Loss of Full-Length GATA1 Expression in Megakaryocytes Is a Sensitive and Specific Immunohistochemical Marker for the Diagnosis of Myeloid Proliferative Disorder Related to Down Syndrome

Loss of Full-Length GATA1 Expression in Megakaryocytes Is a Sensitive and Specific... Abstract Objectives Myeloid proliferative disorders associated with Down syndrome (MPD-DS), including transient abnormal myelopoiesis and myeloid leukemia associated with Down syndrome (DS), harbor mutations of GATA1, a transcription factor essential for erythroid and megakaryocytic development. These mutations result in a N-terminally truncated GATA1 (GATA1s) and prohibit the production of the full-length GATA1 (GATA1f). Here, we demonstrate the utility of immunohistochemical GATA1f reactivity in diagnosing MPD-DS. Methods Immunohistochemical studies for GATA1f expression were performed on bone marrow biopsy specimens. Results In all cases of MPD-DS, megakaryocytes lacked GATA1f expression. In contrast, GATA1f expression was detected in megakaryocytes in all specimen types from patients without DS (normal bone marrows, pediatric myelodysplastic syndrome, juvenile myelomonocytic leukemia, adult acute megakaryocytic leukemia [pediatric and adult; without trisomy 2]), as well as normal bone marrows from patients with DS. Conclusions The lack of GATA1f expression is a sensitive and specific immunohistochemical marker for MPD-DS. GATA1, Down syndrome, Megakaryocytes, Immunohistochemistry, Myeloid proliferative disorder, Transient abnormal myelopoiesis, Myeloid leukemia related to Down syndrome Children with Down syndrome (DS) younger than age 5 years are at an increased risk for developing myeloid proliferative disorders.1,2 Per the 2016 revision to World Health Organization (WHO) classification, myeloid proliferative disorder related to DS (MPD-DS) can be subcategorized into transient abnormal myelopoiesis (TAM) and myeloid leukemia associated with DS (ML-DS).3 Approximately 10% of neonates with DS develop TAM, which exhibits overlapping morphologic and immunophenotypic features with acute myeloid leukemia.4,5 The clinical presentation of TAM can be highly variable, ranging from an asymptomatic presentation to diffuse organ infiltration. Most of the cases undergo spontaneous remission within 4 months of disease onset. However, approximately one in three cases eventually progress to ML-DS with a latency of 1 to 3 years. While the clinicopathologic manifestations resemble that of TAM,2 ML-DS does not undergo spontaneous remission. In both TAM and ML-DS, the neoplastic blasts frequently exhibit megakaryocytic markers (CD61 and CD42), stem cell markers (CD34 and CD117), and aberrant myeloid markers (CD13 and CD33).6,7 GATA binding factor 1 (GATA1) is a transcription factor essential for erythroid and megakaryocytic development.8 Somatic mutations of GATA1, found in nearly all cases of TAM and ML-DS,1 are thought to be the founder mutations of MPD-DS. These mutations result in a premature stop codon in exon 2 or 3 and force the initiation of translation from the second start codon in exon3, resulting in an N-terminally truncated form lacking the first 83 amino acid residues (GATA1s).1 Given that GATA1 is encoded on the X chromosome, these mutations always manifest with the exclusive expression of a short form of GATA1 (GATA1s) in neoplastic cells of MPD-DS. Using experimental models, the lack of full-length GATA1 (GATA1f) and the presence of GATA1s appear to promote megakaryopoieisis and impair erythropoiesis.9-11 Genomic sequencing analysis revealed that ML-DS often harbors additional recurrent mutations affecting components of cohesin complexes, epigenetic regulators, and Ras signaling pathways, representing clonal evolution from TAM.12 N-terminally truncating GATA1 mutations are considered a pathognomonic hallmark in the diagnosis and pathogenesis of TAM and ML-DS.13 In addition, tracking the level of GATA1 mutation has been suggested as a way to monitor minimal residual disease following treatment for ML-DS.7,14 In this study, we demonstrate that it is possible to gauge GATA1f expression by immunohistochemistry using a commercially available rabbit monoclonal antibody. This simple cost-effective method can be incorporated into routine pathology practice for the diagnosis of MPD-DS in adjunct to GATA1 mutation testing. Materials and Methods Antibodies Two commercially available rabbit monoclonal antibodies (mAb) against GATA1 were purchased from Cell Signaling Technology (Danvers, MA): (1) mAb D52H6 (3535) is raised against a peptide centered on Glu13 of GATA1, and (2) mAb D24E4 (4589) is raised against a peptide centered on Ala184 of GATA1. A mouse monoclonal antibody against β-actin clone AC-74 (Sigma-Aldrich, St Louis, MO) was kindly provided by Dr Jon Aster. Mouse mAb against CD61 (clone 2f2) was purchased from Leica Microsystem (Wetzlar, Germany) and mouse mAb against CD71 (clone H68.4) from Invitrogen (Carlsbad, CA). Western Blot Analysis Lysates from the erythroleukemic cell lines (K562 and HEL) were prepared using radioimmunoprecipitation lysis buffer supplemented with protease inhibitors (P2714; Sigma-Aldrich). Protein concentrations of the lysates were determined using Bradford assays per the manufacturer’s instruction (Bio-Rad, Hercules, CA) and normalized in Laemmli sample buffer with 200 mmol/L dithiothreitol. Approximately 5 µg of total protein per sample was submitted for sodium dodecyl sulfate–polyacrylamide gel electrophoresis (4%-12% gradient; Thermo Fisher, Waltham, MA) and electrotransferred onto a polyvinylidene difluoride membrane. The subsequent immunoblotting with anti-GATA1 antibodies and anti–β actin antibody (as a loading control) was performed according to the manufacturer’s instructions. Reactivity to the targeted proteins was visualized using secondary polyclonal anti–rabbit immunoglobulin G (IgG) antibody and polyclonal