εγ-Thalassemia, a New Hemoglobinopathy Category.

BACKGROUND: Large ß-globin gene cluster deletions (hereditary persistence of fetal hemoglobin [Hb] or ß-, δß-, γδß-, and ϵγδß-thalassemia), are associated with widely disparate phenotypes, including variable degrees of microcytic anemia and Hb F levels. When present, increased Hb A2 is used as a surrogate marker for ß-thalassemia. Notably, ϵγδß-thalassemias lack the essential regulatory locus control region (LCR) and cause severe transient perinatal anemia but normal newborn screen (NBS) results and Hb A2 levels. Herein, we report a novel deletion of the ϵ, Aγ, Gγ, and ψß loci with intact LCR, δ-, and ß-regions in 2 women and newborn twins. METHODS: Capillary electrophoresis (CE), high-performance liquid chromatography (HPLC), DNA sequencing, multiplex ligation-dependent probe amplification (MLPA), gap-polymerase chain reaction (gap-PCR), and long-read sequencing (LRS) were performed. RESULTS: NBS showed an Hb A > Hb F pattern for both twins. At 20 months, Hb A2 was increased similarly to that in the mother and an unrelated woman. Unexplained microcytosis was absent and the twins lacked severe neonatal anemia. MLPA, LRS, and gap-PCR confirmed a 32 599 base pair deletion of ϵ (HBE1) through ψß (HBBP1) loci. CONCLUSIONS: This deletion represents a hemoglobinopathy category with a distinct phenotype that has not been previously described, an ϵγ-thalassemia. Both the NBS Hb A > F pattern and the subsequent increased Hb A2 without microcytosis are unusual. A similar deletion should be considered when this pattern is encountered and appropriate test methods selected for detection. Knowledge of the clinical impact of this new category will improve genetic counselling, with distinction from the severe transient anemia associated with ϵγδß-thalassemia.

Immunohistochemistry for LEF1 and SOX11 adds diagnostic specificity in small B-cell lymphomas.

Lymphocyte enhancer-binding factor 1 (LEF1) and SRY-Box 11 (SOX11) are highly sensitive and specific for chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL) and mantle cell lymphoma (MCL) including the cyclin D1-negative subtype, respectively. We assessed the utility of these markers in a large cohort of small B-cell lymphomas (SBCLs) on varied sample types. Immunohistochemistry (IHC) was performed for LEF1 and SOX11 on 354 SBCLs (129 CLL/SLLs, 33 MCLs, 142 marginal zone lymphomas [MZLs]-nodal MZL [NMZL]: 40, extranodal MZL [ENMZL]: 28, splenic MZL [SMZL]: 74 cases-and 50 lymphoplasmacytic lymphomas [LPLs]/Waldenstrom macroglobulinemias [WMs]). Ninety-eight percent of CLL/SLLs were LEF1 positive. SOX11 showed good sensitivity (82%) and excellent specificity for MCL (99%), with only 2 of 142 MZLs (both SMZLs) showing SOX11 expression. The low sensitivity for SOX11 was on account of inclusion of 4 non-nodal cases. All 50 LPL/WMs were negative for both LEF1 and SOX11. The expression of SOX11 and LEF1 was not always mutually exclusive, as 2 confirmed MCLs expressed both markers. LEF1 and SOX11 have excellent utility as diagnostic markers especially for atypical CD5-positive SBCLs.

Improved Characterization of Complex ß-Globin Gene Cluster Structural Variants Using Long-Read Sequencing.

Complex insertion-deletion (indel) events in the globin genes manifest in widely variable clinical phenotypes. Many are incompletely characterized because of a historic lack of efficient methods. A more complete assessment enables improved prediction of clinical impact, which guides emerging therapeutic choices. Current methods have limited capacity for breakpoint assignment and accurate assessment of mutation extent, especially in cases containing duplications or multiple deletions and insertions. Technology, such as long-read sequencing, holds promise for significant impact in the characterization of indel events because of read lengths that span large regions, resulting in improved resolution. Four known complex ß-globin gene cluster indel types were assessed using single-molecule, real-time sequencing technology and showed high correlation with previous reports, including the Caribbean locus control deletion (g.5,305,478_5,310,336del), a large ß-gene duplication containing the Hb S mutation (g.4,640,335_5,290,171dup with g.5,248,232T>A, c.20A>T; variant allele fraction, 64%), and two nested variants (double deletions with intervening inversion): the Indian Gγ(Aγδß)0-thalassemia (g.5,246,804-5,254,275del, g.5,254,276_5,269,600inv, and g.5,269,601_5,270,442del) and the Turkish/Macedonian (δß)0 thalassemia (g.5,235,064_5,236,652del, g.5,236,653_5,244,280inv, and g.5,244,281_5,255,766del). Our data confirm long-read sequencing as an efficient and accurate method to identify these clinically significant complex events. Limitations include high-complexity sample preparation requirements, which hinder routine use in clinical laboratories. Continued improvements in sample and data workflow processes are needed to accommodate volumes in a tertiary clinical laboratory.

