Genotype Hemophilia Screening Program Identified 2 Novel Variants Including a Novel Variant (c.5816-2A > G) Causing a Pathogenic Variant of the Factor 8 Gene.

Establishing a national screening program for hemophilia patients is highly encouraged by the World Health Organization and the World Federation of Hemophilia. Hence, this study aimed to analyze the variant spectrum of F8 and F9 genes in Arab hemophilia patients. Molecular genetic and sequencing studies were performed on a cohort of 135 Saudi hemophilia patients. Out of all screened hemophilia patients (97 hemophilia A and 39 hemophilia B), 15 (11.1%) were positive for inversion 22 and 4 (3%) for inversion 1. Out of a total of 32 (23.7%) substitution/deletion mutations, 2 novel variants were identified: a novel splice acceptor site missense mutation (c.5816-2A > G) causing a pathogenic variant of the F8 gene and another splicing site point mutation in intron/exon 23 (g.164496G > A). The frequent F8 variants were (c.409A > C, p.T137P) in exon 4, (c.760A > G) in exon 6, and (c.1835G > C, p.R612P) in exon 12, while the frequent F9 variants were (c.580A > G) in exon 6 and (c.880C > T) in exon 8. These study data will enrich the spectrum of the genetic databases in the Arab population that could be applied in the future for national genetic counseling.

Successful use of emapalumab in refractory hemophagocytic lymphohistiocytosis in a child with Chédiak-Higashi syndrome: a case report.

BACKGROUND: Hemophagocytic lymphohistiocytosis is a life-threatening disease heralded by fever, cytopenia, hepatosplenomegaly, and multisystem organ failure. Its association with genetic mutations, infections, autoimmune disorders, and malignancies is widely reported. CASE PRESENTATION: A 3-year-old male Arab Saudi patient with insignificant past medical history and parental consanguinity presented with abdominal distension of moderate severity and persistent fever despite receiving antibiotics. This was accompanied by hepatosplenomegaly and silvery hair. The clinical and biochemical profiles were suggestive of Chédiak-Higashi syndrome with hemophagocytic lymphohistiocytosis. The patient received the hemophagocytic lymphohistiocytosis-2004 chemotherapy protocol and had multiple hospital admissions mainly due to infections and febrile neutropenia. After achieving the initial remission, the patient's disease reactivated and did not respond to reinduction with the hemophagocytic lymphohistiocytosis-2004 protocol. Due to the disease reactivation and intolerance of conventional therapy, the patient commenced emapalumab. The patient was successfully salvaged and underwent an uneventful hematopoietic stem cell transplantation. CONCLUSIONS: Novel agents such as emapalumab can be helpful for the management of refractory, recurrent, or progressive disease, while avoiding the toxicities of conventional therapy. Due to a paucity of available data on emapalumab, additional data are needed to establish its role in hemophagocytic lymphohistiocytosis treatment.

Hematologic reference intervals for healthy adult Saudis in Riyadh.

BACKGROUND: Laboratory hematological tests are widely used in clinical practice to assess health and disease conditions. Reference ranges provided by laboratory reports are considered the most authoritative medical tools to assist in the decision-making phase. International standards institutes recommend that reference ranges be established for each region. OBJECTIVES: Provide reference values of routine hematological parameters in Saudi adults according to age and gender. DESIGN: Cross-sectional SETTING: Central province of Saudi Arabia. PATIENTS AND METHODS: Apparently healthy Saudi adults were subjected to laboratory testing of routine hematological parameters (full blood count, hemostatic profile, and serum hematinics), after completing a detailed health medical questionnaire. MAIN OUTCOME MEASURES: Hematological reference values based on the local population. SAMPLE SIZE AND CHARACTERISTICS: 637 after screening 827 potentially healthy Saudi adults with ages ranging from 15 to 65 years. RESULTS: The reference values of routine hematological parameters for the full population and by gender are presented with 90% CI as the lower and upper limits. Reference ranges mostly differed from universal established ranges shown in textbooks. CONCLUSION: The reference ranges of routine hematologic parameters for accurate assessment and appropriate management will help improve the routine clinical care of the adult Saudi population. LIMITATIONS: Difficulty in assessing health status of participants, who could have subclinical illnesses not reflected in the evaluated blood measurement. Lack of ability to eliminate individuals who might be carriers for haemoglobinopathies. Studies with larger sample sizes from different areas of the country are required to achieve a more accurate representation of the whole Saudi population. CONFLICT OF INTEREST: None.