anti–mouse IgG antibodies conjugated with horseradish peroxidase (Cell Signaling Technology), in conjunction with chemiluminescent substrate (Thermo Fisher), following exposure to x-ray films (Labscientific, Highlands, NJ). The cell lines were kindly provided by Drs Li Chai and Jon Aster at Brigham and Women’s Hospital (Boston, MA). Collection of Bone Marrow Core Biopsy Samples Cases were retrieved from the surgical pathology archival files from the Department of Pathology, Boston Children’s Hospital (Boston, MA) and University of Rochester School of Medicine and Dentistry (Rochester, NY). This study was approved by the Brigham and Women’s Hospital Institutional Review Board. H&E-stained slides and pathology reports were reviewed for the confirmation of diagnosis. Inclusion diagnostic criteria are based on the 2016 revision to WHO classification of myeloid neoplasms and acute leukemia.3 Immunohistochemical Studies Immunohistochemical studies for GATA1f were performed using rabbit mAb D52H6 as previously described.15 Double marker studies for CD61/GATA1f and CD71/GATA1f combinations were performed on a Leica Bond III immunostainer (Leica Microsystem) as previously described.15 Antibodies specific for CD61 and CD71 were used at dilutions of 1:1,000 and 1:4,000, respectively. To detect truncated and full-length forms of GATA1, immunohistochemical studies using rabbit mAb D24E4 were performed using the following parameters. Antigen retrieval was performed using EDTA (0.001 mol/L), pH 8.0 (Invitrogen, San Francisco, CA), for 30 minutes in a steamer (model HS80; Black & Decker, Shelton, CT). The slides were allowed to cool to room temperature for 10 minutes, then washed and placed in Tris buffer (Covance, Dedham, MA). The slides were incubated with anti-GATA1 (D24E4) rabbit monoclonal antibody (Cell Signaling Technology) at a 1:100 dilution for 20 hours at room temperature. Slides were washed and then incubated for 30 minutes with a horseradish peroxidase–labeled polymer conjugated to goat anti–rabbit immunoglobulin antibody (PowerVision; Leica Microsystem, Buffalo Grove, IL). Antibody localization was achieved using a peroxidase reaction with DAB+ (Dako, Carpinteria, CA) as the chromogen. The slides were briefly enhanced with 1% copper sulfate solution, washed, counterstained with hematoxylin, dehydrated, and mounted. Results Detection of GATA1f but Not GATA1s by a Commercially Available Rabbit Monoclonal Antibody Wild-type GATA1 transcripts produce both full-length and short forms of GATA1 proteins through the use of the proper and alternative start codons.10,16,17 In MPD-DS, the GATA1 mutations often result in premature stop codons in exons 2 and 3 and prohibit the production of GATA1f. As a result, only GATA1s, which characteristically lacks the first 83 amino acid residues Figure 1A, is produced.1 We reasoned that a monoclonal rabbit antibody (D52H6) raised against a peptide (surrounding Glu13 of human GATA1) within the truncated portion of GATA1 should specifically detect GATA1f but not GATA1s (Figure 1A and Figure 1B ). We previously used this antibody to establish GATA1 as an immunohistochemical erythromegakaryocytic marker.15 Western blot analysis using cell lysates from erythroleukemia cell lines (K562 and HEL), previously shown to contain both forms of GATA1, confirms that this antibody (D52H6) only reacts to the GATA1f, whereas a different antibody (D24E4) derived from a synthetic peptide distal to the truncation detects both forms of GATA1 Image 1.16 Figure 1 View largeDownload slide Schematic diagrams illustrating the relationship between GATA1 mutations, resultant N-terminally truncated GATA1 (GATA1s), and the monoclonal antibody used for detection. A, Nonsense mutations in exons 2 and 3 force the use of an alternative start codon in exon 3 and results in GATA1s. B, A monoclonal antibody (D52H6) is raised against a peptide centered on amino acid residue 13 within the portion of full-length GATA1 (GATA1f) that is not present in GATA1s. A second monoclonal antibody (D24E4) is raised against a peptide centered on amino acid residue 184 of GATA1. Figure 1 View largeDownload slide Schematic diagrams illustrating the relationship between GATA1 mutations, resultant N-terminally truncated GATA1 (GATA1s), and the monoclonal antibody used for detection. A, Nonsense mutations in exons 2 and 3 force the use of an alternative start codon in exon 3 and results in GATA1s. B, A monoclonal antibody (D52H6) is raised against a peptide centered on amino acid residue 13 within the portion of full-length GATA1 (GATA1f) that is not present in GATA1s. A second monoclonal antibody (D24E4) is raised against a peptide centered on amino acid residue 184 of GATA1. Image 1 View largeDownload slide A Western blot with lysates from erythroleukemic cell lines (K562 and HEL) shows D52H6 recognizes full-length GATA1 (GATA1f) only, and D24E4 recognizes both forms. β-Actin reactivity serves as a protein loading control. Image 1 View largeDownload slide A Western blot with lysates from erythroleukemic cell lines (K562 and HEL) shows D52H6 recognizes full-length GATA1 (GATA1f) only, and D24E4 recognizes both forms. β-Actin reactivity serves as a protein loading control. Distribution of GATA1f Reactivity in DS-Related Myeloid Neoplasms and Other Childhood Myeloid Neoplasms Given that the truncating GATA1 mutations are pathognomonic for MPD-DS,7,12,16,18-23 we reasoned that the lack of GATA1f reactivity in megakaryocytes is likely to serve as a sensitive and specific marker for TAM and ML-DS. Using mAb D52H6, we compared GATA1f reactivity in megakaryocytes using bone marrow core biopsy specimens from MPD-DS, including TAM (n = 3) and ML-DS (n = 12), with a number of non-DS-related childhood myeloid neoplasms, including pediatric myelodysplastic syndrome (n = 6) and juvenile myelomonocytic leukemia (n = 6). Ten additional cases of pediatric and