Normal Peripheral Blood Leukocytes Research Articles

Abstract 3454: Cell-free DNA methylation and fragmentomic signatures identify tissue damage and predict cancer risk

Abstract Cell-free DNA (cfDNA) epigenetic and fragmentomic profiling has emerged as prominent non-invasive approaches for early cancer detection and subtyping. However, owing to difficulties in observing the early development of human malignancies, most cancer biomarker and evolution studies to date have primarily examined biologics following a diagnosis. Utilizing cfDNA as a screening tool for early disease detection requires assessment of blood plasma samples collected from asymptomatic individuals prior to the diagnosis of cancers. Here, we leverage the Ontario Health Study (OHS), a prospective longitudinal population cohort, to assess cfDNA profiles in blood plasma collected from participants that developed breast (n=476), prostate (n=342) and pancreatic (n=32) cancers throughout the study follow up time between 2009 and 2019. We performed cell-free methylated DNA immunoprecipitation and high-throughput sequencing (cfMeDIP-seq) on baseline plasma samples from incident cancer cases up to eight years prior to diagnosis, in addition to matched controls (n=404) with no history of cancer through follow-up. Genome-wide plasma cfDNA differential methylation analysis revealed significantly hypermethylated regions, particularly among CpG island and shore regions, were predictive of early breast cancers, and overlapped hypermethylated regions in solid cancer tissues relative to adjacent normal tissues and peripheral blood leukocytes. Using a repeated cross-validation strategy we found that as few as 200 hypermethylated regions can identify individuals with breast cancer in blood up to seven years prior to diagnosis among discovery set samples (AUC=0.725) and are highly generalizable to samples processed in separate batches (AUC = 0.650). Remarkably, integrating biomarker hypermethylated regions with fragmentomic features including fragment length and 5’ tetranucleotide motif proportions further increased accuracy for detecting individuals with early prostate cancers (AUC=0.804). In addition, we observed shared genome-wide cfDNA methylome changes associated with aging and heavy alcohol consumption in cfDNA. We identified increased shedding of tissue-specific methylation markers from liver and pancreatic tissue among individuals consuming more than three drinks a week relative to non-drinkers, reflective of potential signatures of tissue damage. Citation Format: Nicholas Cheng, Kimberly Skead, Tom Oullette, Scott Bratman, Daniel De Carvalho, David Soave, Philip Awadalla. Cell-free DNA methylation and fragmentomic signatures identify tissue damage and predict cancer risk [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3454.

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.

Clinical characteristics of 18 children with chronic nonbacterial osteomyelitis

Objective: To investigate the clinical features of children with chronic nonbacterial osteomyelitis (CNO), and raise awareness among clinicians. Methods: In this retrospective study, 18 patients with CNO who were diagnosed in Children's Hospital of Fudan University from January 2015 to December 2021 were included. Results: Eighteen children with CNO (12 males, 6 females) were identified. Their age at onset was 9 (5, 11) years, the delay in diagnosis was 2 (1, 6) months, and follow-up-was 17 (8, 34) months. The most common symptoms were fever in 14 children, as well as bone pain and (or) arthralgia in 14 children. In terms of laboratory results, normal white blood cell counts were observed at onset in 17 patients; increased erythrocyte sedimentation rate (ESR) in all patients; increased C reactive protein (CRP) over the normal value in 14 patients. Of the 18 patients, 2 had positive antinuclear antibodies, while none had positive human leukocyte antigen-B27 or rheumatoid factor. Imaging examination revealed that all the patients had symmetrical and multifocal skeletal lesions. The number of structural lesions detected by imaging investigation was 8 (6, 11). The most frequently affected bones were tibia in 18 patients and femur in 17 patients. Bone biopsy was conducted in 14 patients and acute or chronic osteomyelitis manifested with inflammatory cells infiltration were detected. Magnetic resonance imaging (MRI) found bone lesions in all the patients and bone scintigraphy were positive in 13 patients. All the patients were treated with nonsteroidal anti-inflammatory drugs, among whom 10 cases also treated with oral glucocorticoids, 9 cases with traditional disease modifying anti-rheumatic drugs, 8 cases with bisphosphonates and 6 cases with tumor necrosis factor inhibitors. The pediatric chronic nonbacterial osteomyelitis disease activity score, increased by 70% or more in 13 patients within the initial 6-month follow-up. Conclusions: The clinical manifestations of CNO are lack of specificity. The first symptom of CNO is fever, with or without bone pain and (or) arthralgia, with normal peripheral blood leukocytes, elevated CRP and (or) ESR. Whole body bone scanning combined with MRI can early detect osteomyelitis at subclinical sites, and improve the diagnostic rate of CNO.