Interpreting sulfhemoglobin and methemoglobin in patients with cyanosis: An overview of patients with M-hemoglobin variants.

INTRODUCTION: Methemoglobin (MetHb) and sulfhemoglobin (SHb) measurements are useful in the evaluation of cyanosis. When one or both values are elevated, additional analysis is important to establish the etiology of the disorder. Methemoglobinemia occurs from acquired or hereditary causes with diverse treatment considerations, while true sulfhemoglobinemia is only acquired and treatment is restricted to toxin removal. Some toxic exposures can result in a dual increase in MetHb and SHb. Hereditary conditions, such as M-Hemoglobin variants (M-Hbs), can result in increased MetHb and/or SHb values but are clinically compensated and do not require treatment if they are cyanotic but otherwise clinically well. METHODS: Herein, we report 53 hemoglobin variant cases that have associated MetHb and SHb levels measured by an adapted Evelyn-Malloy laboratory assay method. RESULTS: Our data indicate M-Hbs cause variable patterns of MetHb and SHb elevation in a fairly reproducible pattern for the particular variant. In particular, α globin chain M-Hbs can mimic acquired sulfhemoglobinemia due to an isolated increased SHb value. CONCLUSION: If the patient appears clinically well other than cyanosis, M-Hbs should be considered early in the evaluation process to differentiate from acquired conditions to avoid unnecessary testing and treatment regimens and prompt genetic counseling.

Genetic and Clinical Studies of Patients With Increased Multinucleated Megakaryocytes in Bone Marrow as an Isolated Finding: A Diagnostic Pitfall for Myelodysplastic Syndrome.

The presence of increased multinucleated megakaryocytes (aka osteoclast-like) is considered a dysplastic feature in myelodysplastic syndrome; however, its clinical significance in isolation is uncertain. Herein, we report the clinicopathologic and genetic features of 18 such cases of 40,539 bone marrow biopsies spanning 10 years. All 18 patients had ≥25% multinucleated megakaryocytes in otherwise normal bone marrow biopsies, which were evaluated for plasma cell neoplasms (n=9), lymphoma (n=4), or anemia/neutropenia (n=5). None of the 17 patients tested showed acquired cytogenetic abnormalities. Sixteen patients underwent targeted gene panel next-generation sequencing: 9 patients had no pathogenic mutations; 3 harbored a single pathogenic mutation with variant allele frequencies of 7.5%, 7.6%, and 10.7%, likely representing clonal hematopoiesis of indeterminate potential; 1 had 2 pathogenic mutations, 1 of which had a variant allele frequency >20%. Fourteen of 18 patients had a follow-up period >6 months (median: 36.5 mo, range: 7 to 110 mo) and no patients developed a new-onset cytopenia, a progressive cytopenia, or a myeloid neoplasm. The patient with 2 mutations had persistent anemia, worrisome for an emerging MDS. However, given the absence of thrombocytopenia, increased multinucleated megakaryocytes in this patient could be an unrelated incidental finding. Our study indicates that increased multinucleated megakaryocytes as an isolated finding is a rare phenomenon, and this sole morphologic finding is not diagnostic of myelodysplastic syndrome. Diagnostic approaches in the presence of increased multinucleated megakaryocytes are proposed based on different clinical and pathologic scenarios.

Further Characterization of Hb Bronovo [α103(G10)His→Leu; HBA2: c.311A>T] and First Report of the Homozygous State.

Hb Bronovo [α103(G10)His→Leu, HBA2: c.311A>T] is an α-globin variant that interferes with and decreases binding efficiency to α hemoglobin (Hb) stabilizing protein (AHSP), a chaperone molecule. The histidine residue at position 103 is integral to the AHSP hydrogen bond formation where disruption results in an increased quantity of cytotoxic free α-globin chains, thereby creating a similar pathophysiology as ß-thalassemia (ß-thal). We report a family with Hb Bronovo, including a homozygous proband, which resulted from maternal uniparental disomy (UPD). Although not detected by routine studies in previous reports, the variant protein is visible by intact mass spectrometry (MS).