Spectrum of Myelodysplastic Syndrome in Patients Evaluated for Cytopenia(s). A Report from a Reference Centre in Saudi Arabia.

BACKGROUND/OBJECTIVE: Myelodysplastic syndrome (MDS) is a clonal disorder of hematopoietic stem cells, characterized by ineffective hematopoiesis, peripheral cytopenias along with hypercellularity of the bone marrow, and marked dysplastic features. Establishing MDS diagnosis is difficult due to nonspecific clinical presentation and imprecise morphological criteria. In anticipation to improve the diagnostic approach in this field, we aimed to characterize the clinical and morphological features of patients presented with cytopenias with a special focus on MDS. METHODS: We comprehensively reviewed all medical record of patients who were referred to the hematology laboratory at KFSH-RC, Riyadh, Saudi Arabia, between January 2009 and March 2016 for evaluation of bone marrow aspirates and trephine biopsies due to severe and persistent cytopenia(s) to rule out MDS. RESULTS: A total of 183 patients, 155 adult and 28 pediatric, were identified. In the adult group, MDS was diagnosed in 82 (52.9%) patients, with a male-to-female (M:F) ratio of 1.6:1 and mean age at diagnosis of 50 years. According to the World Health Organization (WHO) 2017 criteria, MDS subtypes were as follows: MDS with single lineage dysplasia (SLD, 5%), MDS with ring sideroblasts and SLD (MDS-RS-SLD 7%), MDS with multilineage dysplasia (MDS-MLD 21%), MDS with deletion of chromosome 5q (MDS del(5q), 2%), MDS unclassifiable (MDS-U7%), hypoplastic MDS (h-MDS 4%), MDS with excess blasts-1 (MDS-EB1, 20%), MDS with excess blasts-2 (MDS-EB2, 28%), and therapy-related MDS (6%). Laboratory and morphological features were described. In both groups, cytogenetic abnormalities were classified according to the Revised International Prognostic Scoring System cytogenetic risk groups. In adults, the dominating cytogenetic abnormalities were monosomy 5 and monosomy 7 seen in 20.7% and 24.4% of patients, respectively. Peripheral cytopenia not due to MDS was diagnosed in 54 (34.8%) patients, with a mean age of 43 years and M:F ratio of 1:1. The cause of these cytopenias were as follows: bone marrow failure (BMF, 22%), peripheral destruction (20%), drug induced (20%), anemia of chronic disease (16%), B12 deficiency (7%), infection (7%), paroxysmal nocturnal hemoglobinuria (4%), idiopathic cytopenia of undetermined significance (2%), and idiopathic dysplasia of undetermined significance (2%). A definite diagnosis of MDS was not possible in 19 patients due to insufficient clinical data. In the pediatric group, MDS was diagnosed in 14/28 (50%) patients, with M:F ratio of 1.8:1 and mean age at diagnosis of 4 years. MDS subtypes (WHO 2017) in 14 patients were as follows: refractory cytopenia of childhood (RCC, 42.8%), MDS-EB1 (42.8%), and MDS-EB2 (14.2%). Laboratory and morphological features were described. The prevalent cytogenetic abnormality was monosomy 7 in six/14 (42.8%) patients. Cytopenias due to other causes were diagnosed in eight/28 patients (28.5%), with a mean age of 6.5 years and M:F ratio of 1.6:1. The causes of non-MDS related cytopenia were: congenital BMF (4 patients), peripheral destruction (2 patients), immune deficiency (1 patient), and viral infection (1 patient). A definite diagnosis of MDS could not be made in six/28 (21.4%) patients. CONCLUSION: MDS is the cause of cytopenia in a significant number of patients referred for evaluation of cytopenias, appears at younger age, and tends to be more aggressive than that reported in international studies. Anemia, dysplastic neutrophils in the peripheral blood, and dysplastic megakaryocytes in the bone marrow trephine biopsy are the most reliable features in distinguishing MDS from other alternative diagnoses.