Abstract 3385: Pre-diagnosis plasma cell-free DNA methylation profiling reveals signatures of cancers up to7 years prior to clinical detection

Abstract Cancer survival rates are significantly improved when detected at early stages, particularly when the tumour is still localised to the tissue of origin. However, effective screening tools for early cancer detection is currently limited to a subset of cancer types. Profiling cell-free DNA (cfDNA) patterns has emerged as a prominent non-invasive biomarker for detection and subtyping of cancers. However, owing to difficulties in observing the early development of human malignancies as cancers are often detected once they become symptomatic, most cancer biomarker and evolution studies to date have primarily examined the genomics from solid tumour or liquid biopsies following a diagnosis. Utilizing cfDNA as a screening tool for early cancer detection requires profiling of blood plasma samples collected from asymptomatic individuals prior to the diagnosis of cancers to enable assessment of the earliest detectability and predictive performance of potential biomarkers. Here, we leverage the Canadian Partnership for Tomorrow’s Health Project (CanPaTH), to profile blood plasma collected prior to the clinical detection of underlying cancers. Specifically, we use cell-free methylated DNA immunoprecipitation and high-throughput sequencing (cfMeDIP-seq), a highly sensitive assay for profiling cfDNA methylomes, to profile over 300 blood plasma samples collected up to seven years prior to the detection of a breast, prostate or pancreatic cancer, in addition to matched controls with no history of cancer free through follow-up. We identified differentially methylated signatures in pre-diagnosis cfDNA that discriminated cancer-free controls from pre-diagnosis cancer cases up to seven years before diagnosis, and demonstrated that these markers were reflective of differentially methylated in cancer tissue relative to normal tissue and peripheral blood leukocytes. Further, predictive modelling reveals that cfDNA methylation markers in blood are predictive of pre-diagnosis breast cancer cases, achieving an average test AUROC of 0.75 (95% CI: 0.70 - 0.80). Predictive models trained solely with pre-diagnosis cfDNA methylation samples were also predictive of prostate and pancreatic cancer samples collected following diagnosis, achieving an average test AUROCs of 0.95 (95% CI: 0.93-0.96) and 0.96 (95% CI: 0.94-0.97) respectively. In our current studies, we focus specifically on breast, prostate and pancreatic cancer cases, and are extending this to further pan-cancer applications in subsequent investigations. Citation Format: Nicholas Cheng, David Soave, Kimberly Skead, Tom Ouellette, Scott Bratman, Daniel De Carvalho, Philip Awadalla. Pre-diagnosis plasma cell-free DNA methylation profiling reveals signatures of cancers up to7 years prior to clinical detection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3385.

Paclitaxel induces Stathmin 1 phosphorylation, microtubule stability and apoptosis in acute lymphoblastic leukemia cells.

Acute lymphoblastic leukemia (ALL) is a hematological malignancy characterized by abnormal proliferation and accumulation of lymphoblasts in the hematopoietic system. Stathmin 1 is a proliferation marker for normal lymphocytes, which has been described as highly expressed in ALL patients and functionally important for leukemia phenotype. In the present study, we expand our previous observations and aim to investigate Stathmin 1 expression and its impact on laboratory features and clinical outcomes in an independent cohort of ALL patients, and to verify the effects of paclitaxel treatment on Stathmin 1 phosphorylation and cell viability in ALL cell lines. In ALL patients, Stathmin 1 expression was significantly increased, associated with lower age onset and positively correlated with white blood cell counts, but did not impact on clinical outcomes. Functional assays revealed that paclitaxel induces Stathmin 1 phosphorylation at serine 16 (an inhibitory site), microtubule stability and apoptosis in Jurkat and Namalwa cell lines. Paclitaxel treatment did not modulate cell viability of normal peripheral blood leukocytes. In conclusion, our data confirm increased levels of Stathmin 1 in ALL patients and that therapeutic doses of paclitaxel inhibits Stathmin 1 function and promote microtubule stability and apoptosis in ALL cells.

Tracking the interplay between circulating and tumor-infiltrating T cells using AmpliSeq-based Ion Torrent TCRβ immune repertoire sequencing.

e14518 Background: TCRβ immune repertoire analysis by next-generation sequencing is emerging as a valuable tool for research studies of the tumor microenvironment and potential immune responses to future cancer immunotherapy. Here we describe a multiplex PCR-based TCRβ sequencing assay that takes advantage of the exceptionally low base-call error rate and long read capability of the Ion S5 530 chip. The S530 chip provides &gt; 15M reads, allowing for sample sequencing in multiplex. The research assay utilizes AmpliSeq technology and Framework 1 and Constant gene targeting primers to provide comprehensive coverage of TCRβ CDR 1, 2 and 3 from RNA input with a hands-on time of less than 30 minutes. CDR region sequences reveal the antigen specificity of the receptor and may be genetically modified to increase TCR affinity for tumor antigen. Methods: We evaluated assay performance by sequencing TCRβ rearrangements from normal donor peripheral blood leukocyte (PBL) cDNA that had been spiked with 30 reference rearrangements taken from the literature. We then used our assay to evaluate the extent of clonal overlap between matched tumor infiltrating lymphocyte (TIL) and peripheral blood leukocyte repertoires in an individual with squamous cell carcinoma of lung. Results: Results from sequencing of spike-in reference rearrangements indicate that the assay is accurate and sensitive over 5 logs of input template amount while showing minimal amplification bias. Sequencing of matched PBL and TIL repertoires revealed that a subset of the PBL repertoire (8%) consisted of clones also found in TIL. Technical replicates showed high concordance (r &gt; .96) in the frequency of detected clones, indicating that the results were reproducible and samples were sequenced to an appropriate depth. Conclusions: In summary, these data suggest that AmpliSeq-based multiplex PCR and Ion Torrent sequencing provide unbiased, reproducible, scalable, complete, and accurate information for immune repertoire research sequencing applications.