adult acute megakaryocytic leukemia not related to DS (AMKL non-DS; n = 10; including five previously reported cases)15 were also tested for GATA1f reactivity. Finally, marrow cores with maturing trilineage hematopoiesis from patients with DS (NL-DS; n = 2) and without DS (NL; n = 5) were included as positive controls. The results are summarized in Table 1. Megakaryo-cytes that lacked GATA1f reactivity were detected in all cases of MPD-DS. In contrast, the megakaryocytes in other myeloid neoplasms uniformly exhibited intense GATA1f reactivity Image 2. As previously reported, the blasts of AMKL non-DS also exhibited intense GATA1f reactivity.15 In all cases, nucleated erythroid elements showed that robust GATA1f reactivity served as internal positive controls (Image 2). Table 1 Summary of GATA1f Reactivity in MPD-DS and Other Myeloid Neoplasms Diagnosis  Total No. of Cases  GATA1f-Negative Megakaryocytic Progenitors, No.  MPD-DS       TAM  3  3   ML-DS  12  12  NL-DS  2  0  NL  5  0  AMKL non-DS  10a  0  MDS  6  0  JMML  6  0  Diagnosis  Total No. of Cases  GATA1f-Negative Megakaryocytic Progenitors, No.  MPD-DS       TAM  3  3   ML-DS  12  12  NL-DS  2  0  NL  5  0  AMKL non-DS  10a  0  MDS  6  0  JMML  6  0  AMKL, acute megakaryocytic leukemia; DS, Down syndrome; GATA1f, full-length GATA1; JMML, juvenile myelomonocytic leukemia; MDS, myelodysplastic syndrome; ML-DS, myeloid leukemia associated with Down syndrome; MPD-DS, myeloid proliferative disorders associated with Down syndrome; NL, marrow cores with maturing trilineage hematopoiesis from patients without Down syndrome; NL-DS, marrow cores with maturing trilineage hematopoiesis from patients with Down syndrome; TAM, transient abnormal myelopoiesis. aIncludes five previously published cases.13 View Large Image 2 View large Download slide View large Download slide Examples of full-length GATA1 (GATA1f) reactivity (using mAb D52H6) in transient abnormal myelopoiesis (TAM), myeloid leukemia associated with Down syndrome (ML-DS), and other pediatric myeloid malignancies. The conspicuous lack of GATA1f reactivity in the megakaryocytes (arrows) in TAM (A and B, ×100) and ML-DS (C and D, ×100) is contrasted by the intense nuclear staining in megakaryocytes in marrow cores with maturing trilineage hematopoiesis from patients without Down syndrome (E and F, ×100). Image 2 View large Download slide View large Download slide Examples of full-length GATA1 (GATA1f) reactivity (using mAb D52H6) in transient abnormal myelopoiesis (TAM), myeloid leukemia associated with Down syndrome (ML-DS), and other pediatric myeloid malignancies. The conspicuous lack of GATA1f reactivity in the megakaryocytes (arrows) in TAM (A and B, ×100) and ML-DS (C and D, ×100) is contrasted by the intense nuclear staining in megakaryocytes in marrow cores with maturing trilineage hematopoiesis from patients without Down syndrome (E and F, ×100). To ensure that the loss of GATA1f reactivity was not due to a lack of expression or increased degradation of the truncated GATA1 proteins, immunohistochemical studies using mAb D24E4 were performed on a limited sample set, comprising normal (n = 3), AMKL non-DS (n = 3), TAM (n = 1), and ML-DS (n = 6) cases Image 3. In all cases of normal and AMKL non-DS, both antibodies exhibited strong nuclear reactivity in erythroid and megakaryocytic elements, consistent with the expression of wild-type GATA1. In all tested TAM and ML-DS samples, reactivity by D24E4, in contrast to the loss of reactivity by D52H6, was retained in neoplastic megakaryocytes and blasts, consistent with the expression of N-terminally truncated GATA1. This pattern of differential reactivity of this pair of antibodies in MPD-DS cases is consistent with the presence of N-terminally truncated GATA1 proteins. Therefore, the overall findings suggest that the lack of GATA1f reactivity as detected by D52H6 is a sensitive and specific marker for MPD-DS. Image 3 View largeDownload slide Detection of truncated and full-length forms of GATA1 (using mAb D24E4) in myeloid proliferative disorders associated with Down syndrome (MPD-DS) and non–Down syndrome (DS) cases. Strong nuclear reactivity is detected erythroid and megakaryocytic elements in normal marrow (A, ×100) and neoplastic blasts of acute megakaryocytic leukemia non-DS (B, ×100) in a similar pattern as mAb D52H6. However, in contrast to mAb D52H6, the neoplastic megakaryocytes (arrows) in transient abnormal myelopoiesis (C, ×100) and myeloid leukemia associated with Down syndrome (ML-DS) (D, ×100), as well as neoplastic blasts in ML-DS (E, ×100), retained robust nuclear reactivity, consistent with expression of truncated GATA1 proteins. Image 3 View largeDownload slide Detection of truncated and full-length forms of GATA1 (using mAb D24E4) in myeloid proliferative disorders associated with Down syndrome (MPD-DS) and non–Down syndrome (DS) cases. Strong nuclear reactivity is detected erythroid and megakaryocytic elements in normal marrow (A, ×100) and neoplastic blasts of acute megakaryocytic leukemia non-DS (B, ×100) in a similar pattern as mAb D52H6. However, in contrast to mAb D52H6, the neoplastic megakaryocytes (arrows) in transient abnormal myelopoiesis (C, ×100) and myeloid leukemia associated with Down syndrome (ML-DS) (D, ×100), as well as neoplastic blasts in ML-DS (E, ×100), retained robust nuclear reactivity, consistent with expression of truncated GATA1 proteins. Utility of GATA1f/CD61 Double Immuno histochemical Study GATA1f-negative megakaryocytes in MPD-DS can be easily discerned morphologically in single-marker GATA1f immunohistochemical studies, as described above. This approach potentially underestimates the extent of involvement as some of the dysplastic small hypolobated GATA1f-negative megakaryocytes can be difficult to recognize. This problem is highlighted by two cases of TAM, in which heterogeneous populations of GATA1f-positive