Sequential Peripheral Blood Samples Research Articles

PF442 TREATMENT WITH 5‐AZACYTIDINE RESULTS IN EXPRESSION OF LONG TERMINAL REPEAT (LTR) ELEMENTS IN PERIPHERAL BLOOD MONONUCLEAR CELLS OF PATIENTS WITH MDS AND AML: FIRST PILOT STUDY

Background:Epigenetic drugs are used for the treatment of several hematologic malignancies, but their pharmacological mechanism remains poorly understood. By genome wide analysis of transcription start sites (TSS), methylation status and chromatin dynamics, we showed for DNA methyltransferase and histone deacetylase inhibitors (DNMTi and HDACi) global reactivation of long terminal repeat elements (LTRs) in vitro (cell line models) and in vivo (neuroblastoma mouse xenograft model). The LTR reactivation is resulting in numerous fusion transcripts that encode novel protein isoforms which have the potential to influence cell proliferation, seem to be an explanation for the priming effect of epigenetic therapy and might be used as a potential marker for epigenetic treatment response.Aims:To investigate the reactivation of LTRs in sequential peripheral blood samples of AML and MDS patients upon epigenetic drug treatment with 5‐Azacytidine (DNMT‐inhibitor) by qRT‐PCR and ddPCR and to correlate the expression results with treatment response.Methods:Sequential RNA samples from peripheral blood mononuclear cells (PBMCs) of three AML and four MDS patients treated with 5‐Azacytidine were analyzed. Consensus primers covering the transcript variants of LTR12C as well as particular primers for specific LTR elements were used. Expression of LTRs was analyzed by qRT‐PCR technique with SYBRGreen as well as ddPCR technique with EvaGreen. Expression results were associated with treatment response.Results:In AML and MDS patients, we could detect LTR expression in sequential samples from PBMCs over time with 5‐Azacytidine treatment. LTR12C expression values measured by qRT‐PCR were confirmed by ddPCR and allowed to assess specific LTR elements. Patients with response to 5‐Azacytidine showed increased LTR12C expression whereas patients without treatment response had no increase in LTR12C expression. In contrast to the results from our previous NSCLC cell line model, specific LTR elements were only partially expressed in sequential PBMC samples from AML patients treated with 5‐Azacytidine.Summary/Conclusion:In this first in‐vivo pilot study, we detected LTR expression in sequential PBMC samples of AML and MDS patients treated with 5‐Azacytidine. In this group of patients, treatment response correlated with LTR expression over several treatment cycles with 5‐Azacytidine. These are the first results in AML and MDS patients showing expression of LTRs upon epigenetic drug treatment. Larger patient cohorts are necessary to confirm these results by either PCR‐ or genome wide‐analysis of transcription start sites (TSS) (Cap analysis of gene expression (CAGE) sequencing) as well as to evaluate the association of LTR expression and treatment response.

Immune Biomarkers Identify Sustained Quantitative and Functional Immune Reconstitution in the Setting of Adjunctive Lenalidomide Following T-Depleted RIC-Allo SCT for Multiple Myeloma