Uptake of lymphoma-derived exosomes by peripheral blood leukocytes.

Exosomes are nanosized lipid vesicles secreted into blood and other body fluids and serve as vehicles for intercellular communication. Despite being an important component of the tumor microenvironment (TME), exosomal targeting and uptake into recipient cells are still not fully understood. Few studies have looked at lymphoma exosomes and their interactions with circulating blood cells. In this study, we examine the exosomal uptake distribution among peripheral blood leukocytes (PBLs) using vesicles derived from a diffuse large B cell lymphoma cell line, WSU-DLCL2. Lymphoma cells survive, proliferate, and are protected from the cytotoxic effects of chemotherapeutic agents by soluble factors or by direct contact with inflammatory and stromal cells within the TME. In an attempt to close the gap in knowledge concerning lymphoma TME immunosuppression, we have treated normal human PBLs with PKH67-labeled lymphoma exosomes and monitored the uptake by measuring fluorescence at different time points using flow cytometry and fluorescent microscopy. Our results show that of the four populations examined, B cells and monocytes demonstrated uptake of PKH67-labeled exosomes, while T cells and NK cells displayed significantly less uptake.

Stearidonic acid, a plant-based dietary fatty acid, enhances the chemosensitivity of canine lymphoid tumor cells

Lymphoma is the most common hematopoietic tumor in dogs and humans, with similar pathogenesis and therapeutic responses. Anticancer drugs like vincristine (VCR) and doxorubicin (DOX) are often used in treating lymphoma. However, the cure rate is generally poor due to chemoresistance. Here, we sought to determine whether stearidonic acid (SDA), a plant-based dietary fatty acid, sensitizes chemoresistant canine lymphoid-tumor cells. GL-1 B-cell lymphoid-tumor cells were found to be highly sensitive to the antitumor-activity of VCR and DOX, while OSW T-cell and 17-71 B-cell lymphoid-tumor cells were moderately and fully resistant, respectively. SDA, at its non-toxic concentrations, significantly promoted the antitumor action of VCR and DOX in both OSW and 17-71 cells. SDA-mediated chemosensitization was associated with SDA inhibition of P-glycoprotein (P-gp) function. This was confirmed in HEK293 cells stably expressing P-gp as well as by increased binding-affinity of SDA to P-gp in P-gp docking analysis. SDA at its chemosensitizing concentrations did not affect the viability of healthy dog peripheral blood mononuclear cells, suggesting that SDA is non-toxic to normal dog peripheral blood leucocytes at its chemosensitizing concentrations. Our study identifies a novel dietary fatty acid that may be used as a dietary supplement in combination with chemotherapy to promote the antitumor efficacy of the chemotherapy drugs in dogs and possibly in humans with chemoresistant lymphoma.

Identification of Somatic KRAS Mutation in a Korean Baby with Nevus Sebaceus Syndrome

Identification of Somatic <i>KRAS</i> Mutation in a Korean Baby with Nevus Sebaceus Syndrome

Decreased SATB1 Expression in ALT and Promoted Invasion of Jurkat Cell in Vitro

BACKGROUND: Special AT-rich sequence-binding protein-1 (SATB1) is critical for genome organizer that reprograms chromatin organization and transcription profiles, and associated with tumor growth and metastasis in several cancer types cancer. However, their expression patterns and function values for adult T-cell leukemia (ALT) are still largely unknown.METHODS: Expression of SATB1 in normal and ALT peripheral blood leucocytes (n=50 for normal, n=30 for ALT) were evaluated by RT-qPCR in mRNA level. Expression of protein SATB1 in leukemia cell lines were evaluated by Weston Blot.SATB1-shRNA was constructed in psuper-retro-puro retrovirus vector. Jurkat-SATB1-sh and control stable cell lines were established after virus infection and puromycin selection. Cellular proliferation and invasion of Jurkat-ctr and SATB1 knockdown Jurkat cells were evaluated by CCK-8 kit and transwell matrigel invasion assay, respectively.RESULTS: SATB1 mRNA expression was decreased in ALT (p<0.001) compared with normal peripheral blood leucocytes cells. SATB1 protein expression in Jurkat cell was higher than other leukemia cell lines. SATB1-shRNA was constructed successfully. Weston Blot show Jurkat-SATB1-sh and control stable cell lines were established successfully. Knockdown of SATB1 expression significantly enhanced invasion of Jurkat cell in vitro but no effect in proliferationCONCLUSIONS: Decreased expression of SATB1 in ALT was associated with cell invasion. DisclosuresNo relevant conflicts of interest to declare.

V‑set and transmembrane domain‑containing 1 is silenced in human hematopoietic malignancy cell lines with promoter methylation and has inhibitory effects on cell growth.