and GATA1f-negative megakaryocytes were present in a patchy distribution Image 4E. To address this concern, we explored the approach of using double markers—CD61, a megakaryocytic marker, in combination with GATA1f immunostain on all cases of TAM, ML-DS, NL non-DS, and NL-DS. In cases with GATA1f expression, costaining of CD61 and GATA1f in megakaryocytes are seen Image 4A and Image 4B . In cases with extensive involvement, we found that CD61/GATA1f double staining performed similar to single GATA1f staining Image 4C and Image 4D . However, in cases with patchy involvement, the CD61/GATA1f double staining highlighted many of the GATA1f negative small hypolobated megakaryocytes that were difficult to discern using the GATA1f single immunostain (Image 4E). Image 4 View largeDownload slide Examples of double immunostain for full-length GATA1 (GATA1f) (brown) and megakaryocytic marker, CD61 (red), highlight the specificity of the GATA1f immunostain. GATA1f reactivity in CD61-positive megakaryocytes is retained in marrow cores with maturing trilineage hematopoiesis from patients without Down syndrome (A, x100) and marrow cores with maturing trilineage hematopoiesis from patients with Down syndrome (B, ×100) and is lost in transient abnormal myelopoiesis (TAM) (C, ×100) and myeloid leukemia associated with Down syndrome (D, ×100). The double immunostain is also useful in detecting patchy involvement, as illustrated in this case of TAM (E, ×100). Image 4 View largeDownload slide Examples of double immunostain for full-length GATA1 (GATA1f) (brown) and megakaryocytic marker, CD61 (red), highlight the specificity of the GATA1f immunostain. GATA1f reactivity in CD61-positive megakaryocytes is retained in marrow cores with maturing trilineage hematopoiesis from patients without Down syndrome (A, x100) and marrow cores with maturing trilineage hematopoiesis from patients with Down syndrome (B, ×100) and is lost in transient abnormal myelopoiesis (TAM) (C, ×100) and myeloid leukemia associated with Down syndrome (D, ×100). The double immunostain is also useful in detecting patchy involvement, as illustrated in this case of TAM (E, ×100). Discussion The most striking finding in this study is the lack of GATA1f reactivity in megakaryocytes in all cases of MPD-DS, which is in contrast with the robust reactivity in erythroid elements. Our understanding of the pathogenesis of MPD-DS began with the discovery of the founder GATA1 mutations, which force the expression of GATA1s while prohibiting the production of GATA1f.13,16,24 The interplay of the GATA1f loss of function, the GATA1s gain of function, and the effect of trisomy 21 has just begun to unravel through a number of studies using transgenic mouse and patient-derived induced pluripotent stem cell models. In these experimental models, trisomy 21 alone can result in varying degrees of aberrant hematopoiesis with a skewing toward erythromegakaryocytic differentiation, providing the backdrop for MPD-DS.25,26 The lack of GATA1f severely disrupts erythroid development and results in aberrant megakaryocytic differentiation, while the overexpression of GATA1s, which appears to preferentially drive genes for megakaryocytic differentiation, further exacerbates aberrant megakaryocytic proliferation.7,9-11,25,26 Therefore, our observations of retained GATA1f reactivity in erythroid elements and the lack of GATA1f reactivity in megakaryocytes are consistent with our current understanding of MPD-DS pathogenesis. Given that GATA1 is encoded on the X-chromosome, erythroid progenitors giving rise to GATA1f-positive erythroid elements must carry a functional wild-type GATA1 gene. As substantial numbers of GATA1f-positive erythroid elements are observed in all cases of MPD-DS, one would expect that these progenitors with wild-type GATA1 also would give rise to GATA1f-positive megakaryocytes even in samples with extensive involvement. Indeed, rare GATA1f-positive megakaryocytes are almost always found using the CD61/GATA1f double stain. Truncating GATA1 mutations have also been described in two other settings. First, a single case of adult acute megakaryocytic leukemia not associated with DS was reported to harbor a 20-nucleotide insertion in exon 2 of GATA1, resulting in a premature stop codon.27 In this case, the neoplastic megakaryocytic progenitors are expected to lack GATA1f reactivity, similar to MPD-DS. However, we have previously observed robust GATA1f expression in neoplastic megakaryoblasts in all cases of adult megakaryocytic leukemia tested at our institutions using the same monoclonal antibody.15 This discrepancy likely reflects the rare occurrence of truncating GATA1 mutations in adult acute megakaryocytic leukemia. Second, rare cases of Diamond-Blackfan anemia with an X-linked recessive pattern of inheritance were reported to harbor mutations involving the exon 2 donor splice site, leading to the deletion of exon 2 in GATA1 messenger RNA and the sole production of GATA1s.17,19 One would expect a lack of GATA1f reactivity in both erythroid and megakaryocytic elements. Both scenarios may represent additional applications for the immunohistochemical detection of GATA1f. We believe that this method can be useful in the diagnostic study of MPD-DS. In our experience, MPD-DS often can be diagnosed based on the appropriate clinicopathologic correlation, given its relatively unique clinical presentation. However, in challenging cases with atypical presentations, demonstration of GATA1 mutations becomes pertinent in the diagnostic workup of MPD-DS. Currently, targeted sequencing (either based on next-generation sequencing technology or Sanger’s method) remains to be the only way to demonstrate GATA1 mutations. These sequencing methods require nondecalcified samples and, in most practices, are send-out tests. Therefore, our immunohistochemistry-based method has several advantages. First, our