Mechanisms of central nervous system disease in childhood acute lymphoblastic leukaemia

Acute lymphoblastic leukaemia (ALL) is the commonest childhood malignancy. Once a universally fatal disease, modern therapy has achieved excellent outcome and the majority of children achieve long term cure. Yet relapse remains a challenge. One of the major hurdles in achieving complete cure is the relapse of ALL at extramedullary sites such as the central nervous system (CNS). Despite significant advances in understanding leukaemia biology, most predictors of leukaemic behaviour are accurate for the bone marrow disease only. The precise timing, frequency and properties of CNS infiltrating leukaemic cells are not elucidated. Therefore, the broad aim of this thesis is to develop a better understanding of the mechanisms of leukaemic entry and infiltration patterns of leukaemic cells in the CNS. In order to address the frequency and pattern of CNS infiltration, a xenograft model using primary leukaemic cells from children with B-cell precursor (BCP) ALL in NOD/Scid IL2Rγ null (NSG) mice was established. The majority of samples from children with and without overt CNS disease were able to infiltrate the CNS in NSG mice. CNS infiltration was seen in mice engrafted with small numbers of cells and distinct immunophenotypic subpopulations. The leukaemic samples followed a distinct and reproducible pattern of CNS infiltration with leukaemic infiltrates in the meninges while sparing the CNS parenchyma. To investigate whether a distinct set of leukocyte trafficking molecules provided tissue specificity for entering the CNS, leukaemic cells retrieved from the bone marrow and the CNS were assessed for expression levels of selected chemokine receptors and P-selectin glycoprotein ligand-1 (PSGL1). Additionally, chemotaxis assays were utilized to investigate the function of the chemokine receptor CXCR4. Despite surface expression of chemokine receptors on leukaemic cells and presence of chemokines in the CNS, no evidence for positive selection of a high-expressing subpopulation was seen. Overall, it appears that, unlike the bone marrow, chemokine receptors do not direct leukaemic cell trafficking to the CNS. To investigate the published observation that interleukin-15 (IL-15) expression in leukaemic samples correlates with the risk of CNS disease, the effects of IL-15 stimulation on BCP-ALL cells were assessed. IL-15 and IL-15 receptor subunits were noted to be expressed at mRNA level in samples from BCP-ALL patient and cell lines. Exogenous IL-15 stimulated leukaemic cell proliferation and upregulated genes associated with migration and invasion in SD1 cells. A higher proliferative advantage was observed at low-serum conditions which mimics conditions found in the CNS. Therefore a plausible mechanistic link was established for the association of CNS disease with high IL-15 expression levels. In cerebrospinal fluid (CSF) samples from patients, quantitative PCR (qPCR) was utilized to detect submicroscopic levels of CNS disease. In approximately 40% patients, qPCR using patient specific primers tested positive for the presence of leukaemic DNA. Therefore this test is much more sensitive than conventional diagnostic techniques which only detect CNS disease in 2-5% of patients. CSF supernatants were also tested to assess whether the levels of chemokines could be used to diagnose patients with qPCR positive disease. Although differences in the levels of chemokines between qPCR positive and negative patients were noted, the values are not sufficiently discriminatory to be clinically useful. In conclusion, CNS entry appears to be a much more frequent property of leukaemic cell than previously appreciated. Leukocyte trafficking molecules do not appear to play an instructive role in the CNS entry and therefore, it is unlikely that expression levels of leukocyte trafficking molecules or the levels of chemokines in the CSF will be useful biomarkers of CNS disease. In addition, CNS disease appears to be present at diagnosis in at least 40% of patients. Therefore, attempts at blocking leukaemic entry into the CNS are unlikely to be therapeutically useful. Instead, analysing and targeting factors that allow long-term survival of leukaemic cells in the CNS may be a better strategy to eradicate CNS disease and prevent leukaemic relapse