The Diagnostic Value of New Additional Antiphospholipid Antibodies in Antiphospholipid Syndrome.

BACKGROUND: The antiphospholipid syndrome (APS) is an autoimmune disease that is characterized by thrombosis and/or pregnancy failure and associated with the presence of all or at least one of three standard antibodies (anti-phospholipid (aPL) antibodies, including lupus anticoagulant (LA), anti-cardiolipin (aCL), and anti-ß2-glycoprotein I (anti-ß2GPI)). A growing body of evidence recommends adding additional aPL antibodies, such as anti-phosphatidylserine (aPS), anti-prothrombin (aPT), and anti-annexin A5 (aAA5), to conventional laboratory tests (revised Sapporo criteria), especially in seronegative APS cases. OBJECTIVES: We aimed to compare the diagnostic value, utility, and performance of these three additional antibodies along with the standard aPL antibodies in cases with confirmed and non-criteria APS (seronegative). METHODS: This was a prospective observational study on 59 patients who presented with clinical features of APS at the hematology, medical, rheumatology, and obstetric clinics. LA was detected by standard coagulation tests, while other aPL, IgG, and IgM antibodies (aCL, aß2GPI, aPS, aPT, aAA5) were detected with enzyme-linked immunosorbent assay (ELISA). RESULTS: Anti-PS antibody was more frequent compared to aPT and aAA5 in both confirmed cases (84.6%) and non-criteria (seronegative) (15.4%) APS. As a single test, the aPS antibody was significantly better (P<0.05) than the aPT and aAA5 antibodies in the detection of APS cases. Seven non-criteria patients were confirmed using additional aPL antibodies. Among these patients, four, two, and one patient was confirmed with aPS, aPT, and aAA5 antibodies, respectively. CONCLUSION: Our data support the findings of previously published studies and attribute the clinical significance of additional aPL antibodies, particularly aPS, in identifying non-criteria APS cases. In the future, along with conventional aPL antibodies, these additional antibodies should be included as standard laboratory tests in the revised Sapporo criteria.

Disseminated histoplasmosis in a heart transplant recipient from Saudi Arabia: A case report.

Histoplasma is endemic in North and Central America. We describe a case of disseminated histoplasmosis in a heart transplant recipient outside the known endemic areas. A 68-year-old gentleman known to have dilated cardiomyopathy. He underwent left ventricular assist device (LVAD) implantation in India and 2 years later did heart transplant in King Faisal Specialist and Research Center Hospital. Six weeks post-transplant he presented with headache and fever. All investigations were negative, and he was discharged home. Four days after discharge he presented with headache, fever, blurred vision, and an episode of loss of consciousness. Examination showed an ill looking patient who is highly febrile. Repeated work up showed pancytopenia. A repeat LP was negative. Bone marrow biopsy showed Small intracellular organisms. Extended work up revealed a positive Histoplasma urinary antigen, positive Histoplasma PCR from the bone marrow biopsy. Patient was started on Liposomal Amphotericin followed by Itraconazole with marker clinical improvement. This is the first reported case of disseminated Histoplasmosis in Saudi Arabia. We postulate that the patient had reactivation of a latent infection acquired at the time of LVAD insertion in India rather than donor derived infection by the negative fungal culture and PCR done on the donor's lung granuloma tissue.

Early T precursor acute lymphoblastic leukaemia/lymphoma shows differential immunophenotypic characteristics including frequent CD33 expression and in vitro response to targeted CD33 therapy.