method is compatible with decalcified bone marrow core biopsy specimens, from which extraction of high-quality DNA is difficult. Second, our method can be implemented using existing immunohistochemistry setups, allowing for on-site testing and more rapid turnaround. Third, this is only method available where one can observe the loss of full-length GATA1 at the single cell level, allowing correlation for lineage specificity. Therefore, we believe that our method for detecting truncated GATA1 proteins can be useful to practicing pathologists in the diagnostic workup of MPD-DS. In summary, this study demonstrates that the lack of GATA1f reactivity in megakaryocytes is a sensitive and specific immunohistochemical marker for truncating GATA1 mutations in the diagnosis of DS-related myeloid proliferative disorder. In most cases, GATA1f-negative megakaryocytes can be readily discerned using single-color immunohistochemistry in conjunction with morphologic studies. However, in cases with partial involvement, combination with CD61 can further increase the sensitivity of detection. This work was supported in part by National Institutes of Health–Supported Training Grant Award T32 (HL007627 to W.Y.L.). Acknowledgments: We thank Jon Aster, MD, PhD, and Li Chai, MD (Brigham and Women’s Hospital, Boston, MA), for providing cell lines and materials necessary for Western blot analysis. We also thank Alyson Campbell for expert technical assistance. References 1. Gruber TA, Downing JR. The biology of pediatric acute megakaryoblastic leukemia. Blood . 2015; 126: 943- 949. Google Scholar CrossRef Search ADS PubMed  2. Roy A, Roberts I, Norton A et al.   Acute megakaryoblastic leukaemia (AMKL) and transient myeloproliferative disorder (TMD) in Down syndrome: a multi-step model of myeloid leukaemogenesis. Br J Haematol . 2009; 147: 3- 12. Google Scholar CrossRef Search ADS PubMed  3. Arber DA, Orazi A, Hasserjian R et al.   The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia. Blood . 2016; 127: 2391- 2405. Google Scholar CrossRef Search ADS PubMed  4. Langebrake C, Creutzig U, Reinhardt D. Immunophenotype of Down syndrome acute myeloid leukemia and transient myeloproliferative disease differs significantly from other diseases with morphologically identical or similar blasts. Klin Padiatr . 2005; 217: 126- 134. Google Scholar CrossRef Search ADS PubMed  5. Karandikar NJ, Aquino DB, McKenna RW et al.   Transient myeloproliferative disorder and acute myeloid leukemia in Down syndrome: an immunophenotypic analysis. Am J Clin Pathol . 2001; 116: 204- 210. Google Scholar CrossRef Search ADS PubMed  6. Bombery M, Vergilio JA. Transient abnormal myelopoiesis in neonates: GATA get the diagnosis. Arch Pathol Lab Med . 2014; 138: 1302- 1306. Google Scholar CrossRef Search ADS PubMed  7. Alford KA, Reinhardt K, Garnett C et al.  ; International Myeloid Leukemia–Down Syndrome Study Group. Analysis of GATA1 mutations in Down syndrome transient myeloproliferative disorder and myeloid leukemia. Blood . 2011; 118: 2222- 2238. Google Scholar CrossRef Search ADS PubMed  8. Cantor AB, Orkin SH. Transcriptional regulation of erythropoiesis: an affair involving multiple partners. Oncogene . 2002; 21: 3368- 3376. Google Scholar CrossRef Search ADS PubMed  9. Byrska-Bishop M, VanDorn D, Campbell AE et al.   Pluripotent stem cells reveal erythroid-specific activities of the GATA1 N-terminus. J Clin Invest . 2015; 125: 993- 1005. Google Scholar CrossRef Search ADS PubMed  10. Li Z, Godinho FJ, Klusmann JH et al.   Developmental stage-selective effect of somatically mutated leukemogenic transcription factor gata1. Nat Genet . 2005; 37: 613- 619. Google Scholar CrossRef Search ADS PubMed  11. Birger Y, Goldberg L, Chlon TM et al.   Perturbation of fetal hematopoiesis in a mouse model of Down syndrome’s transient myeloproliferative disorder. Blood . 2013; 122: 988- 998. Google Scholar CrossRef Search ADS PubMed  12. Yoshida K, Toki T, Okuno Y et al.   The landscape of somatic mutations in Down syndrome–related myeloid disorders. Nat Genet . 2013; 45: 1293- 1299. Google Scholar CrossRef Search ADS PubMed  13. Roberts I, Izraeli S. Haematopoietic development and leukaemia in Down syndrome. Br J Haematol . 2014; 167: 587- 599. Google Scholar CrossRef Search ADS PubMed  14. Pine SR, Guo Q, Yin C et al.   GATA1 as a new target to detect minimal residual disease in both transient leukemia and megakaryoblastic leukemia of Down syndrome. Leuk Res . 2005; 29: 1353- 1356. Google Scholar CrossRef Search ADS PubMed  15. Lee WY, Weinberg OK, Pinkus GS. GATA1 is a sensitive and specific nuclear marker for erythroid and megakaryocytic lineages. Am J Clin Pathol . 2017; 147: 420- 426. Google Scholar CrossRef Search ADS PubMed  16. Wechsler J, Greene M, McDevitt MA et al.   Acquired mutations in GATA1 in the megakaryoblastic leukemia of Down syndrome. Nat Genet . 2002; 32: 148- 152. Google Scholar CrossRef Search ADS PubMed  17. Hollanda LM, Lima CS, Cunha AF et al.   An inherited mutation leading to production of only the short isoform of GATA-1 is associated with impaired erythropoiesis. Nat Genet . 2006; 38: 807- 812. Google Scholar CrossRef Search ADS PubMed  18. Cabelof DC, Patel HV, Chen Q et al.   Mutational spectrum at GATA1 provides insights into mutagenesis and leukemogenesis in Down syndrome. Blood . 2009; 114: 2753- 2763. Google Scholar CrossRef Search ADS PubMed  19. Sankaran VG, Ghazvinian R, Do R et al.   Exome sequencing identifies GATA1 mutations resulting in Diamond-Blackfan anemia. J Clin Invest . 2012; 122: 2439- 2443. Google Scholar CrossRef Search ADS PubMed  20. Nikolaev SI, Santoni F, Vannier A et al.   Exome sequencing identifies putative drivers of progression of transient myeloproliferative disorder to AMKL in infants with Down syndrome. Blood . 2013; 122: 554- 561. Google Scholar CrossRef Search ADS PubMed  21. Thiollier C, Lopez CK, Gerby B et al.   