and reported in the manuscript.

Acknowledgments

We thank Bahadir Yildiz and Omar Aljitawi (University of Rochester) for discussion, and three anonymous reviewers for their highly valuable comments on the manuscript.

Author Contributions

S.E.H. and Z.N.O. conceptualized the project, reviewed and analyzed the data, created the figures and tables, and wrote the manuscript with input from A.G.E., A.N.J., P.G.R., and J.L.L. The chimerism, TCRG-PCR, and NGS experiments were performed by N.L., J.M.F., and M.B., respectively. The expanded panel NGS was performed and interpreted by J.P.S. All authors read the manuscript and approved its final version.

Competing Interest Statement

The authors have declared no competing interest.

Supplementary Material

Supplemental Material
supp_9_2_a006241__DC1.html (1.1KB, html)

Footnotes

[Supplemental material is available for this article.]

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental Material
supp_9_2_a006241__DC1.html (1.1KB, html)
supp_mcs.a006241_Supplemental_Figure_3.pdf (408.3KB, pdf)
supp_mcs.a006241_Supplemental_Figure_4.pdf (201.9KB, pdf)
supp_mcs.a006241_Supplemental_Figure_1.jpg (177KB, jpg)
supp_mcs.a006241_Supplemental_Figure_2.jpg (110.6KB, jpg)

Data Availability Statement

The consent documentation signed by the patient does not expressly allow submission of full sequencing data (FASTQ, BAM/BAI, VCF) to external data repositories. The variants were deposited to ClinVar and can be found under accession numbers SCV003840279–SCV003840281.

Articles from Cold Spring Harbor Molecular Case Studies are provided here courtesy of Cold Spring Harbor Laboratory Press

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