Numerous leukocyte differentiation antigens act as important markers for research, diagnosis, triage and eventually treatment targets for hematopoietic malignancies. V‑set and transmembrane domain‑containing 1 (VSTM1) was identified by immunogenomic analysis as a potential leukocyte differentiation antigen gene. VSTM1 is located at 19q13.4 on human chromosomes, an important genomic region prone to genetic and epigenetic modifications in numerous hematopoietic malignancies. VSTM1‑v1, a primary splicing form encoded by VSTM1, is a type I transmembrane molecule with an extracellular immunoglobulin V‑like domain and two cytoplasmic immunoreceptor tyrosine-based inhibitory motifs. In the present study, VSTM1 expression was examined in normal human peripheral leukocytes and hematopoietic tumor cell lines; in addition, the aberrant methylation of the VSTM1 gene was evaluated using methylation‑specific polymerase chain reaction (MSP). The results of the present study demonstrated that VSTM1 was widely expressed in normal human peripheral blood leukocytes, including granulocytes and monocytes, in concurrence with previous studies, as well as lymphocytes; in addition, the molecular size and expression levels of VSTM1 varied considerably between leukocytes. However, VSTM1 was undetectable in numerous hematopoietic tumor cell lines following promoter hypermethylation. The effects of pharmacologically‑induced demethylation of the VSTM1 gene and promoter region were analyzed using MSP and biosulfite genomic sequencing, and the results revealed that VSTM1 expression was restored in methylation‑silenced Jurkat cells. In addition, CKK‑8 assays revealed that VSTM1‑v1 overexpression in Jurkat cells resulted in growth suppression. Furthermore, the inhibitory effect on cell growth was enhanced following antibody‑induced cross‑linking of VSTM1‑v1. In conclusion, the results of the present study indicated that promoter methylation silenced VSTM1 and negatively regulated cell growth in human hematopoietic malignancy cell lines.

Multiple gastrointestinal stromal tumors with novel germline c-kit gene mutation, K642T, at exon 13

Multiple gastrointestinal stromal tumors (GISTs) caused by germline c-kit gene mutations are an extremely rare autosomal dominant disorder. A 57-year-old Japanese woman was referred to a hospital for appetite loss and severe weight loss. She had 2 large abdominal masses around the stomach, which were surgically resected. Histological examination revealed that these tumors were GISTs. Multiple microscopic GISTs and diffuse hyperplasia of the interstitial cells of Cajal were also seen in the background gastric and small intestinal walls. Characteristically, the GISTs showed severe hyalinization with calcification and partial heterotopic ossification, which may have caused the patient's severe dysphagia. Mutational analysis of the c-kit gene revealed a substitution at codon 642 in exon 13 (K642T) in the tumor, normal ileal mucosa and peripheral blood leukocytes, indicating that the mutation is in the germline. This is the first case of multiple GISTs with novel germline c-kit gene mutation at exon 13.

Combined ROR1 and CD160 Detection For Improved Minimal Residual Disease In Patients With Chronic Lymphocytic Leukemia (CLL)