The differential immunophenotypic characteristics of early T precursor (ETP) acute lymphoblastic leukaemia/lymphoma (ALL) remain incompletely characterized. The study group (n = 142) included 106 (74·7%) men and 36 (25·3%) women with a median age of 34·9 years (range, 2-79) at diagnosis. Patients were subtyped by flow cytometry immunophenotyping as follows: 33 (23·2%) ETP; 32 (22·5%) early non-ETP; 60 (42·2%) thymic; and 17 (12·1%) mature. Excepting definitional markers, there was a significant differential expression of the markers CD2, CD10, CD33 and TdT between ETP-ALL and non-ETP-ALL. Positive CD33 expression (≥20% of leukaemic blasts) was detected in 21/33 (63%) ETP-ALL compared with 17/95 (17·9%) non-ETP-ALL (P < 0·001). Notably, targeted anti-CD33 therapy with IMGN779 resulted in significant growth inhibition and increased apoptosis in ETP-ALL cells in vitro. An 11-marker T-ALL immunophenotype score discriminated reliably between ETP and non-ETP ALL. Longitudinal analysis of ETP-ALL cases in this study demonstrated that the immunophenotype may be occasionally dynamic but is largely stable over the disease course. In summary, identification of ETP-ALL might be enhanced by using an 11-marker T-ALL immunophenotype score. CD33 expression is frequent in ETP-ALL, and in vitro data suggest that exploring anti-CD33 therapy in ETP-ALL is warranted.

CD123 expression patterns and selective targeting with a CD123-targeted antibody-drug conjugate (IMGN632) in acute lymphoblastic leukemia.

The potential of CD123-targeted therapies in acute lymphoblastic leukemia/lymphoma remains largely unexplored. We examined CD123 expression levels in a large cohort of patients with acute lymphoblastic leukemia/lymphoma and assessed the in vitro impact of IMGN632, a conjugate of CD123-binding antibody with a novel DNA-alkylating payload. CD123 expression on leukemic blasts was surveyed using multicolor/multiparameter flow cytometry. The in vitro effect of IMGN632 was evaluated on B acute lymphoblastic leukemia/lymphoma cell lines and primary B acute lymphoblastic leukemia/lymphoma blasts. The study cohort (n=213) included 183 patients with B acute lymphoblastic leukemia/lymphoma and 30 with T acute lymphoblastic leukemia/lymphoma. CD123 expression was more prevalent in B acute lymphoblastic leukemia/lymphoma than in T acute lymphoblastic leukemia/lymphoma (164/183, 89.6% versus 13/30, 43.3%; P<0.0001), and within B acute lymphoblastic leukemia/lymphoma CD123 expression was more prevalent in Philadelphia chromosome-positive patients than in Philadelphia chromosome-negative patients (96.6% versus 86.3%; P=0.033). In T acute lymphoblastic leukemia/lymphoma, 12/13 (92.3%) patients with CD123-positive blasts had either early T precursor (ETP) or early non-ETP immunophenotype. IMGN632 was highly cytotoxic to B acute lymphoblastic leukemia/lymphoma cell lines, with half maximal inhibitory concentrations (IC50) between 0.6 and 20 pM. In five of eight patients' samples, low picomolar concentrations of IMGN632 eliminated more than 90% of the B acute lymphoblastic leukemia/lymphoma blast population, sparing normal lymphocytes. In conclusion, CD123 expression is prevalent across acute lymphoblastic leukemia/lymphoma subtypes, and the CD123-targeted antibody-drug conjugate IMGN632 demonstrates promising selective activity in preclinical models of B acute lymphoblastic leukemia/lymphoma.

Ultra-Rapid Reporting of GENomic Targets (URGENTseq): Clinical Next-Generation Sequencing Results within 48 Hours of Sample Collection.