Characterization of novel genomic alterations and therapeutic approaches using acute megakaryoblastic leukemia xenograft models. J Exp Med . 2012; 209: 2017- 2031. Google Scholar CrossRef Search ADS PubMed  22. Roberts I, Alford K, Hall G et al.  ; Oxford-Imperial Down Syndrome Cohort Study Group. GATA1-mutant clones are frequent and often unsuspected in babies with Down syndrome: identification of a population at risk of leukemia. Blood . 2013; 122: 3908- 3917. Google Scholar CrossRef Search ADS PubMed  23. Toki T, Kanezaki R, Kobayashi E et al.   Naturally occurring oncogenic GATA1 mutants with internal deletions in transient abnormal myelopoiesis in Down syndrome. Blood . 2013; 121: 3181- 3184. Google Scholar CrossRef Search ADS PubMed  24. Bhatnagar N, Nizery L, Tunstall O et al.   Transient abnormal myelopoiesis and AML in Down syndrome: an update. Curr Hematol Malig Rep . 2016; 11: 333- 341. Google Scholar CrossRef Search ADS PubMed  25. Banno K, Omori S, Hirata K et al.   Systematic cellular disease models reveal synergistic interaction of trisomy 21 and GATA1 mutations in hematopoietic abnormalities. Cell Rep . 2016; 15: 1228- 1241. Google Scholar CrossRef Search ADS PubMed  26. Kazuki Y, Yakura Y, Abe S et al.   Down syndrome–associated haematopoiesis abnormalities created by chromosome transfer and genome editing technologies. Sci Rep . 2014; 4: 6136. Google Scholar CrossRef Search ADS PubMed  27. Harigae H, Xu G, Sugawara T et al.   The gata1 mutation in an adult patient with acute megakaryoblastic leukemia not accompanying Down syndrome. Blood . 2004; 103: 3242- 3243. Google Scholar CrossRef Search ADS PubMed  © American Society for Clinical Pathology, 2018. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png American Journal of Clinical Pathology Oxford University Press

Loss of Full-Length GATA1 Expression in Megakaryocytes Is a Sensitive and Specific Immunohistochemical Marker for the Diagnosis of Myeloid Proliferative Disorder Related to Down Syndrome

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Abstract

Abstract Objectives Myeloid proliferative disorders associated with Down syndrome (MPD-DS), including transient abnormal myelopoiesis and myeloid leukemia associated with Down syndrome (DS), harbor mutations of GATA1, a transcription factor essential for erythroid and megakaryocytic development. These mutations result in a N-terminally truncated GATA1 (GATA1s) and prohibit the production of the full-length GATA1 (GATA1f). Here, we demonstrate the utility of immunohistochemical GATA1f reactivity in diagnosing MPD-DS. Methods Immunohistochemical studies for GATA1f expression were performed on bone marrow biopsy specimens. Results In all cases of MPD-DS, megakaryocytes lacked GATA1f expression. In contrast, GATA1f expression was detected in megakaryocytes in all specimen types from patients without DS (normal bone marrows, pediatric myelodysplastic syndrome, juvenile myelomonocytic leukemia, adult acute megakaryocytic leukemia [pediatric and adult; without trisomy 2]), as well as normal bone marrows from patients with DS. Conclusions The lack of GATA1f expression is a sensitive and specific immunohistochemical marker for MPD-DS. GATA1, Down syndrome, Megakaryocytes, Immunohistochemistry, Myeloid proliferative disorder, Transient abnormal myelopoiesis, Myeloid leukemia related to Down syndrome Children with Down syndrome (DS) younger than age 5 years are at an increased risk for developing myeloid proliferative disorders.1,2 Per the 2016 revision to World Health Organization (WHO) classification, myeloid proliferative disorder related to DS (MPD-DS) can be subcategorized into transient abnormal myelopoiesis (TAM) and myeloid leukemia associated with DS (ML-DS).3 Approximately 10% of neonates with DS develop TAM, which exhibits overlapping morphologic and immunophenotypic features with acute myeloid leukemia.4,5 The clinical presentation of TAM can be highly variable, ranging from an asymptomatic presentation to diffuse organ infiltration. Most of the cases undergo spontaneous remission within 4 months of disease onset. However, approximately one in three cases eventually progress to ML-DS with a latency of 1 to 3 years. While the clinicopathologic manifestations resemble that of TAM,2 ML-DS does not undergo spontaneous remission. In both TAM and ML-DS, the neoplastic blasts frequently exhibit megakaryocytic markers (CD61 and CD42), stem cell markers (CD34 and CD117), and aberrant myeloid markers (CD13 and CD33).6,7 GATA binding factor 1 (GATA1) is a transcription factor essential for erythroid and megakaryocytic development.8 Somatic mutations of GATA1, found in nearly all cases of TAM and ML-DS,1 are thought to be the founder mutations of MPD-DS. These mutations result in a premature stop codon in exon 2 or 3 and force the initiation of translation from the second start codon in exon3, resulting in an N-terminally truncated form lacking the first 83 amino acid residues (GATA1s).1 Given that GATA1 is encoded on the X chromosome, these mutations always manifest with the exclusive expression of a short form of GATA1 (GATA1s) in neoplastic cells of MPD-DS. Using experimental models, the lack of full-length GATA1 (GATA1f) and the presence of GATA1s appear to promote megakaryopoieisis and impair erythropoiesis.9-11 Genomic sequencing analysis revealed that ML-DS often harbors additional recurrent mutations affecting components of cohesin complexes, epigenetic regulators, and Ras signaling pathways, representing clonal evolution from TAM.12 N-terminally truncating GATA1 mutations are considered a pathognomonic hallmark in the diagnosis and pathogenesis of TAM and ML-DS.13 In addition, tracking the level of GATA1 mutation has been suggested as a way to monitor minimal residual disease following treatment for ML-DS.7,14 In this study, we demonstrate that it is possible to gauge GATA1f