IntroductionOver the past decade, treatments for patients with chronic lymphocytic leukemia (CLL) have produced complete remissions (CR) without evidence for minimal residual disease (MRD), particularly for younger and/or fitter patients. In this setting, achieving an MRD-negative CR has prognostic implications, yielding longer progression free survival (PFS) and overall survival (OS) than for patients who achieve a CR with persistent MRD. Complicating efforts to incorporate testing for MRD in clinical practice has been the lack of a defined consensus on the methods of MRD detection. In this study, we report on a novel combination of mAbs for MRD detection by flow cytometry based on two antigens: the NK-cell receptor and tumor specific antigen, CD160; and the tumor associated antigen, receptor tyrosine kinase-like orphan receptor 1 (ROR-1). ObjectiveTo compare a novel single-tube, tumor-specific (CD160+ROR1) targeted approach to MRD detection against the previously published CD160 flow cytometric assay (CD160FCA) (Farren et al, 2011) and the new, single-tube 8-color ERIC assay. MethodsBetween October 2012 and July 2013, prospective assessment of MRD was performed on peripheral blood in 56 patients (86 samples). We developed a flow cytometric assay using mAbs specific for CD160 or ROR1 (Fukuda et al, 2008). For this we used the following mAb from BD Biosciences: CD2 FITC, CD5 Pe-Cy7, CD19 PerCP5.5, CD45V500, CD160PE, ROR-1 AF647 (“ROR-160FCA”) and a sequential gating strategy. This was compared with CD160FCA and the 8-color ERIC consortium protocol (unpublished). Light chain analysis (LCR) was performed in all cases and reported where detectable. A proof of concept spiking experiment simulating MRD was prepared by mixing CLL and normal peripheral blood leukocytes in a serial dilution to a level of 10-5(n=3). Statistical analysis was performed using Spearman Rank correlation coefficients, Mann-Whitney t-test, and Bland-Altman method comparison. Significance was set at <0.05%. ResultsTo establish the proof of principle, MRD levels ranging from 0.001% to 100% (Neat CLL) were prepared by serial dilutions, in which MRD levels could be established by the ROR-160FCA to 10-5 (n=3). Assessment of the observed incidence against expected incidence of CLL MRD demonstrated a highly significant correlation (R2=0.96, p=0.01). In the study, the range of detectable disease went from <0.01% to 38.59%, of which 37% of samples had levels below <0.01%. Analyzing all flow cytometric methods, a highly significant correlation was observed between all three: CD160FCA vs ERIC: Spearman R=0.96 (95%CI: 0.93 - 0.97, p<0.001); CD160FCA vs ROR-160FCA: Spearman R=0.97 (95%CI: 0.96 – 0.99, p<0.001); ROR-160FCA vsERIC: Spearman R=0.97 (95%CI: 0.94 – 0.98, p<0.001).54 samples had levels of disease <1%. Bland-Altman assay comparison in these patients again demonstrated significant associations between the assays (CD160FCA vs ERIC: mean 0.08 ±0.15; CD160FCA vs ROR-160FCA: mean 0.04 ±0.19; ROR-160FCA vs ERIC: mean 0.03 ±0.25). Light chain restriction was detectable in 24 patients (size of the restricted population ranged from 0.2% to 47% of all cellular events). This sub-group of patients also demonstrated excellent correlation between level of LCR and detectable disease by CD160FCA (Spearman R=0.96, 95%CI: 0.92-0.98, p<0.001), ERIC (R=0.95, 95%CI: 0.92-0.98, p<0.01) and ROR-160FCA (R=0.97, 95%CI 0.93-0.98, p<0.001). ConclusionMonitoring minimal residual disease in CLL is a key focus for clinical trials, as MRD is an important prognostic marker in CLL in terms of PFS and OS. Here we provide a single tube assay, ROR-160FCA, which is unique by targeting two antigens restricted to malignant B-cells, CD160 and ROR1. ROR-160FCA is equivalent in MRD detection compared to both CD160FCA and the current ERIC assay under development. The two tumor-specific antigens give ROR-160FCA the potential for improved sensitivity, particularly where limited sample is available. Furthermore, it only requires a simple sequential gating strategy, is rapid, and appears more cost effective than other Methods.ReferencesFarren TW, Giustiniani J, Liu FT et al. Blood. 2011;118 (8):2174-2183.Fukuda T, Chen L, Endo T et al. Proc Natl Acad Sci U S A. 2008;105 (8):3047-3052. Disclosures:Farren:BD Biosciences: Research Funding. Warner:BD Biosciences: Employment, Research Funding. Agrawal:BD Biosciences: Research Funding.

Single Cell Analysis Of Protein Phosphorylation In Chronic Myeloid Leukemia Treated With Dasatinib, Eltrombopag, and Pegfilgrastim

IntroductionTyrosine kinase inhibitor (TKI) induced neutropenia and thrombocytopenia may be relieved with G-CSF and trombopoietin analogs. To characterize the signaling response in a CML patient treated with hematopoietic growth factors and TKI, we employed single cell analysis of phosphoproteins in serial samples. MethodsWe characterized the single cell phosphoprotein responses to treatment with dasatinib, eltrombopag, and pegfilgrastim in CML-like cell lines K562 and KCL-22, normal peripheral blood leukocytes and leukocytes from a patient starting on nilotinib. These data formed a basis for comparison with serial samples (Figure 1) from a CML patients after extensive wash-out before restarted on third-choice TKI, combined with eltrombopag and pegfilgrastim. For characterization of in vivo transformational potential CML patient bone marrow cells were engrafted in duplicate in NOG mice by femoral injection. Basal phosphorylation was determined using 17 phosphorylation specific antibodies: All staining panels contain the same 4 surface antibodies and a live-dead discriminator; CD33(P67.6) PerCP-Cy5.5, CD38(HB7) PE-Cy7, CD34(581) PE, CD45(MEM-28) PE-Dynamics590 and phospho c-PARP(Asp214) AlexaFlour-700. Two phosphospecific antibodies were added to each panel with the respective direct conjugated dye Alexa Flour 488 and Alexa Flour 647. The lowest median signal for each phosphoprotein in CML cells and lymphocytes, respectively, were used as reference value for calculation of basal phosphorylation. [Display omitted] ResultsA 56 y old male was diagnosed with CML (Sokal RR 0.75, Hasford RR 929, karyotype 46,XY,t(9;22)(q34;q11)[20], FISH 9q34 ABL/22q11.2 BCR dual fusion 62% of cells, PCR positive BCR-ABL e13e2/e14a2), developed prolonged thrombocytopenia and neutropenia on various dose levels of imatinib and nilotinib (neutrophile <1x109/L, <30x109/L) during the first 7 months after diagnosis. After a 6 months pause of TKI therapy: quantitative PCR BCR-ABL1/ABL1 25%IS, bone marrow biopsy with reduced normal megakaryocytes. Platelet supportive therapy with eltrombopag 50 mg daily was initiated 14 days before start with dasatinib 50 mg daily keeping platelet level above 50. After two more weeks pegfilgastim 6 mg was started, increasing neutrophils above 1.0x109/L.The CML bone marrow engrafted without symptomatic disease in NOG mice over 6 months monitoring, in contrast to engraftment with disseminated disease in an accelerated phase CML patient sample and the KCL-22 cell line. Phosphorylation of patient myeloid cells indicated STAT3(pY705) and STAT5(pY694) phosphorylation 2 and 3 hours after intake of eltrombopag. Dasatinib modestly inhibited SRC(pY418) and STAT3(pY705), but only transiently blunted the effect of eltrombopag. Pegfilgrastim strongly induced STAT3(pY705), STAT3(pS727), and SRC(pY418). Further analyses of cellular subsets and phospho-Abl will be presented. ConclusionEltrombopag kept stable levels of platelets on dasatinib treatment, and cell phosphorylation reflected persisting signaling induced by eltrombopag after seven days on combination therapy. Pegfilgrastim induced strong phosphorylation of STAT3(pY705) during dasatinib co-therapy. Phosphoprotein signal analysis of patient leukemic cells reflected the hematological response of dasatinib, eltrombopag, and pegfilgrastim and may be used to monitor therapy responses of and growth factors and targeted therapy.Phosphoprotein modulated early after start of treatment. (A) Each day 1-23 is analysed separately with its pretreatment sample as control and signal baseline. (B) Various treatment days are compared, using pretreatment control at day 1 used as baseline. (A) and (B) were analysed by flow cytometry at the same day, using bar code labelling, sample processing and analysis as previously described (Skavland et al. Blood Cancer J 2012). On day 16 dasatinib and eltrombopag is administrated concomitantly. Signalling responses in lymphocytes were distinct different from myeloid cells. Further determination of cellular subsets and their signalling responses will be presented. Disclosures:Popa:KinN Therapeutics AS: Employment.