Cancer biomarker development from basic science to clinical practice

The amount of published literature on biomarkers has exponentially increased over the last two decades. Cancer biomarkers are molecules that are either part of tumour cells or secreted by tumour cells. Biomarkers can be used for diagnosing cancer (tumour versus normal and differentiation of subtypes), prognosticating patients (progression free survival and overall survival) and predicting response to therapy. However, very few biomarkers are currently used in clinical practice compared to the unprecedented discovery rate. Some of the examples are: carcino-embryonic antigen (CEA) for colon cancer; prostate specific antigen (PSA) for prostate; and estrogen receptor (ER), progesterone receptor (PR) and HER2 for breast cancer. Cancer biomarkers passes through a series of phases before they are used in clinical practice. First phase in biomarker development is identification of biomarkers which involve discovery, demonstration and qualification. This is followed by validation phase, which includes verification, prioritisation and initial validation. More large-scale and outcome-oriented validation studies expedite the clinical translation of biomarkers by providing a strong ‘evidence base’. The final phase in biomarker development is the routine clinical use of biomarker. In summary, careful identification of biomarkers and then validation in well-designed retrospective and prospective studies is a systematic strategy for developing clinically useful biomarkers

Determinants of response at 2 months of treatment in a cohort of Pakistani patients with pulmonary tuberculosis

Mycobacterium tuberculosis infection continues to be a major global challenge. All patients with pulmonary tuberculosis are treated with a standard 6-month treatment regimen. Historical data suggest that even with shortened treatment, most patients achieve long-term remission. Risk stratification is a goal for reducing potentially toxic prolonged treatment. This study aimed to determine the factors associated with the early clearance of sputum acid-fast bacilli (AFB). A total of 297 freshly diagnosed patients with pulmonary tuberculosis were included and enrolled in this study. Information related to their ethno-demographic and anthropometric characteristics was collected. We also assessed their complete blood counts, and blood iron, folate, and vitamin B12 levels. We found that the presence of higher levels of acid-fast bacilli (AFB) in diagnostic sputum microscopy was the single most significant prognostic factor associated with early clearance of sputum AFB after 2 months of treatment. All of our patients achieved treatment success after 6 months of treatment and were disease free. Our results support the data obtained from previous studies indicating that AFB clearance at 2 months is unlikely to be a clinically useful biomarker or indicator for therapeutic stratification. Furthermore, demographic, anthropometric, and nutritional factors are not clinically useful biomarkers

Use of quantitative polymerase chain reaction (qPCR) for the diagnosis and monitoring of CNS leukaemia

No abstract available

Cancer biomarker development from basic science to clinical practice

The amount of published literature on biomarkers has exponentially increased over the last two decades. Cancer biomarkers are molecules that are either part of tumour cells or secreted by tumour cells. Biomarkers can be used for diagnosing cancer (tumour versus normal and differentiation of subtypes), prognosticating patients (progression free survival and overall survival) and predicting response to therapy. However, very few biomarkers are currently used in clinical practice compared to the unprecedented discovery rate. Some of the examples are: carcino-embryonic antigen (CEA) for colon cancer; prostate specific antigen (PSA) for prostate; and estrogen receptor (ER), progesterone receptor (PR) and HER2 for breast cancer. Cancer biomarkers passes through a series of phases before they are used in clinical practice. First phase in biomarker development is identification of biomarkers which involve discovery, demonstration and qualification. This is followed by validation phase, which includes verification, prioritisation and initial validation. More large-scale and outcome-oriented validation studies expedite the clinical translation of biomarkers by providing a strong ‘evidence base’. The final phase in biomarker development is the routine clinical use of biomarker. In summary, careful identification of biomarkers and then validation in well-designed retrospective and prospective studies is a systematic strategy for developing clinically useful biomarkers

Trajectory of anti-SARS-COV-2 IgG antibodies from onset of symptoms to 6 months in a cohort of Pakistani patients