expression by immunohistochemistry using a commercially available rabbit monoclonal antibody. This simple cost-effective method can be incorporated into routine pathology practice for the diagnosis of MPD-DS in adjunct to GATA1 mutation testing. Materials and Methods Antibodies Two commercially available rabbit monoclonal antibodies (mAb) against GATA1 were purchased from Cell Signaling Technology (Danvers, MA): (1) mAb D52H6 (3535) is raised against a peptide centered on Glu13 of GATA1, and (2) mAb D24E4 (4589) is raised against a peptide centered on Ala184 of GATA1. A mouse monoclonal antibody against β-actin clone AC-74 (Sigma-Aldrich, St Louis, MO) was kindly provided by Dr Jon Aster. Mouse mAb against CD61 (clone 2f2) was purchased from Leica Microsystem (Wetzlar, Germany) and mouse mAb against CD71 (clone H68.4) from Invitrogen (Carlsbad, CA). Western Blot Analysis Lysates from the erythroleukemic cell lines (K562 and HEL) were prepared using radioimmunoprecipitation lysis buffer supplemented with protease inhibitors (P2714; Sigma-Aldrich). Protein concentrations of the lysates were determined using Bradford assays per the manufacturer’s instruction (Bio-Rad, Hercules, CA) and normalized in Laemmli sample buffer with 200 mmol/L dithiothreitol. Approximately 5 µg of total protein per sample was submitted for sodium dodecyl sulfate–polyacrylamide gel electrophoresis (4%-12% gradient; Thermo Fisher, Waltham, MA) and electrotransferred onto a polyvinylidene difluoride membrane. The subsequent immunoblotting with anti-GATA1 antibodies and anti–β actin antibody (as a loading control) was performed according to the manufacturer’s instructions. Reactivity to the targeted proteins was visualized using secondary polyclonal anti–rabbit immunoglobulin G (IgG) antibody and polyclonal anti–mouse IgG antibodies conjugated with horseradish peroxidase (Cell Signaling Technology), in conjunction with chemiluminescent substrate (Thermo Fisher), following exposure to x-ray films (Labscientific, Highlands, NJ). The cell lines were kindly provided by Drs Li Chai and Jon Aster at Brigham and Women’s Hospital (Boston, MA). Collection of Bone Marrow Core Biopsy Samples Cases were retrieved from the surgical pathology archival files from the Department of Pathology, Boston Children’s Hospital (Boston, MA) and University of Rochester School of Medicine and Dentistry (Rochester, NY). This study was approved by the Brigham and Women’s Hospital Institutional Review Board. H&E-stained slides and pathology reports were reviewed for the confirmation of diagnosis. Inclusion diagnostic criteria are based on the 2016 revision to WHO classification of myeloid neoplasms and acute leukemia.3 Immunohistochemical Studies Immunohistochemical studies for GATA1f were performed using rabbit mAb D52H6 as previously described.15 Double marker studies for CD61/GATA1f and CD71/GATA1f combinations were performed on a Leica Bond III immunostainer (Leica Microsystem) as previously described.15 Antibodies specific for CD61 and CD71 were used at dilutions of 1:1,000 and 1:4,000, respectively. To detect truncated and full-length forms of GATA1, immunohistochemical studies using rabbit mAb D24E4 were performed using the following parameters. Antigen retrieval was performed using EDTA (0.001 mol/L), pH 8.0 (Invitrogen, San Francisco, CA), for 30 minutes in a steamer (model HS80; Black & Decker, Shelton, CT). The slides were allowed to cool to room temperature for 10 minutes, then washed and placed in Tris buffer (Covance, Dedham, MA). The slides were incubated with anti-GATA1 (D24E4) rabbit monoclonal antibody (Cell Signaling Technology) at a 1:100 dilution for 20 hours at room temperature. Slides were washed and then incubated for 30 minutes with a horseradish peroxidase–labeled polymer conjugated to goat anti–rabbit immunoglobulin antibody (PowerVision; Leica Microsystem, Buffalo Grove, IL). Antibody localization was achieved using a peroxidase reaction with DAB+ (Dako, Carpinteria, CA) as the chromogen. The slides were briefly enhanced with 1% copper sulfate solution, washed, counterstained with hematoxylin, dehydrated, and mounted. Results Detection of GATA1f but Not GATA1s by a Commercially Available Rabbit Monoclonal Antibody Wild-type GATA1 transcripts produce both full-length and short forms of GATA1 proteins through the use of the proper and alternative start codons.10,16,17 In MPD-DS, the GATA1 mutations often result in premature stop codons in exons 2 and 3 and prohibit the production of GATA1f. As a result, only GATA1s, which characteristically lacks the first 83 amino acid residues Figure 1A, is produced.1 We reasoned that a monoclonal rabbit antibody (D52H6) raised against a peptide (surrounding Glu13 of human GATA1) within the truncated portion of GATA1 should specifically detect GATA1f but not GATA1s (Figure 1A and Figure 1B ). We previously used this antibody to establish GATA1 as an immunohistochemical erythromegakaryocytic marker.15 Western blot analysis using cell lysates from erythroleukemia cell lines (K562 and HEL), previously shown to contain both forms of GATA1, confirms that this antibody (D52H6) only reacts to the GATA1f, whereas a different antibody (D24E4) derived from a synthetic peptide distal to the truncation detects both forms of GATA1 Image 1.16 Figure 1 View largeDownload slide Schematic diagrams illustrating the relationship between GATA1 mutations, resultant N-terminally truncated GATA1 (GATA1s), and the monoclonal antibody used for detection. A, Nonsense mutations in exons 2 and 3 force the use of an alternative start codon in exon 3 and results in GATA1s. B, A monoclonal antibody (D52H6) is raised against a peptide centered on amino acid residue 13 within the portion of full-length GATA1 (GATA1f) that is not present in GATA1s. A second monoclonal antibody (D24E4) is raised against a peptide centered on amino acid residue 184 of