Leukocytosis with left-shifted myeloid maturation in a peripheral blood specimen: a clue to the lymphoid blast phase of CML

Abstract Background: The Philadelphia chromosome negative myeloproliferative neoplasms (MPN) includePolycythemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF). These stem cell disorders carry a propensity to evolve into acute myeloid leukemia (MPN-blast phase [BP] or post-MPN AML) with a dismal prognosis not meaningfully improved by conventional anti-leukemia therapy. Thus, MPN-BP is an urgent unmet clinical need. Responses in patients with MPN-BP to hypomethylating agents and single agent ruxolitinib have been reported. More recently, combination of ruxolitnib and decitabine has demonstrated synergistic activity in vitro in cells derived from patients with MPN-BP and from a murine model of MPN-BP (Rampal et al PNAS 2014). These observations led us to explore the safety of combined decitabine and dose escalation of ruxolitinib in MPN-BP. Objective: To establish the maximum tolerated dose (MTD) of ruxolitinib in combination with a fixed dose of decitabine (DEC-RUX). Methods: We conducted an open label Phase I trial in patients with MPN acceleration phase (AP) as defined by 10%-19% blasts in the peripheral blood or bone marrow or a diagnosis of MPN-BP as defined by ≥ 20% blasts in the blood or bone marrow, following a previous diagnosis of ET, PV or PMF. Patients were enrolled in a standard 3+3 phase I design with an MTD defined as a dose <33% DLT. Ruxolitinib was administered at doses of 10mg, 15mg, 25mg, or 50mg every 12 hours in combination with concurrent decitabine at a dose of 20mg/m2 daily intravenously over 5 days and repeated every 28 days. Adverse events were assessed using the NCI CTCAE v. 4.0. DLTs were defined as Grade 3 or higher non-hematologic toxicity events not clearly related to disease and grade 4 hematologic events with a bone marrow cellularity of ≤5% and no evidence of leukemia. Response assessment was carried out every cycle using modified Cheson criteria: CR required 0% peripheral blood blasts, WBC ≥4x109/L, hemoglobin ≥10g/L, and platelets ≥100x109/L; CRi required 0% peripheral blood blasts with incomplete count recovery; and PR required ≥50% decrease in peripheral blood blasts regardless of blood counts. Results: A total of 21 patients were accrued to study (Table 1). The median age was 63 years (range 48-88). 52% carried a diagnosis of MPN-AP, and 48% carried a diagnosis of MPN-BP. 29% of patients and 24% of patients had prior exposure to ruxolitinb and decitabine, respectively. The median number of cycles received varied from 10.5 cycles in the 10mg BID cohort to 2 and 2.5 cycles in the 25mg BID and 50mg BID cohorts, respectively (Table 2). The most common Grade 3/4 non-hematologic AEs observed were due to infection in all dosing cohorts. In terms of hematologic toxicity, treatment emergent Grade 3/4 anemia was observed in 1 patient in each of the 10mg BID, 15mg BID, and 50mg BID cohorts. Grade 3/4 leukopenia was observed in only 1 patient at the 50mg BID cohort, and Grade 3/4 thrombocytopenia was observed in 2 patients in the 10mg BID cohort and 1 patient in the 15mg BID cohort. DLT rate was below 33% for all dose levels so the MTD was not reached. The most common reason for ending study treatment was toxicity/adverse events (33%) followed by disease progression (22%). 9 patients died during study or follow-up. Of those, 5 (56%, 2 in 10mg BID cohort, 1 in 15mg BID cohort, 2 in 50mg BID cohort) died of infection, 3 (33%, 1 in each of 10mg, 25mg, and 50mg BID cohorts) of progressive disease, and 1 (11%, 25mg BID cohort) of hemorrhage. The median overall survival for patients on study was 10.4 months (95% CI 3.3 mo - not reached). CR/CRi as best response was observed in 7/21 patients (33%, 95% CI 15-57%; 2 CR, 5 CRi; Table 2). Conclusions: DEC-RUX combination therapy was safely administered to patients with MPN-AP/BP and an MTD was not reached. Based on pre-clinical data, observed safety profile, duration of treatment, and clinical responses in this phase I trial, the Recommended Phase II Dose of RUX was selected as 25mg BID for an induction cycle followed by 10mg BID in all ensuing cycles. Molecular and bone marrow pathology responses will be presented at the meeting. Disclosures Mascarenhas: Promedior: Research Funding; CTI Biopharma: Research Funding; Novartis: Other: DSMB , Research Funding; Janssen: Research Funding; Roche: Research Funding; Incyte: Other: Clinical Trial Steereing Committee, Research Funding. Hexner:Blueprint medicines: Consultancy; Novartis: Research Funding. Abboud:Alexion: Honoraria; Takeda: Honoraria; Novartis: Research Funding; Teva: Research Funding, Speakers Bureau; Pfizer: Research Funding; Merck: Research Funding; Pharmacyclics: Honoraria; Baxalta: Honoraria; Seattle Genetics: Research Funding; Gerson and Lehman Group: Consultancy; Cardinal: Honoraria. Levine:Novartis: Consultancy; Qiagen: Membership on an entity's Board of Directors or advisory committees. Mesa:Promedior: Research Funding; Novartis: Consultancy; Incyte: Research Funding; Celgene: Research Funding; Galena: Consultancy; Ariad: Consultancy; Gilead: Research Funding; CTI: Research Funding.