Background: Immune reconstitution after AlloSCT is a highly complex process influenced by both graft & recipient-related factors including post-graft therapies. Delayed T-cell reconstitution remains a source of morbidity & mortality and may contribute to limited anti-tumour effect. We sought to evaluate the immune reconstitution kinetics in the setting of post-graft Lenalidomide (Revlimid®) to augment the graft-versus-myeloma effect, by defining key immune biomarkers, in the NCRI LenaRIC phase II trial.Material & Methods: All patients recruited into the study underwent a reduced intensity conditioned (RIC) AlloSCT using Fludarabine & 2Gy TBI with ATG (Fresenius) as in vivo T-cell depletion, following a high dose melphalan ASCT (planned tandem SCT programme). Patients were scheduled to receive Lenalidomide (5mg-10mg/day) on day+35 if stable donor engraftment in the absence of GvHD. Lenalidomide discontinuation was planned at 12 months post-transplant, with donor lymphocyte infusions (DLI) given to patients with evidence of residual disease, relapse or mixed chimerism. Sequential peripheral blood samples were drawn at predetermined time frames before (baseline) and after graft infusion (D+30,+60,+90,+180,+270 & D+360) in all trial registrants. Using multi-parameter flow cytometry (MFC), a comprehensive panel of immune subset & functional/activation markers were used to define characteristics of T-cell, B-cell, regulatory T-cell & NK cells. The inflammatory proteome (IL-1b, IL-4, IL-5, IL-6, IL-10, IFNg, TNFa, CXCL8 & GM-CSF) and soluble immune checkpoints (PD-1 & ICOS) were analyzed using LUMINEX assays on serial serum samples at parallel points. PB cell subset-specific Complete Donor Chimerism (CDC) was analysed by single tandem repeat PCR at day (D)+100,+180,+270 and D+365.Results: From January 2011 to May 2015, 40 patients with myeloma ≥VGPR after 1st or 2nd ASCT, were recruited into the LenaRIC protocol. The median age was 49 years (range 35-65) with PB as the graft source (Matched Related Donor n=16, 10/10 Matched Unrelated Donor n=23; 1 patient not transplanted). Viable samples were received from 37/40 patients (92.5%), with no cycles of Lenalidomide received by 3/37 (8.1%) patients, 1-6 cycles received by 13/37 (35.1%) patients & >6 cycles received by 21/37 (56.8%) patients. 5 patients completed 12 cycles as per protocol treatment; of those completing >= 10 cycles, 5/16 received DLI.Only 10/37 (27%) of patients achieved a CD4+ cell count >200/µl in a median of 180 days compared with 30/37 (81%) of patients who achieved a CD8+ cell count >200/µl in a median of 60 days. In patients who did not receive any doses of Lenalidomide, higher levels of CD8+ cells (p=0.013) but lower NK cell levels (p=0.071) were evident at D+30 compared with those eligible to proceed with Lenalidomide. No difference was noted between Lenalidomide exposure subgroups in relation to CD8+ or CD4+ cell recovery. Treg cells were increased at D=270 & D+365 in those who received >6 cycles of Lenalidomide, though this corresponded with a higher level of HLA-DR expression on CD8+ T cells representing global immune activation. Significant differences between Lenalidomide exposure subgroups were evident in the inflammatory proteome readout at D+90. CXCL8 levels peaked at D+270, especially in those who received >6 cycles of Lenalidomide. Of 30 evaluable patients, T-cell CDC was seen in 63.3% at D100, with 84% of evaluable patients having CDC at D365. No difference between subgroups was evident in relation to rapidity of CDC and stability of CDC.Conclusions: The sequential monitoring of immune biomarkers in the setting of post-graft Lenalidomide following a T-deplete RIC-AlloSCT highlights the rapid and sustained quantitative and functional reconstitution of donor immune homeostasis. The clinical significance of these findings requires correlation with both the anti-tumour immune effect and theimmunopathy ofAlloSCT (GvHD).Unrestricted educational grants were provided by Cancer Research UK and byCelgene.Lenalidomideprovided free of charge byCelgene. The support and time of participating patients and their families is gratefully acknowledged [Display omitted] DisclosuresCook:Glycomimetics: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Research Funding, Speakers Bureau. Brock:AstraZeneca: Equity Ownership; GlaxoSmithKline: Equity Ownership. Cavenagh:Amgen: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau. Cook:Celgene: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Amgen: Honoraria; Cancer Research UK: Research Funding; Takeda: Honoraria.

Sequential Serum Let-7 Is a Novel Biomarker to Predict Accelerated Reproliferation During Fractional Radiotherapy in Lung Cancer

BackgroundAccelerated reproliferation, usually deemed to occur late during the course of radiation therapy, is an important factor resulting in radiotherapy failure. The identification of strategies that accurately reflect accelerated reproliferation might help to determine radiotherapy strategy, and thus implementation of individualized treatment. Patients and MethodsPatients with lung cancer were enrolled from Shandong Cancer Hospital and Institute in China between March 2014 and March 2015. Tumor tissue and sequential peripheral blood samples were obtained before treatment and during radiotherapy to detect Ki-67 and let-7 expression. ResultsWe found a strong correlation between serum let-7 and tissue Ki-67 before treatment (r = −0.773; P = .003). Patients with a high level of baseline serum let-7 expression level had significantly better overall survival (the overall survival were 100% and 27.3%, respectively; P = .024). For patients with a relatively low expression level of baseline serum let-7, there was a peak of expression levels at week 4 (the mean expression levels were 1.40, 5.01, and 1.36, respectively). However, for patients with relatively high expression levels of baseline serum let-7, the expression levels were constantly downregulated at week 4 and 6 compared with the original (the mean expression levels were 6.16 vs. 1.34 vs. 0.80, respectively). ConclusionThe study showed that serum let-7 expression level could reflect the proliferation of tumor tissue reliably in patients with lung cancer. Furthermore, the study might change conventional views of accelerated reproliferation. We showed that initial accelerated proliferation exists in patients with a relatively slow proliferation rate before radiotherapy, and late course accelerated reproliferation exists in patients with relatively fast proliferation before radiotherapy.