Two rare cases of osteomyelitis

circulatory collapse, severe hypoglycemia, metabolic acidosis, severe anemia or hemolysis, renal failure, hemoglobinuria, liver failure, coagulopathy. Patients with complicated or severe malaria may have hyperparasitemia (≥100,000 parasites/μL of blood; ≥5 to 10% of parasitized RBCs) and/ormajor signs of organ dysfunction. The criteria for complicated malaria are acidemia/acidosis (arterial pH b 7.25; plasma bicarbonate b15 mmol/L); venous lactate (N5 mmol/L); severe anemia (Hct b 15% parasitemia levels N 100.000/μL); kidney failure with serum creatinine N 3.0 mg/dL; pulmonary edema/ARDS; hypoglycemia (plasma glucose b 40 mg/dL); hypotension/shock; hemorrhage/CID; convulsions; hemoglobinuria not associated with drug effects oxidizing or enzymatic deficits. Hyperparasitism increases mortality. Case report: Male 61 years old. In December/2010, the patient presented a clinical condition characterized by fever, chills, myalgias, arthralgias, frontal headache, asthenia, and anorexia. At the emergency room, the clinical examination revealed sub-jaundiced; sweaty skin, painful hepatomegaly, without palpable lymphadenopathy; hypotension and tachycardia fever (40 °C). Analytically: normal peripheral blood leukocyte count, Hb 16.5 g/dL, leukocytes 7800 (g/L), creatinine 1.80 mg/dL; hyperbilirubinemia and Plasmodium positive (P. falciparum parasitemia 7.2%). Hewas admitted in the intensive care unit due to severity criteria. The patient developed multiorganic dysfunction — MODS: pulmonary (mechanical ventilation); hematologic with thrombocytopenia and severe anemia requiring transfusional support, liver dysfunction (total bilirubin up to maximum 3.9 g/dL); renal dysfunction (AKI stage I) and cardiovascular dysfunction. The treament was initiated with doxycycline 100 mg 8/8 h and quinine sulfate 600 mg 8/8 h for 7 days. He had favorable clinical course with total recovery of all disorders. Conclusion: According to the WHO, malaria is by far the tropical and parasitic disease that causes more social and economic problems in the world and is only surpassed in number of deaths by AIDS. Severe malaria can progress extremely rapidly and cause death within hours or days. The authors emphasize that timely diagnosis and organ support in intensive care unit allowed the favorable outcome of this case report. More studies are needed to combat the disease.

Oral administration of an HSP90 inhibitor, 17-DMAG, intervenes tumor-cell infiltration into multiple organs and improves survival period for ATL model mice