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Clinical Evolution to Primary Myelofibrosis - Blast Phase: An International Working Group for Myelofibrosis Research and Treatment (IWG-MRT) Collaborative Retrospective Analysis.

Blood ◽

10.1182/blood.v110.11.682.682 ◽

2007 ◽

Vol 110 (11) ◽

pp. 682-682 ◽

Cited By ~ 2

Author(s):

Ruben A. Mesa ◽

Francisco Cervantes ◽

Srdan Verstovsek ◽

Constantine Tam ◽

Brigitte Dupriez ◽

...

Keyword(s):

Peripheral Blood ◽

Clinical Course ◽

Clonal Evolution ◽

Group Versus ◽

Primary Myelofibrosis ◽

Prognostic Scores ◽

Control Group ◽

Study Group ◽

Blast Phase ◽

Clinical Evolution

Abstract Background: We sought to retrospectively analyze the clinical differences, at diagnosis and throughout the disease course, between patients with primary myeloifbrosis whom undergo blastic transformation (PMF-BP) and those whom expire from PMF complications without undergoing transformation. Methods: An international collaborative database of patients with PMF which progressed to PMF BP, and a control group of individuals with PMF who expired secondary to PMF was created. Data regarding clinical course, bone marrow, karyotypic, quantitative JAK2V617F analysis (when able), laboratory values, and therapy at the diagnosis of PMF, PMF BP and up to 4 return visits (R1-4) in between these milestones was abstracted. Results: COMPARISON AT DIAGNOSIS OF PMF: 136 cases of PMF who eventually underwent PMF-BP were included, with a control group of 42 PMF patients, were analyzed. Both groups of patients had similar demographic (median age at diagnosis 59 years (range 23–83)/ 59.1 (15–93); and disease parameters (median hemoglobin 10 g/dL (range 2–15.7) /9.8 (4.9–14.6); median platelet count 181 x 109/L (range 7–1400)/168 (16–1916)), and Lille PMF prognostic scores (p = n.s.) for PMF-BP vs. PMF control group respectively. Additionally, blasts (peripheral blood /marrow) were a median of 0% (range 0–18%)/ 2% (range 0–16%) for the study group versus 0.25% (range 0–5%)/ 3% (range 0–4%) (p = n.s. for both) for the control group, respectively. Both groups had similar rates of requiring therapy (86%; 93%) and survival from diagnosis (median 34 months (range 2–441); 29 months (1–236)) (p = n.s.). However, lactate dehydrogenase (LDH) (although high in both groups) was higher at diagnosis (median 802 IU/L (range 83–10353) vs. 416 IU/L (124–2197)) (p=0.04), as well as an abnormal marrow karyotype (85% vs. 25%; p<0.001) amongst the PMF BP group. SUBSEQUENT CLINICAL COURSE: Analyzing the clinical evolution between groups demonstrated persistently higher LDH and worsening thrombocytopenia in the PMF-BP cohort (see figure). Decreasing Platelets Approaching PMF BP Decreasing Platelets Approaching PMF BP Peripheral blood blasts increased in both groups, but values above 10% were unique to the PMF BP group. Additionally marrow karyotype displayed clonal evolution developed in 56% of the PMF-BP patients as opposed to 14% of controls (p<0.001). Initial and serial quantitative JAK2V617F mutation analysis (available from 20 /19 patients from the PMF-BP and control group at diagnosis, with 11 / 12 serial samples) showed no difference between the groups, nor a pattern of mutation burden elevation with transformation to leukemia in the study group. Conclusions: Increasing LDH, peripheral blood blast percentages >10%, and clonal evolution are features commonly seen in PMF patients whom evolve to blast phase and may be a harbinger of movement towards PMF BP.