Next-generation sequencing (NGS)-based mutation panels profile multiple genes simultaneously, allowing the reporting of numerous genes while saving labor and resources. However, one drawback of using NGS is that the turnaround time is often longer than conventional single gene tests. This delay can be problematic if molecular results are required to guide therapy in patients with clinically aggressive diseases, such as acute myeloid leukemia. To overcome this limitation, we developed a novel custom platform designated as Ultra-rapid Reporting of GENomic Targets (URGENTseq), an integrated solution that includes workflow optimization and an innovative custom bioinformatics pipeline to provide targeted NGS results on fresh peripheral blood and bone marrow samples within an actionable time period. URGENTseq was validated for clinical use by determining mutant allelic frequency and minimum coverage in silico to achieve 100% concordance for all positive and negative calls between the URGENTseq and conventional sequencing approach. URGENTseq enables the reporting of selected genes useful for immediate diagnosis (CALR, CSF3R, JAK2, KRAS, MPL, NPM1, NRAS, SF3B1) and treatment decisions (IDH1, IDH2) in hematologic malignancies within 48 hours of specimen collection. In addition, we summarize the molecular findings of the first 272 clinical test results performed using the URGENTseq platform.

Chronic Myelomonocytic Leukemia With Fibrosis Is a Distinct Disease Subset With Myeloproliferative Features and Frequent JAK2 p.V617F Mutations.

A subset of patients with chronic myelomonocytic leukemia (CMML) presents with significance myelofibrosis. In myelodysplastic syndromes, significant myelofibrosis has been associated with adverse outcomes and p53 dysregulation. However, in CMML the clinical and molecular correlates of significant myelofibrosis at presentation remain poorly understood. From a cohort of 651 CMML patients, we identified retrospectively 20 (3.1%) cases with moderate to severe reticulin fibrosis (CMML-F) detected at diagnosis, and we compared them to CMML patients without fibrosis (n=631) seen during the same period. Patients with CMML-F had a median age of 69.8 years (range, 24.8 to 91.2 y) and most (13; 65%) were men. Patients with CMML-F differed significantly from other CMML patients across the following parameters: white blood count, absolute monocyte count, serum lactate dehydrogenase level, splenomegaly, and bone marrow blast percentage. Notably, the frequency of JAK2 p.V617F mutation was higher in CMML-F patients compared with other CMML patients (P<0.001). Most CMML-F patients (12/20; 60%) had myeloproliferative CMML. Dysregulation of p53 was uncommon in CMML-F. CMML-F patients tended to have a shorter median overall survival compared with other CMML patients (P=0.079). Multivariate analysis using the Cox proportional hazards model showed an independent association between CMML-F and overall survival (P=0.047). In summary, unlike typical CMML, CMML-F is commonly associated with JAK2 p.V617F. The high frequency of myeloproliferative features and JAK2 p.V617F mutation, and the low frequency of p53 dysregulation, suggest that fibrosis in the context of CMML has a different pathogenesis from that previously reported in myelodysplastic syndrome.

Chronic lymphoproliferative disorder of NK-cells: A single-institution review with emphasis on relative utility of multimodality diagnostic tools.

BACKGROUND: Chronic lymphoproliferative disorder of NK-cells (CLPD-NK) manifests as a persistent increase (≥2 × 109 /L, for > 6 months) of mature NK-cells in peripheral blood with an indolent clinical course. The disease is rare, and only limited case series have been published. METHODS: We retrospectively studied 11 patients with CLPD-NK diagnosed at our institution between 2005 and 2017. RESULTS: Patients included 7 men and 4 women with a median age of 60 years (range, 25-89 years). Ten patients (91%) had cytopenias. Bone marrow involvement by CLPD-NK ranged from 5-15%. The most commonly detected antigenic aberrancies by  low cytometry immunophenotyping were as follows: CD7decreased/dim (30%), CD8uniform+ (36%), CD56-/partial (73%), CD94bright (55%), and KIR restriction (100%). JAK/STAT pathway mutations were detected in 8 of 10 (80%) patients and involved STAT3 (n = 7) and JAK3 (n = 1). The presence of mutations tended to correlate with the occurrence of other cytopenias (anemia/thrombocytopenia) and requirement for treatment. Seven patients received single-agent therapy, with amelioration of symptoms; 4 patients were observed. There were no disease-associated deaths or progression to more aggressive disease during the follow-up interval (median, 17 months). CONCLUSIONS: Patients with CLPD-NK have an indolent clinical course and frequent hematologic manifestations that are responsive to single-agent therapy. Mutations in STAT3 are common and portend more pronounced clinical manifestations.