In the peripheral blood leukocytes (PBLs) from the carriers of the human T-lymphotropic virus type-1 (HTLV-1) or the patients with adult T-cell leukemia (ATL), nuclear factor kappaB (NF-κB)-mediated antiapoptotic signals are constitutively activated primarily by the HTLV-1-encoded oncoprotein Tax. Tax interacts with the I κB kinase regulatory subunit NEMO (NF-κB essential modulator) to activate NF-κB, and this interaction is maintained in part by a molecular chaperone, heat-shock protein 90 (HSP90), and its co-chaperone cell division cycle 37 (CDC37). The antibiotic geldanamycin (GA) inhibits HSP90's ATP binding for its proper interaction with client proteins. Administration of a novel water-soluble and less toxic GA derivative, 17-dimethylaminoethylamino-17-demethoxygeldanamycin hydrochloride (17-DMAG), to Tax-expressing ATL-transformed cell lines, C8166 and MT4, induced significant degradation of Tax. 17-DMAG also facilitated growth arrest and cellular apoptosis to C8166 and MT4 and other ATL cell lines, although this treatment has no apparent effects on normal PBLs. 17-DMAG also downregulated Tax-mediated intracellular signals including the activation of NF-κB, activator protein 1 or HTLV-1 long terminal repeat in Tax-transfected HEK293 cells. Oral administration of 17-DMAG to ATL model mice xenografted with lymphomatous transgenic Lck-Tax (Lck proximal promoter-driven Tax transgene) cells or HTLV-1-producing tumor cells dramatically attenuated aggressive infiltration into multiple organs, inhibited de novo viral production and improved survival period. These observations identified 17-DMAG as a promising candidate for the prevention of ATL progression.

Abstract 4240: A novel approach to adjust for immune cell distribution in blood-based epigenome-wide association studies of cancer.

Abstract Epigenome-wide association studies (EWAS) promise to advance our understanding of epigenetic variation in cancer and are made possible by the recent emergence of cost-effective high-throughput technologies. Blood is the most widely available source of genomic DNA that can be used for study of cancer-associated alterations in DNA methylation (DNAm). These analyses are complicated by the heterogeneity of blood, which represents multiple cell types with unique DNAm signatures. Shifts in immune profile could confound results since altered leukocyte numbers are common in response to exposures or disease. We applied a novel approach to blood-based EWAS, adjusting for leukocyte composition estimated by epigenetic deconvolution of blood. Normal human peripheral blood leukocytes (n = 47) were isolated by magnetic activated cell sorting and purity was confirmed by fluorescence activated cell sorting. DNAm was interrogated using the Infinium HumanMethylation27 BeadArray (Illumina) on the sorted leukocyte samples and peripheral blood samples from 3 independent case-control studies of bladder cancer (223 cases, 205 controls), head and neck squamous cell carcinoma (HNSCC; 92 cases, 92 controls), and ovarian cancer (131 cases, 274 controls). Differentially methylated loci associated with leukocyte lineage were identified using a series of linear mixed effects models fit to the DNAm data for each of the 26,486 autosomal CpGs for the sorted leukocytes, yielding an F-statistic for each locus. The subject-specific leukocyte distribution (vector ω) was estimated using constrained projections, as previously described by Houseman et al (2012). Several regression parameters were estimated: βj, representing the association of case-status and DNAm at CpG j, unadjusted for ω; αj, representing the corresponding association adjusted for ω, and Γ, the association of case-status and ω. Γ, β and α were each adjusted for age, sex and smoking for bladder cancer and HNSCC, and age for ovarian cancer. Statistical inference was achieved by permutation, where null distributions were obtained by permuting case-status with respect to DNAm values and other covariates, using an omnibus test adjusted for multiple-comparisons, constructed by comparing the observed average F-statistic across all CpGs to the corresponding quantity obtained from the permutation distribution. After adjusting for leukocyte composition (α), the association between DNAm and case-status was significant for all 3 studies (bladder cancer: p = 0.047; HNSCC: p = 0.013; ovarian cancer: p = 0.0002). Subsequent analyses revealed that cancer-associated pathways were overrepresented among significant loci. These results indicate cancer-specific variation in DNAm of peripheral blood, independent of immune cell shifts. Further research is indicated for elucidation of mechanisms driving these observations. Citation Format: Scott M. Langevin, E. A. Houseman, William P. Accomando, Devin C. Koestler, Brock C. Christensen, Heather H. Nelson, Margaret R. Karagas, Carmen J. Marsit, John K. Wiencke, Karl T. Kelsey. A novel approach to adjust for immune cell distribution in blood-based epigenome-wide association studies of cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4240. doi:10.1158/1538-7445.AM2013-4240

Abstract LB-374: A novel method for the detection of circulating NK cells in archived blood reveals a decrease in NK cells in head and neck squamous cell carcinoma