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Prevalence of Large Granular Lymphocyte Proliferation in Chronic Myeloid Leukemia (CML) Patients Treated with Dasatinib.

Blood ◽

10.1182/blood.v114.22.1111.1111 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1111-1111

Author(s):

Jason Valent ◽

Charles A. Schiffer

Keyword(s):

Flow Cytometry ◽

Side Effects ◽

Peripheral Blood ◽

Chronic Phase ◽

Cell Transplant ◽

Pleural Effusions ◽

Blast Phase ◽

Number Of Patients ◽

Dasatinib Treatment

Abstract Abstract 1111 Poster Board I-133 Dasatinib is a potent inhibitor of the BCR-ABL tyrosine kinase which is effective in the treatment of imatinib refractory CML. While hematologic toxicities of neutropenia and thrombocytopenia are well known, large granular lymphocytosis has been reported in only a small number of patients without prior allogeneic stem cell transplant treated with dasatinib for CML. During routine follow up of leukocyte counts in 15 consecutive patients (age range 27-77 years) treated with dasatinib, 4 patients (2 chronic-phase, 1 accelerated phase with clonal cytogenetic progression, 1 blast-phase) developed a lymphocytosis (> 3800/mm3). Peripheral blood smear and peripheral blood flow cytometry revealed a population of large granular lymphocytes (LGLs) expressing CD3, CD8, CD57, and variable expression of CD56. Lymphocytosis was first noted between 1 and 9 months after initiation of dasatinib and has persisted in 3 of the patients with a median follow up of 33 months from the onset of lymphocytosis. Peak absolute lymphocyte count ranged from 5000/mm3 to 6900/mm3 and approximately 40 to 60% of the lymphocytes were LGLs by flow cytometry with the remainder being predominantly T lymphocytes. These 4 patients with LGL lymphocytosis have all have major molecular responses and the patient with blast-phase has remained in a complete cytogenetic remission with a major molecular response 44 months after initiation of dasatinib. The 11 other patients (6 chronic-phase, 2 accelerated-phase, 3 blast-phase) treated with dasatinib for CML have not developed lymphocytosis. These patients have been followed for a median of 25 months (range 3-50 months) although some were treated with dasatinib for a relatively short period of time because of poor response of their advanced CML. Review of the peripheral blood smears from 3 of the 6 chronic phase patients without lymphocytosis who remain on dasatinib treatment did not reveal any LGLs. All of these 6 patients have had complete, sustained cytogenetic responses. A persistent pleural effusion developed in the blast phase patient with lymphocytosis approximately 12 months after lymphocytosis developed; no significant side effects were noted in the other 3 patients although one remains thrombocytopenic. Pleural effusions developed in 2 of the 6 patients without lymphocytosis who remain on dasatinib treatment. Previous reports have suggested an increased incidence of “inflammatory” type side effects such as pleural effusions and pneumonitis in patients with dasatinib related LGL proliferation, although the small number of patients in this series precludes analysis of this association. In summary, LGL proliferation was detected in a minimum of 27% of dasatinib recipients and may be associated with a beneficial response. While the mechanism of LGL proliferation has not been fully explained, it has been suggested that dasatinib mediated inhibition of immunoregulatory kinases such as Src is permissive of LGL proliferation. Further evaluation of the frequency and clinical impact of this phenomenon in the large clinical trials of patients treated with dasatinib is warranted. Disclosures Schiffer: Bristol Myers: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Mitigation of Channel Clogging in a Microfluidic Device for Capturing Circulating Tumor Cells