An open-label, single-group, phase 2 study of brentuximab vedotin as salvage therapy for males with relapsed germ-cell tumors (GCT): Results at the end of first stage (FM12GCT01).

480 Background: Prognosis of patients (pts) failing multiple chemotherapy (CT) regimens is quite dismal. CD30 is expressed and prognostic in embryonal carcinoma, hence it is a rational target for treatment. Brentuximab Vedotin (BV) is an antibody-drug conjugate consisting of the chimeric anti-CD30 antibody SGN-30 conjugated to an antitubulin synthetic analog (MMAE). A phase 2 trial is ongoing in GCT (NCT01851200). Methods: 24 pts with biopsy-proven CD30+ GCT will receive BV 1.8 mg/Kg IV q3 weeks until disease progression or onset of unacceptable toxicity. Eligibility will include failure of 2 or 3 platinum-based CT (including HDCT). All pts will undergo measurement of serum tumor markers (STM), a computed tomography and a PET scan q6 weeks. An optimal Simon’s 2-stage design will be applied. The primary endpoint is the objective response-rate (ORR; H0: ≤ 5%, H1: ≥ 25%, α and β = 10%). In stage 1, 9 evaluable patients will be accrued. Sequential peripheral blood samples are being collected for immune profiling by flow cytometry of immune cell subsets. Results: From 07/13 to 04/15, 9 pts have been treated, 3 in third-line, and 6 beyond the third-line. 5 had received HDCT. STM decline was obtained in 7 pts (77.8%) after the first dose and in 4 pts (44.4%) after 2 doses (with normalized [1] or still positive [3] STM). ORR was 22.2% (1 CR+1 PR) according to RECIST v1.1, and there were 3 metabolic PR. 3-month PFS was 11.1% (95%CI: 0.6-38.8), 6-month OS was 85.7% (95%CI: 33.4-97.9). 1 case of reversible G3 hyperglycemia was recorded. No BV discontinuation due to toxicity occurred. In 7 evaluable pts, a trend to increasing expression of activation (HLA-DR), proliferation (Ki67), and functional differentiation (Granzyme B) markers in CD4 and CD8 T cells was observed during BV. In CR patient, an increased frequency of naive and central memory T cells was observed after the first cycle of BV, as well as a reduction in the fraction of PD1+/CD8+ T cells. Conclusions: Brentuximab is endowed with potent antitumor activity and meaningful immunomodulatory effects in GCT. The rapid development of resistance is a concern. ORR is pending confirmation in the whole sample size. Clinical trial information: NCT01851200.

JAK Inhibition Reduces CD25 high CD27+ FOXp3+ T Regulatory Cells and Causes a Silencing Of T Effector Cells In Patients With Myeloproliferative Neoplasms Whilst Promoting a TH17 Phenotype