Mixed-phenotype large granular lymphocytic leukemia: a rare subtype in the large granular lymphocytic leukemia spectrum.

Large granular lymphocytic leukemia (LGLL) is a chronic proliferation of cytotoxic lymphocytes in which more than 70% of patients develop cytopenia(s) requiring therapy. LGLL includes T-cell LGLL and chronic lymphoproliferative disorder of natural killer (NK) cells. The neoplastic cells in LGLL usually exhibit a single immunophenotype in a patient, with CD8-positive/αß T-cell type being the most common, followed by NK-cell, γδ T-cell, and CD4-positive/αß T-cell types. We investigated a total of 220 LGLL cases and identified 12 mixed-phenotype LGLLs (5%): 7 cases with coexistent αß T-cell and NK-cell clones and 5 with coexistent αß and γδ T-cell clones. With a median follow-up of 48 months, the clinicopathological characteristics of these patients seemed similar to those of typical LGLL patients. Treatment was instituted in 9 patients, and 5 patients (55%) attained complete hematologic response or partial response. The therapeutic response rate of this cohort is comparable to the reported overall response rate of 40% to 60% in typical LGLL patients. Three patients who did not receive any treatment had progressive or persistent cytopenias. Interestingly, inverted proportions of 2 clones at disease recurrence were identified in 4 patients (36%) and stable clonal proportions in 7 patients (64%). Mixed-phenotype LGLL is rare, and this study underscores the importance of recognizing this rare type of LGLL in patients who may benefit from LGLL treatment.

Nodular Lymphocyte Predominant Hodgkin Lymphoma of the Ileum.

Nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) is a rare lymphoma derived from germinal center B lymphocytes that typically presents with localized lymph node involvement and can mimic a variety of both reactive and other neoplastic conditions. Extranodal involvement is uncommon in NLPHL and typically occurs in the context of previously documented or synchronous nodal disease. Involvement of the gastrointestinal tract is exceedingly rare. Here, we present the first case to our knowledge of NLPHL involving the ileum that was discovered incidentally on routine screening colonoscopy in an asymptomatic patient. An awareness of the spectrum of clinical presentations, careful morphologic evaluation, and a comprehensive panel of immunohistochemical stains are essential for correct diagnosis of NLPHL presenting in unusual anatomic sites.

BCR-JAK2 fusion in a myeloproliferative neoplasm with associated eosinophilia.

Janus kinase 2 (JAK2) is located on chromosome 9 at band p24 and JAK2V617F is the most common mutation in Philadelphia chromosome-negative myeloproliferative neoplasms (Ph-MPN). However, rearrangement of JAK2 is a rare event. We report a case of myeloproliferative neoplasm, unclassifiable (MPN-U) with BCR-JAK2 fusion confirmed by molecular studies. Conventional chromosome analysis (CC) revealed t(9;22)(p24;q11.2) and fluorescence in situ hybridization (FISH) showed a JAK2 gene rearrangement in 88% of interphase nuclei. The BCR-JAK2 fusion was confirmed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) and demonstrated two in-frame 5'BCR/3'JAK2 transcripts with BCR exon 1 juxtaposed to JAK2 exon 15 and exon 17, respectively. Our results, together with literature review, reveal BCR-JAK2 fusions as oncogenic genetic alterations that are associated with myeloid or lymphoid neoplasms and are frequently characterized by eosinophilia. Further, patients with BCR-JAK2 are candidates for JAK2 inhibitor therapy. Given the distinct clinical and pathological characteristics, we believe that hematological neoplasms harboring BCR-JAK2 should be included as an additional distinct entity to the current WHO category of "myeloid and lymphoid neoplasms with eosinophilia and abnormalities of PDGFRA, PDGFRB, or FGFR", and testing for a JAK2 fusion should be pursued in neoplasms with a karyotypic 9p24 abnormality.

Clinical and laboratory issues associated with persistent low-level elevations of hCG in a female of childbearing age.

Persistent low-level elevations of serum concentrations of hCG in a non-pregnant female of childbearing age were investigated by a number of laboratory techniques including heterophile blocking reagents, polyethylene glycol precipitation, serial dilutions and hCG measurements on several different instrument/reagent systems. The results of these studies indicated that this patient had immunoreactive hCG in her serum that was not the intact hCG molecule but primarily the free ß-hCG subunit. Differential diagnoses are discussed along with recommendation for continued surveillance of serum hCG concentrations.