Abstract Patients with head and neck squamous cell carcinoma (HNSCC) frequently exhibit abnormal immune cell levels and activities, leading many to believe that progression of the disease is linked to evasion of immune surveillance. A more complete understanding of immune cell alterations could be obtained from analysis of archival samples, but this type of study is not possible using current immunodiagnostic methods. Combining information on cell lineage specific differentially methylated regions (DMRs) with methylation specific quantitative PCR (MS-qPCR) enables highly sensitive and accurate counts of individual cell types in archival blood and tissues. DNA methylation and mRNA expression in magnetic microbead (MACS) isolated, flow cytometry (FACS) validated normal human peripheral blood leukocytes were assessed using the Infinium HumanMethylation27 and Whole-Genome DASL HT (Illumina) microarrays. Cell-specific DMRs were identified by comparative analysis of DNA methylation and mRNA expression patterns between different cell lineages, and validated by MS-qPCR and pyrosequencing. Analysis of the microarray data revealed CpG loci significantly demethylated in NK cells compared to all other leukocyte lineages (q &amp;lt; 0.1) within genes exhibiting significantly increased mRNA expression in NK cells (q &amp;lt; 0.1). Pyrosequencing and MS-qPCR assays confirmed a NK cell specific demethylated region. The MS-qPCR assay was calibrated using known quantities of NK cell DNA to facilitate quantification of these cells in biological samples, and was then applied to archival peripheral blood DNA samples from 122 control donors and 122 HNSCC patients. Demethylation of the candidate region was significantly lower in HNSCC patient bloods compared with control bloods (p &amp;lt; 0.001), but was not significantly associated with age, gender, HPV16 (E6 and/or E7) serology, cigarette smoking, alcohol consumption, BMI, or disease treatment. Measurements from control samples were used to establish normal demethylation tertiles. Proportion of total HNSCC cases decreased significantly with increasing tertile (p &amp;lt; 0.001). Multivariate logistic regression controlling for age, gender, cigarette smoking, alcohol consumption, BMI, and HPV16 serology confirmed increased HNSCC risk for individuals in the lowest tertile (OR = 5.6, 95% CI: 2.0, 17.4) and second lowest tertile (OR = 4.9, 95% CI: 1.8, 16.1). The advent of DNA methylation based immunodiagnostic methods will make assessment of immune cell profiles in archived samples that have not undergone rigorous special handling requirements possible. Using a novel NK cell specific demethylated region to detect and quantify NK cells in the blood, we have detected a HNSCC associated decrease in circulating NK cells that is independent of disease treatments, known exposures and other putative risk factors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-374. doi:1538-7445.AM2012-LB-374

Taxol-oligoarginine conjugates overcome drug resistance in-vitro in human ovarian carcinoma

ObjectiveMultidrug resistance is the major cause of failure of many chemotherapeutic agents. While resistance can arise from several factors, it is often dominated by drug efflux mediated by P-glycoprotein (P-gp), a membrane-bound polysubstrate export pump expressed at high levels in resistant cells. While co-administration of pump inhibitors and a drug could suppress efflux, this two-drug strategy has not yet advanced to therapy. We recently demonstrated that the reversible attachment of a guanidinium-rich molecular transporter, polyarginine, to a drug provides a conjugate that overcomes efflux-based resistance in cells and animals. This study is to determine whether this strategy for overcoming resistance is effective against human disease. MethodsTumor samples from ovarian cancer patients, both malignant ascites cells and dissociated solid tumor cells, were exposed to Taxol-oligoarginine conjugates designed to release free drug only after cell entry. Cell viability was determined via propidium-iodide uptake by flow cytometry. To analyze bystander effect, toxicity of the drug conjugates was also tested on peripheral blood leucocytes. ResultsHuman ovarian carcinoma specimens resistant to Taxol in vitro demonstrated increased sensitivity to killing by all Taxol-transporter conjugates tested. These studies also show that the drug conjugates were not significantly more toxic to normal human peripheral blood leukocytes than Taxol. ConclusionsThese studies with human tumor indicate that oligoarginine conjugates of known drugs can be used to overcome the efflux-based resistance to the drug, providing a strategy that could improve the treatment outcomes of patients with efflux-based drug-resistance.

Changes in leukocyte gene expression profiles induced by antineoplastic chemotherapy

In the present study, we studied changes in gene expression induced by chemotherapy (CT) on normal peripheral blood leukocytes (PBLs), at baseline and following three CT cycles, in order to identify which genes were specifically affected and were potentially useful as biomarkers for a personalised prognosis and follow-up. A PBL subtraction cDNA library was constructed from four patients undergoing CT with paclitaxel and carboplatin (PC). mRNA from the PBLs was isolated prior to the patients receiving the first cycle and following the completion of the third cycle. The library was screened and the expression of the identified genes was studied in PBLs obtained from patients suffering from cancer prior to and following three cycles of PC and a reference group of patients undergoing treatment with Adriamycin-cyclophosphamide (AC). From the 1,200 screened colonies, 65 positive clones showed varied expression intensity and were sequenced; 27 of these were mitochondrial DNA and 38 clones (27 different) were coded for cytosolic and nuclear proteins. The genes that were studied in patients undergoing CT were ATM (ataxia-telangiectasia mutated gene), eIF4B (translation initiation factor 4B), MATR3 (Matrin 3), MORC3 (microrchidia 3), PCMTD2 (protein-L-isoaspartate O-methyltransferase), PDCD10 (programmed cell death gene 10), PSMB1 (proteasome subunit type β), RMND5A (required for meiotic nuclear division 5 homologue A), RUNX2 (runt-related transcription factor 2), SACM1L (suppressor of actin mutations 1-like), TMEM66 (transmembrane protein 66) and ZNF644 (zinc finger protein 644). Certain variations were observed in the expression of the genes that are involved in drug resistance mechanisms, some of which may be secondary to non-desirable effects and others of which may cause the undesired effects of CT. The expression of genes with a dynamic cellular role showed a marked positive correlation, indicating that their upregulation may be involved in a specific pattern of cell survival versus apoptosis in response to the cell damage induced by CT. Whether these CT-induced changes are random or directed in a specific selection-evolution manner needs to be elucidated.