BackgroundThe myeloproliferative neoplasms (MPN), in particular myelofibrosis, are associated with elevated levels of inflammatory cytokines and constitutional symptoms. Treatment with JAK inhibitors (JAKi) have lead to marked improvement in symptoms and splenomegaly. Signaling through the JAK pathway is critical for T cell development and differentiation. However the baseline immune signature remains largely undescribed in MPN as does the effect of JAK inhibition on the immune subsets in this disease. Materials and MethodsThe % and absolute number of CD4+ T cell subsets (TH1, TH2 and TH17 and Foxp3+ T regulatory cells) in peripheral blood (PB) were investigated by flow cytometry. T cells were stimulated and stained intracellularly for IFNg, IL-4, IL-17 & TNFα. Tregs were defined as CD4+ CD25highCD27+FOXp3+. The serum level of 30 cytokines was also measured by Luminex. Patients received either ruxolitinib (n=21) or SAR302503 (n=13) as JAKi. ResultsWe analysed 50 MPN patients (30 Myelofibrosis, 15 Polycythemia Vera, 5 Essential Thrombocythemia) and 14 healthy donors (HD). 34 patients were treated with JAKi and sequential PB samples were obtained at 1, 3, 6 and 12 month intervals (median follow up 6 months). Tregs are significantly lower in MPN patients compared to HD and drop further following treatment (p<0.0001 and p=0.0049 respectively). There was no difference at baseline in the T effector subsets between the groups including TH1, TH2 and TH17 secreting cells but there was a significant increase in TH17 following JAKi therapy (fig 1a). JAKi resulted in a significant decrease (p=0.03) in CD4 T cells secreting pro-inflammatory cytokines at 3 months follow up although this was less evident at 6 months follow up and occurred irrespective of disease response to treatment. This silencing was confirmed by both intracellular staining and luminex assay of supernatants including a significant decrease in Interleukin-2 receptor (IL-2r) p=0.0007, Interferon gamma induced protein (IP-10) p=0.0006, monokine induced by gamma interferon (MIG) p=0.0008 and hepatocyte growth factor (HGF) p=0.0009. [Display omitted] This finding was reproduced in-vitro in healthy peripheral blood mononuclear cells (PBMCs). PBMCs were treated with the JAKi ruxolitinib (100-300NM) in the presence or absence of plate bound anti-CD3/28 stimulation and cultured for 5 days. Tregs were reduced in number and there was a considerable increase in the percentage of “cytokine negative” or “silent” T effector cells by FACS analysis compared to untreated or vehicle treated cells (median of 42 % of CD4 to 91% of CD4) (fig 1b). This finding was reproduced by Luminex cytokine assay of supernatants. Western blot demonstrated a reduction in pSTAT3 in ruxolitinib treated cells. To assess the effect of JAKi on Treg function, healthy isolated Tregs were treated with ruxolitinib and co cultured with CFSE labeled autologous T effector cells. Short term JAKi treated Tregs were unable to suppress the proliferation of T effector cells compared to Tregs treated with vehicle alone. Similarly, proliferation rate and function of Tregs was reduced following 4 weeks expansion in the presence of ruxolitinib compared to expanded Tregs in the presence of ATRA and rapamycin as a control. [Display omitted] ConclusionsTregs are significantly lower in MPN patients compared to healthy controls in keeping with the inflammatory environment of MPN and decrease further with JAKi. Surprisingly, T effector numbers were not significantly different to healthy controls at baseline and TH1 and TH2 subsets did not change with therapy. However, secretion of proinflammatory cytokines from these cells was blocked with JAKi both invivo and invitro resulting in a functional silencing of T effectors. Interestingly, TH17 subsets increase with treatment possibly representing a polarization from a Treg phenotype to a TH17 phenotype, suggesting the re-establishment of immune-surveillance against the malignant clone. Further investigation is required to confirm the hypothesis that these expanded TH17 cells originate from Tregs or previously “silenced” CD4 T cells. Disclosures:Harrison:Novartis: Honoraria; Sanofi : Honoraria.

Short-term HIV-1 treatment interruption is associated with dysregulated TLR-stimuli responsiveness

Viremia during human immunodeficiency virus type-1 (HIV-1) infection results in progressive impairment of several components of the immune system. Here a unique model of repeated treatment interruptions (TIs) was used with the aim to reveal the effect of controlled short-term viremia on innate stimuli responsiveness and circulating dendritic cells (DCs). Sequential peripheral blood samples from HIV-1-infected patients on combination antiretroviral therapy, subjected to repeated TI cycles as part of a therapeutic DNA vaccination study, were analyzed. In vitro responsiveness of peripheral blood mononuclear cells to toll-like receptor (TLR) stimuli was analyzed by cytokine secretion, and frequencies of plasmacytoid DCs (pDCs) and myeloid DCs (mDCs) were monitored by flow cytometry. These parameters were found not to be significantly different between the vaccinated and placebo groups. Instead, independent of vaccination altered in vitro TLR responsiveness was observed in parallel with TI cycles. TLR7/8-triggered secretion of IL-12 and IFN-α, as well as TLR9-triggered secretion of IL-12, was hyperactivated. In contrast, expression of IFN-α after TLR9 stimulation decreased during the initial cycle of TI. Reduced frequencies of pDCs and mDCs, compared with baseline, were noted before and during the second TI, respectively. Furthermore, spontaneous ex vivo release of IL-12 from PBMC was noted during cycles of TI. In conclusion, these results suggest that consequences of short-term TI include dysregulated TLR responses and fluctuations in the frequencies of circulating DCs. Knowledge of these immunological factors may influence the continuation of stringent treatment schedules during HIV infections.