International Journal of Automation Technology ◽

10.20965/ijat.2020.p0109 ◽

2020 ◽

Vol 14 (1) ◽

pp. 109-116

Author(s):

Tomoki Konishi ◽

Yuki Jingu ◽

Tatsuya Yoshizawa ◽

Masaru Irita ◽

Toshihiro Suzuki ◽

...

Keyword(s):

Platelet Aggregation ◽

Tumor Cells ◽

Circulating Tumor Cells ◽

Propidium Iodide ◽

Peripheral Blood ◽

Lateral Displacement ◽

Microfluidic Devices ◽

Cell Detachment ◽

Deterministic Lateral Displacement ◽

Blood Specimen

Deterministic lateral displacement (DLD) based microfluidic devices have been developed for capturing circulating tumor cells (CTCs) from the peripheral blood. There was frequent and problematic channel clogging around the micro-post array formed on a microchannel of the device. In this study, various agents were dispersed into the blood specimen to avoid clogging. At first, platelet aggregation was considered to be the cause of the clogging, but even plasmin, which was assumed to decompose platelet aggregations, did not show obvious inhibition of the clogging. Then, enzymes used for cell detachment from tissue were examined and decomposition of the clogging residue was observed. Finally, dispersion of deoxyribonuclease into a blood specimen was found to be effective for the inhibition of clogging. The existence of DNA in the clogging residue was also confirmed by propidium iodide (PI) staining, suggesting DNA adhering to the micro-post.

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Validation of Next Generation Sequencing-Based Clonality Assays for Diagnosis and Minimal Residual Disease Tracking in Lymphoid Malignancies

Blood ◽

10.1182/blood-2021-150151 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2959-2959

Author(s):

Haiyan Wu ◽

Feng Li ◽

Jiacheng Cai ◽

Ke Zhang ◽

Xin Zheng ◽

...

Keyword(s):