Aberrant crypt focus and fragile histidine triad protein in sporadic colorectal carcinoma.

AIM: To characterize aberrant crypt focus (ACF) in adjoining mucosa in sporadic colorectal carcinoma and to evaluate fragile histidine triad (Fhit) protein and Ki67. METHODS: ACF was identified grossly and classified histologically in 75 resected specimens. ACF was typed into hyperplastic ACF (HACF) and dysplastic ACF (DACF). Sections of ACF, carcinoma and normal colonic mucosa as control were studied for Fhit and Ki67 expressions by immunohistochemistry and were grouped according to staining intensity and the number of positive stained cells observed in different histological groups. Comparison was done between the different groups by Pearson's χ(2) test and γ test for the ordinal data. P value < 0.05 was considered as significant. RESULTS: Age range was 40 to 86 years in males (mean = 43.36) and 45 to 70 years in females (mean = 56). HACF was identified in all cases studied in the non-tumorous colonic mucosa; ACF was observed as non-contiguous scattered foci, which supports the hypothesis of acquisition of single focus monoclonality by colonic epithelial cells in tumor generation. Twenty-four (32%) had DACF and were observed as closure to carcinoma foci. Intensity of Fhit expression: (1) HACF - 40% exhibited strong intensity, similar to normal, moderate in 36% and weak in 24%; (2) DACF - strong in 25%, moderate in 37.5% and weak in 37.5%; and (3) carcinoma - negative in 16%, strong in 43% and moderate and weak in 28.5% each. Significant difference was observed in intensity of the Fhit protein expressions by HACF and DACF (P < 0.05). Tumor in older patients showed a stronger Fhit intensity compared to younger patients (P = 0.036). Vegetarian diet intake and non-smokers showed stronger Fhit intensities. Advanced stage tumor, non-vegetarian diet and younger age was associated with loss of Fhit protein. Ki67 positivity was an extended crypt pattern in HACF and DACF showed extension up to the neck region of the crypts and surface epithelium. Carcinomas showed a marked increase in Ki67 expression (P < 0.05). Fhit protein had an inverse association with Ki67 expression. CONCLUSION: Weaker Fhit intensity was associated with smoking, non-vegetarian diet intake and increasing Ki67 expression. Loss of Fhit protein expression is possibly influenced by environmental factors like smoking and non-vegetarian diet intake.

ß-Thalassemia mutations in subjects with borderline HbA2 values: a pilot study in North India.

BACKGROUND: Interpreting hemoglobin high performance liquid chromatograms with borderline HbA2 values is often problematic, especially in antenatal cases if the partner is a known thalassemia trait. METHODS: We tested for underlying ß-thalassemia mutations in 25 subjects with borderline HbA2 values (between 3.0%-4.0%). Amplification refractory mutation system (ARMS-PCR) was used to detect the five common Indian ß-thalassemia mutations: (IVS-I-5 (G>C), IVS-I-1 (G>T), codons 8/9 (+G), codons 41/42 (-TTCT) and 619 bp deletion). ß-Globin gene sequencing was performed if no mutation was detected. RESULTS: A ß-globin gene defect was identified in 8 (32%) of the 25 cases with HbA2 levels ranging from 3.5%-3.9%. ARMS-PCR revealed IVS-I-5 (G>C) in three, 619 bp deletion in two and codons 41/42 (-TTCT) in one case. Two cases had CAP +1 (A>C) mutation on gene sequencing. IVS-I-1 (G>T) and codons 8/9 (+G) were not found in this small cohort. CONCLUSIONS: Mutation analysis should be offered to all at-risk couples with borderline HbA2, especially those with values between 3.5% and 4.0% and microcytic hypochromic indices. Significant mutations different from those in other ethnic populations were seen in this small institution-based study.

Florid plasmacytosis in a case of acute myeloid leukemia: A diagnostic dilemma.

The association of acute myeloid leukemia (AML) with plasmacytosis is a known, although rare event. There are very few case reports documenting an increase in the number of plasma cells at the time of AML diagnosis. Here, we present the case of a 65-year-old male diagnosed as acute myelomonocytic leukemia with exuberant plasmacytosis, which posed a difficulty in diagnosis. Paracrine interleukin-6 production by leukemic blast cells is thought to contribute to this associated reactive plasma cell proliferation.