Abstract 1761: Extracellular telomeres can be detected in serum following cellular apoptosis in vitro and after chemotherapy in acute myelogenous leukemia (AML) patients.

Abstract Telomeres are repeated DNA sequences that cap the ends of each chromosome. Telomeres are temporally among the earliest genomic sequences to be degraded during apoptosis. We quantified telomeres in cell-free DNA (CF-DNA) in vitro and in vivo. CF-DNA was obtained by differential centrifugation of media or serum to remove intact cells and cellular debris. Telomere sequence in CF-DNA was measured with a quantitative PCR-based assay. We initially studied human breast cancer and brain cancer cell lines in vitro. We found that following treatment with doxorubicin, telomere sequences were rapidly detected in CF-DNA in the media (within 24-48 hours). We call these CF-DNA telomere sequences “extracellular telomeres” (ETs). ETs were preferentially secreted after chemotherapy. The telomere sequence molar fraction in the CF-DNA was up to 50,000-fold higher compared with other genomic sequences such as exons or structural DNA sequences such as alpha-satellite DNA. CF-DNA from the serum of 80 patients with a history of breast cancer and 40 normal female volunteers were analyzed. The patients with a history of metastatic breast cancer (n = 40) had 8-fold higher median value for ETs compared to those with a history of localized breast cancer (p=0.006). 20 normal women had ET levels similar to the local breast cancer patients. We thus hypothesized that the serum extracellular telomere assay could be used as a surrogate marker for in vivo apoptotic cell death. We collected sequential peripheral blood samples of newly diagnosed AML patients with a minimum peripheral circulating blast count of 2500/mm3 or more who received standard induction chemotherapy (anthracycline and cytarabine). These sequential peripheral blood samples were collected before, during and after standard chemotherapy at 12 hours interval for consecutive 21 days. The samples were immediately centrifuged to isolate CF DNA. Subsequently, ETs were quantitatively measured by qPCR telomere assay. In our preliminary data analysis of seven patients, we routinely observed two peaks of ETs about 10- fold higher than baseline that occurred about 2-3 and 4-6 days after initiating standard chemotherapy respectively. We conclude that 1) Telomere DNA is preferentially released from cancer cells in response to chemotherapy-induced apoptosis, 2) ETs can be detected and measured in normal patients and patients with a history of cancer, and 3) ETs are released after chemotherapy in AML patients. We are further investigating the relationship between ETs and other prognostic features in AML including initial white blood cell count, cytogenetics, disease free survival and overall survival. Citation Format: Amitkumar Mehta, Mallick Hossain, Christine Pressey, Johanna Tuomela, Varun Dhulipala, Sunil Rangarajan, Robert Crescentini, Arja Jukkola-Vuorinen, Katri Selander, David Graves, Kevin Harris, Uma Borate. Extracellular telomeres can be detected in serum following cellular apoptosis in vitro and after chemotherapy in acute myelogenous leukemia (AML) patients. [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 1761. doi:10.1158/1538-7445.AM2013-1761

Modulation of in vivo and in vitro cytokine production over the course of pregnancy in allergic and non‐allergic mothers

Cytokines secreted during pregnancy may influence immune development of the foetus. This study aimed to determine if maternal allergy alters patterns of systemic cytokine production throughout and after pregnancy. Maternal plasma cytokines and allergen-specific production of interleukin (IL)-10, IL-13 and interferon (IFN)-gamma were measured in allergic (n = 63) and non-allergic (n = 70) pregnant women who had a full set of sequential peripheral blood samples collected at 20-, 30-, 36-wk gestation and 6-wk post-partum. Maternal allergy was strictly defined by both allergen sensitization and doctor-diagnosed asthma, eczema or rhinitis. IL-13 responses to allergen were higher for allergic mothers at all time-points (20 wk: p < 0.001; 30 wk: p = 0.001; 36 wk: p < 0.001; post-partum: p < 0.001). For the non-allergic group, IL-13 levels to house dust mite decreased from 20- to 36-wk gestation (Friedman ANOVA p = 0.012) and were significantly lower at 36 wk compared with post-partum (p = 0.002). In contrast, IL-13 production by allergic mothers did not change from 20 wk through to post-partum. For both allergic and non-allergic mothers, in vitro IFN-gamma production was lower at all pregnancy time-points compared with post-partum levels. Allergic women had an increased propensity for peripheral blood allergen-specific T helper-2 responses during pregnancy, and failed to downregulate these responses in comparison with non-allergic women. This may be a factor that contributes to the increased risk of atopy in infants born to allergic mothers.