Cell Lines ◽

Peripheral Blood ◽

Residual Disease ◽

Clinical Samples ◽

Blast Phase ◽

Lymphoid Malignancies ◽

Mixed Reaction ◽

Generation Sequencing ◽

Clonality Assessment ◽

Minimal Residual

Abstract Introduction: B or T cell receptor (BCR/TCR) clonal rearrangements have served as important diagnostic markers and minimal residual disease (MRD) tracking markers to guide treatment decisions in lymphoid malignancies. Next generation sequencing (NGS)-based BCR/TCR clonality assessment allows easier sample preparation, higher sensitivity and simpler standardization than flow cytometry and polymerase chain reaction (PCR)-based assays. Here, we developed NGS-based BCR/TCR clonality assays to identify and track disease-associated clonotypes of IGH, IGK, IGL, TCRB and TCRG rearrangements and BCL1/2-IGH translocations in lymphoid malignant cells. Our studies validated the analytical performance of the assays using genomic DNA (gDNA) from cell lines and patient samples diagnosed with acute lymphoblastic leukemia (ALL), multiple myeloma (MM), chronic lymphocytic leukemia (CLL), lymphoma and lymphoid blast phase chronic myeloid leukemia (BP-CML), as well as peripheral blood gDNA and circulating tumor DNA (ctDNA) from lymphoma patients. Methods: Our BCR/TCR clonality assays were based on two rounds of multiplex PCR followed by NGS. In the first-round PCR, the sequences of complementary determining region 3 (CDR3) in rearranged immune receptor genes were amplified by multiplex primers, and sample-specific index and NGS adapters were then added in the second-round PCR. Sequencing was performed on NovaSeq 6000 System and processed with customized bioinformatics pipelines. BCR clonality assays could identify IGH (V H-D H-J H or D H-J H), IGK (V κ-J κ, V κ-Kde and intronRSS-Kde) and IGL (V λ-J λ) rearrangements, as well as BCL1-IGH and BCL2-IGH translocations. TCR clonality assays could detect TCRB (V β-D β-J β) and TCRG (V γ-J γ) rearrangements. To evaluate the performance of our assays, we detected BCR or TCR clonality in gDNA from 4 cell lines and 40 clinical samples with ALL, MM, CLL and lymphoma, as well as paired chronic- and blast-phase samples of CML. Limit-of-detection (LOD) was estimated by clinical samples and cell lines with a background of peripheral blood gDNA from healthy donors. Linearity of detection was established with gDNA of cell lines spiked into normal gDNA to generate across orders of magnitude of clonal frequencies. To ensure consistent performance of the assays, we tested separate reactions and a single mixed reaction for IGH VDJ, IGH DJ, IGK and IGL. In addition, peripheral blood gDNA matching aforementioned lymphoma cases and ctDNA samples were also tested by our assays. Results: Both BCR clonality of B-cell lymphoid malignancies (B-ALL, MM, CLL and lymphoma) and TCR clonality of T-cell lymphoid malignancies (T-ALL and lymphoma) showed above 90% positive detection rate and mostly positive in more than one receptor gene. Chronic and blast phase samples from the same CML patient showed an identical dominant clonotype. The assays had high sensitivity, with LoD defined between 1 to 2 malignant cells in both BCR and TCR clonality assessment. Linearity was observed with clonal frequencies from 1 to 10 -6, which indicated consistence between observed and expected frequencies. The sequencing results with a single adjusted mixed reaction for IGH VDJ, IGH DJ, IGK and IGL were comparable to that with separate reactions, suitable for both diagnosed samples and MRD samples, suggesting the robustness of the assays. Our testing results also showed that peripheral blood gDNA of lymphoma patients carried identical clonotypes found in malignant tissues and ctDNA. Conclusion: We characterized and validated the performance of our NGS -based BCR/TCR clonality assays. We also demonstrated its potential application as a highly sensitive tool for diagnosis and MRD tracking for lymphoid malignancies, including ALL, MM, CLL, lymphoma and even CML at risk of lymphoid blast transformation. Disclosures No relevant conflicts of interest to declare.

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The Growth of Herpesvirus mulatto in Human Fibroblast

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100051232 ◽

1974 ◽

Vol 32 ◽

pp. 244-245

Author(s):

Glennelle Washington ◽

Philip P. McGrath ◽

Peter R. Graze ◽

Ivor Royston

Keyword(s):

Rhesus Monkey ◽

Microscopic Study ◽

Electron Microscopic Study ◽

Peripheral Blood ◽

Human Fibroblast ◽

Rhesus Monkeys ◽

Human Fibroblasts ◽

Electron Microscopic ◽

Specific Antisera ◽

Peripheral Blood Leucocytes

Herpes-like viruses were isolated from rhesus monkey peripheral blood leucocytes when co-cultivated with WI-38 cells. The virus was originally designated rhesus leucocyte-associated herpesvirus (LAHV) and subsequently called Herpesvirus mulatta (HVM). The original isolations were from juvenile rhesus monkeys shown to be free of antibody to rhesus cytomegalic virus. The virus could only be propagated in human or simian fibroblasts. Use of specific antisera developed from HVM showed no relationship between this virus and other herpesviruses. An electron microscopic study was undertaken to determine the morphology of Herpesvirus mulatta (HVM) in infected human fibroblasts.