No abstract available

Clinical and laboratory relevance of JAK2 V617F and BCR-ABL co-existence in Philadelphia positive CML patients

Chronic Myeloid Leukaemia (CML) is characterized by BCR-ABL1 mutation. A number of research studies have published reports of concomitant JAK2-V617F mutation in BCR-ABL positive Chronic Myeloid Leukaemia. This study aims to investigate the frequency of JAK2-V617F mutation in BCR-ABL positive CML cases. After approval from ethical committee, participants were enrolled in the study. A total of 103 samples from CML patients were analysed for the presence of JAK2-V617F mutation using real-time polymerase chain reaction. Patients were monitored for treatment response using real-time quantitative PCR for BCR-ABL1 mutation. Out of 103 samples analysed, 2 patients tested positive for JAK2-V617F mutation. These two patients when treated with standard Tyrosine Kinase Inhibitors (TKI) therapy achieved molecular response and normalized the haemoglobin and white cell counts. However, one patient has sustained thrombocytosis. JAK2 remained positive throughout the treatment course. We could not follow the second patient till the end of the study. JAK2 mutation in BCR-ABL1 mutated CML appears to be rare. Treatment with TKI does not appear to reduce JAK2 mutation burden despite a decrease in BCR-ABL1 copy numbers

Co-existing iron deficiency/overload in beta-thalassemia trait

To identify the co-existence of iron deficiency and iron overload in individuals with beta thalassaemia trait. The cross-sectional study was conducted at Rehman Medical Institute and Khyber Medical University, Peshawar, Pakistan, September 1, 2015, to December 31, 2017, and comprised individuals with hypochromic microcytic blood picture. Haemoglobin electrophoresis was performed on their blood samples. Serum ferritin levels of subjects with Haemoglobin Subunit Alpha 2 levels between 3.5% and 7% were checked. Data were analysed using analysed using GraphPad Prism v6. Of the 292 subjects, 159(54.5%) were males and 133(45.5%) were females. Of these, 240 (82.2%) were anaemic and 52 (17.8%) had haemoglobin within the normal range. Serum ferritin level of 55(18.8%) subjects was low and 207(70.9%) were iron-replete. Notably, 30(10.3%) subjects had serum ferritin levels higher than the reference range, and this was more common among adults (p<0.001). Ferritin levels in beta thalassaemia trait can be low, normal or higher than the normal values.

Seroprevalence of Sars-Cov-2 antibodies among eligible blood donors of Peshawar, Pakistan

To determine the seroprevalence of SARS-CoV-2 antibodies and the associated risk factors among healthy blood donors from Peshawar Pakistan, during the second and third waves of the COVID-19 pandemic. The study was conducted on 4047 healthy (with no history or symptoms of COVID-19) blood donors attending regional blood center Peshawar between Nov 2020 and June 2021. Demographic data was collected and donors were screened for the presence of anti-SARS-CoV-2 antibodies using electrochemiluminescence immunoassay (ECLIA). The mean age of the participants was 27.27±7.13 and the majority (99%) were males. Overall, 59% (2391/4047) of the blood donors were reactive for SARS-CoV-2 antibodies. An increasing trend in seropositivity was observed from 45.5% to 64.8% corresponding to the second and third wave of the pandemic in Pakistan. Logistic regression analysis revealed significantly higher odds of seropositivity among male donors compared to females. Similarly, in multivariable analysis, the odds ratio for seropositivity among blood types AB, A, and B were, 1.6, 1.4, and 1.3 (CI 95%) times higher compared to blood group O ( -value ≤0.0001). Seropositivity of SARS-CoV-2 antibodies among blood donors gradually increased during the second and third wave of the pandemic in Pakistan indicating a widespread prevalence of Covid-19 in the general population. Susceptibility to SARS-CoV-2 varies with ABO blood types, with blood group O associated with low risk of infection. [Abstract copyright: AJBR Copyright © 2022.