Early Therapy in HIV-1-Infected Children: Effect on HIV-1 Dynamics and HIV-1-Specific Immune Response

Perinatal HIV-1 infection is acquired in the milieu of a developing immune system, leading to high levels of uncontrolled viral replication. Few data have been reported that address the viral dynamics and immunological response in infants who initiated aggressive antiretroviral therapy (ART) shortly after birth. Six HIV-1-infected infants who started ART within 3 months of age were studied. The median followup was 61 months. Plasma HIV-1 RNA, cell-associated HIV-1 DNA, unspliced and multiply spliced HIV-1 mRNAs, HIV-1 antibodies, and CD4+ and CD8+ T-cell subsets were assessed in sequential peripheral blood samples. HIV-1 cellular immune response was measured by EliSpot assay. All children showed a decline in plasma viraemia to undetectable levels. HIV-1 DNA persisted in four children, but only two of these had detectable HIV-1 mRNA. All viral parameters remained persistently negative in two children. Only two children produced HIV-1 antibodies, while the others, after having lost maternal antibodies, remained seronegative. No HIV-1 cellular immune response was observed in any child. Therapy interruption was performed in two children: one HIV-1-seropositive and one HIV-1-seronegative with persistently undetectable levels of all viral parameters. Rebound of HIV-1 plasma viraemia in the seronegative child was more rapid and higher than that observed in the seropositive child. Early ART treatment in infants modifies the natural course of infection by controlling HIV-1 replication and reducing viral load to below the threshold levels required for onset of HIV-1 immune response, but does not prevent the establishment of a reservoir of latently infected cells that precludes virus eradication.

The Prevalence of Chronic Lymphoproliferative Diseases in Patients with Borderline Lymphocytosis in Community Practice.

Introduction: The standard criterion for diagnosing chronic lymphocytic leukemia (CLL) is clonal lymphocytosis of >5×10 9/L. Cases of CLL with normal lymphocyte count have been diagnosed by flow cytometry based on the presence of clonal CD19+/CD5+/CD23+ cells. Therefore, it is not unexpected that a proportion of patients with borderline lymphocytosis (>3.5 and <5.0x10 9/L) will have CLL. The aim of our study is to establish the prevalence of CLL in patients with borderline “4.0 to 5.0x109/L” lymphocytosis in the adult population (age >40 years) seen in community practice.Methods: Using flow cytometry we analyzed a total of 157 sequential peripheral blood samples collected from patients older than 40 years presented with borderline lymphocytosis (4 to 5 x109/L). Majority of these patients (#106) were detected incidentally during routine CBC and 51 samples were submitted to rule out lymphoproliferative diseases.Results: Forty of the 157 (26%) patients had clonal B-cell disease meeting the criteria for chronic lymphoproliferative disease. The disease was classified as CLL in 35 patients (87.5%), hairy cell leukemia in 1 patient (2.5%), Waldenstrom's macroglobulinemia in 1 patient (2.5%) and marginal zone B-cell lymphoma in 3 patients (7.5%). This data suggests that patients older than 40 year with lymphocyosis >4x10 9/L have high probability of having chronic lymphoproliferative disease. This disease could be other than CLL and should be thoroughly investigated.DISCUSSION: In our study the prevalence of low grade lymphoproliferative disorders in patients with borderline lymphocytosis (4–5 x109/L) above the age of 40 is 26%. This number may be positively skewed considering our selection criteria (including a subset of patients retrospectively included).Currently, there is no data to support that early intervention is beneficial for CLL, even for patients with unfavorable prognosis (e.g., those with ATM and P53 deletions). Early diagnosis of CLL will create more opportunity to study the disease in its early stages.[Shanafelt TD, Geyer SM, Kay NE: Prognosis at diagnosis: integrating molecular biologic insights into clinical practice for patients with CLL. Blood. 2004 Feb 15;103(4):1202–10. Epub 2003 Oct 23. Review.], [ Hamblin TJ: Achieving optimal outcomes in chronic lymphocytic leukemia. Drugs . 2001; 61(5):593–611. Review.]. Since lymphocyte doubling time (LDT) is a prognostic factor (the prognostic utility of LDT is most important for patients with early stage disease who typically are treated by watchful waiting [ Shanafelt TD, Call TG. Current approach to diagnosis and management of chronic lymphocytic leukemia. Mayo Clin Proc. 2004 Mar;79(3):388–98. Review. ] ). early diagnosis may help to better segregate low from high-risk patients. Finally, in today's cost containment pressures, it is beneficial to have an expectation for the cost/benefit ratio of performing a test. Knowing the prevalence of disease at decision limits may help us to better justify establishing testing guidelines.