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A Quantitative Ultrastructural Study of Peripheral Blood Lymphocytes Containing Parallel Tubular Arrays in Epstein-Barr Virus and Cytomegalo-Virus Mononucleosis

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100098769 ◽

1981 ◽

Vol 39 ◽

pp. 454-455

Author(s):

C. M. Payne ◽

P. M. Tennican

Keyword(s):

Peripheral Blood ◽

Lymphocyte Count ◽

Epstein Barr Virus ◽

Absolute Lymphocyte Count ◽

Peripheral Blood Lymphocytes ◽

Clinical State ◽

Barr Virus ◽

The Absolute ◽

Epstein Barr ◽

Tubular Arrays

In the normal peripheral circulation there exists a sub-population of lymphocytes which is ultrastructurally distinct. This lymphocyte is identified under the electron microscope by the presence of cytoplasmic microtubular-like inclusions called parallel tubular arrays (PTA) (Figure 1), and contains Fc-receptors for cytophilic antibody. In this study, lymphocytes containing PTA (PTA-lymphocytes) were quantitated from serial peripheral blood specimens obtained from two patients with Epstein -Barr Virus mononucleosis and two patients with cytomegalovirus mononucleosis. This data was then correlated with the clinical state of the patient.It was determined that both the percentage and absolute number of PTA- lymphocytes was highest during the acute phase of the illness. In follow-up specimens, three of the four patients' absolute lymphocyte count fell to within normal limits before the absolute PTA-lymphocyte count.In one patient who was followed for almost a year, the absolute PTA- lymphocyte count was consistently elevated (Figure 2). The estimation of absolute PTA-lymphocyte counts was determined to be valid after a morphometric analysis of the cellular areas occupied by PTA during the acute and convalescent phases of the disease revealed no statistical differences.

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Neutrophil pseudoplatelets in subgingival plaque and crevicular fluid samples from periodontal diseased sites

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100156298 ◽

1989 ◽

Vol 47 ◽

pp. 862-863 ◽

Cited By ~ 1

Author(s):

J Hanker ◽

E.J. Burkes ◽

G. Greco ◽

R. Scruggs ◽

B. Giammara

Keyword(s):

Electron Microscopy ◽

Bone Marrow ◽

Peripheral Blood ◽

Gingival Crevicular Fluid ◽

Light And Electron Microscopy ◽

Subgingival Plaque ◽

Staining Reaction ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Crevicular Fluid

The mature neutrophil with a segmented nucleus (usually having 3 or 4 lobes) is generally considered to be the end-stage cell of the neutrophil series. It is usually found as such in the bone marrow and peripheral blood where it normally is the most abundant leukocyte. Neutrophils, however, must frequently leave the peripheral blood and migrate into areas of infection to combat microorganisms. It is in such areas that neutrophils were first observed to fragment to form platelet-size particles some of which have a nuclear lobe. These neutrophil pseudoplatelets (NPP) can readily be distinguished from true platelets because they stain for neutrophil myeloperoxidase. True platelets are not positive in this staining reaction because their peroxidase Is inhibited by glutaraldehyde. Neutrophil pseudoplatelets, as well as neutrophils budding to form NPP, could frequently be observed in peripheral blood or bone marrow samples of leukemia patients. They are much more prominent, however, in smears of inflammatory exudates that contain gram-negative bacteria and in gingival crevicular fluid samples from periodontal disease sites. In some of these samples macrophages ingesting, or which contained, pseudoplatelets could be observed. The myeloperoxidase in the ingested pseudoplatelets was frequently active. Despite these earlier observations we did not expect to find many NPP in subgingival plaque smears from diseased sites. They were first seen by light microscopy (Figs. 1, 3-5) in smears on coverslips stained with the PATS reaction, a variation of the PAS reaction which deposits silver for light and electron microscopy. After drying replicate PATS-stained coverslips with hexamethyldisilazane, they were sputter coated with gold and then examined by the SEI and BEI modes of scanning electron microscopy (Fig. 2). Unstained replicate coverslips were fixed, and stained for the demonstration of myeloperoxidase in budding neutrophils and NPP. Neutrophils, activated macrophages and spirochetes as well as other gram-negative bacteria were also prominent in the PATS stained samples. In replicate subgingival plaque smears stained with our procedure for granulocyte peroxidases only neutrophils, budding neutrophils or NPP were readily observed (Fig. 6).

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