Age-dependent changes in peripheral blood lymphocyte subpopulations in cattle: A longitudinal study

Immunofluorescence and flow cytometric analyses were used to study the age-dependent changes in the peripheral blood lymphocyte (PBL) subpopulations in cattle. Four healthy Holstein heifer calves (A, B, C and D), 1–2 months of age, were used in this study. Sequential peripheral blood samples were collected once a month for up to 2–2.5 years, and once at approximately 4 years of age. For the first 6 months of age, the calves had similar proportions of CD2 +, CD4 +, CD8 + T lymphocytes, CD20 + B lymphocytes and MHC class II + lymphocytes. From 2 months of age up to 2–2.5 years of age, all animals had similar proportions of CD5 + cells; but during the same period, animals A and B had significantly lower proportions of WC1 +γδ T cells than animals C and D. After 7 months of age, however, the proportions of CD2 +, CD4 + and CD8 + T cells in PBL of animals A and B significantly decreased, whereas the proportions of both CD20 + B lymphocytes and MHC class II + lymphocytes significantly increased. In contrast, the proportions of the various PBL subpopulations in animals C and D remained virtually unchanged after 7 months of age. For the first 6 months of age, all the calves showed similar absolute counts of PBL. Thereafter, the absolute counts of PBL in animals A and B significantly increased, but remained virtually unchanged in animals C and D. Throughout the study, from 1–2 months up to 2–2.5 years of age the absolute counts of CD2 +, CD4 +, CD8 + and WC1 +γδ T cells in PBL of the four animals were not significantly different from each other. Up to 6 months of age, the CD4 +/ CD8 + ratio in all calves was 2.38 ± 0.46, but significantly decreased thereafter to 1.81 ± 0.34. However, there were no significant differences in the CD4 + CD8 + ratios among individual animals. The increase in the absolute counts of PBL in animals A and B, after 7 months of age, was due to an increase in the absolute counts of CD5 + cells, CD20 + B lymphocytes and MHC class II + lymphocytes. Thus, changes in the percent, but not the absolute counts of T lymphocytes, were due to high percent and absolute counts of B lymphocytes, expressing the CD5 and MHC class II antigens.

Genomic variation of human immunodeficiency virus type 1 (HIV-1): molecular analyses of HIV-1 in sequential blood samples and various organs obtained at autopsy.

Length polymorphisms and partial nucleotide sequences were determined for the hypervariable regions, V1 to V5, of the human immunodeficiency virus type 1 (HIV-1) env gene obtained from proviral DNA of sequential peripheral blood samples, from viral RNA in plasma, and from proviral DNA obtained from different organs of individuals at autopsy. The lengths of several env regions of HIV-1 proviral DNA differed markedly when obtained from different organs of an individual. Nucleotide sequences of the hypervariable V3 region of HIV-1 obtained from different organs of one patient demonstrated distinct viral variants. Most proviral DNA sequences found in organs were also present in viral RNA obtained from plasma. The majority of HIV-1 V3 variants present in the lymph tissue could be found in the plasma viral population obtained at autopsy and in the sequential blood samples obtained before death, but were absent from the cardiac blood provirus population obtained at autopsy. However, sequence variants found in the brain proviral DNA were not detected in either plasma or the sequential blood samples. Sequence differences were observed at the apex of the V3 loop between HIV-1 variants present in sequential blood samples and in blood lymphocytes and nervous tissue, lymph tissue and plasma obtained post-mortem. The potential effect of lymph tissue on the long-term persistence of different viral variants is discussed. Virus obtained from the two sequential blood samples produced syncytia in primary cultures and was easily transmitted to the continuous JM cell line. Consensus (majority) V3 loop sequences determined for the adapted viruses demonstrated that some, but not all, sequences were represented within the in vivo viral population.