GATA1. Figure 1 View largeDownload slide Schematic diagrams illustrating the relationship between GATA1 mutations, resultant N-terminally truncated GATA1 (GATA1s), and the monoclonal antibody used for detection. A, Nonsense mutations in exons 2 and 3 force the use of an alternative start codon in exon 3 and results in GATA1s. B, A monoclonal antibody (D52H6) is raised against a peptide centered on amino acid residue 13 within the portion of full-length GATA1 (GATA1f) that is not present in GATA1s. A second monoclonal antibody (D24E4) is raised against a peptide centered on amino acid residue 184 of GATA1. Image 1 View largeDownload slide A Western blot with lysates from erythroleukemic cell lines (K562 and HEL) shows D52H6 recognizes full-length GATA1 (GATA1f) only, and D24E4 recognizes both forms. β-Actin reactivity serves as a protein loading control. Image 1 View largeDownload slide A Western blot with lysates from erythroleukemic cell lines (K562 and HEL) shows D52H6 recognizes full-length GATA1 (GATA1f) only, and D24E4 recognizes both forms. β-Actin reactivity serves as a protein loading control. Distribution of GATA1f Reactivity in DS-Related Myeloid Neoplasms and Other Childhood Myeloid Neoplasms Given that the truncating GATA1 mutations are pathognomonic for MPD-DS,7,12,16,18-23 we reasoned that the lack of GATA1f reactivity in megakaryocytes is likely to serve as a sensitive and specific marker for TAM and ML-DS. Using mAb D52H6, we compared GATA1f reactivity in megakaryocytes using bone marrow core biopsy specimens from MPD-DS, including TAM (n = 3) and ML-DS (n = 12), with a number of non-DS-related childhood myeloid neoplasms, including pediatric myelodysplastic syndrome (n = 6) and juvenile myelomonocytic leukemia (n = 6). Ten additional cases of pediatric and adult acute megakaryocytic leukemia not related to DS (AMKL non-DS; n = 10; including five previously reported cases)15 were also tested for GATA1f reactivity. Finally, marrow cores with maturing trilineage hematopoiesis from patients with DS (NL-DS; n = 2) and without DS (NL; n = 5) were included as positive controls. The results are summarized in Table 1. Megakaryo-cytes that lacked GATA1f reactivity were detected in all cases of MPD-DS. In contrast, the megakaryocytes in other myeloid neoplasms uniformly exhibited intense GATA1f reactivity Image 2. As previously reported, the blasts of AMKL non-DS also exhibited intense GATA1f reactivity.15 In all cases, nucleated erythroid elements showed that robust GATA1f reactivity served as internal positive controls (Image 2). Table 1 Summary of GATA1f Reactivity in MPD-DS and Other Myeloid Neoplasms Diagnosis  Total No. of Cases  GATA1f-Negative Megakaryocytic Progenitors, No.  MPD-DS       TAM  3  3   ML-DS  12  12  NL-DS  2  0  NL  5  0  AMKL non-DS  10a  0  MDS  6  0  JMML  6  0  Diagnosis  Total No. of Cases  GATA1f-Negative Megakaryocytic Progenitors, No.  MPD-DS       TAM  3  3   ML-DS  12  12  NL-DS  2  0  NL  5  0  AMKL non-DS  10a  0  MDS  6  0  JMML  6  0  AMKL, acute megakaryocytic leukemia; DS, Down syndrome; GATA1f, full-length GATA1; JMML, juvenile myelomonocytic leukemia; MDS, myelodysplastic syndrome; ML-DS, myeloid leukemia associated with Down syndrome; MPD-DS, myeloid proliferative disorders associated with Down syndrome; NL, marrow cores with maturing trilineage hematopoiesis from patients without Down syndrome; NL-DS, marrow cores with maturing trilineage hematopoiesis from patients with Down syndrome; TAM, transient abnormal myelopoiesis. aIncludes five previously published cases.13 View Large Image 2 View large Download slide View large Download slide Examples of full-length GATA1 (GATA1f) reactivity (using mAb D52H6) in transient abnormal myelopoiesis (TAM), myeloid leukemia associated with Down syndrome (ML-DS), and other pediatric myeloid malignancies. The conspicuous lack of GATA1f reactivity in the megakaryocytes (arrows) in TAM (A and B, ×100) and ML-DS (C and D, ×100) is contrasted by the intense nuclear staining in megakaryocytes in marrow cores with maturing trilineage hematopoiesis from patients without Down syndrome (E and F, ×100). Image 2 View large Download slide View large Download slide Examples of full-length GATA1 (GATA1f) reactivity (using mAb D52H6) in transient abnormal myelopoiesis (TAM), myeloid leukemia associated with Down syndrome (ML-DS), and other pediatric myeloid malignancies. The conspicuous lack of GATA1f reactivity in the megakaryocytes (arrows) in TAM (A and B, ×100) and ML-DS (C and D, ×100) is contrasted by the intense nuclear staining in megakaryocytes in marrow cores with maturing trilineage hematopoiesis from patients without Down syndrome (E and F, ×100). To ensure that the loss of GATA1f reactivity was not due to a lack of expression or increased degradation of the truncated GATA1 proteins, immunohistochemical studies using mAb D24E4 were performed on a limited sample set, comprising normal (n = 3), AMKL non-DS (n = 3), TAM (n = 1), and ML-DS (n = 6) cases Image 3. In all cases of normal and AMKL non-DS, both antibodies exhibited strong nuclear reactivity in erythroid and megakaryocytic elements, consistent with the expression of wild-type GATA1. In all tested TAM and ML-DS samples, reactivity by D24E4, in contrast to the loss of reactivity by D52H6, was retained in neoplastic megakaryocytes and blasts, consistent with the expression of N-terminally truncated GATA1. This pattern of differential reactivity of this pair of antibodies in MPD-DS cases is consistent with the presence of N-terminally truncated GATA1 proteins. Therefore, the overall findings suggest that the lack of GATA1f reactivity as detected by D52H6 is a sensitive and specific marker for MPD-DS. Image 3 View largeDownload slide Detection of truncated and full-length forms of GATA1 (using mAb D24E4) in myeloid proliferative disorders associated with Down syndrome (MPD-DS) and non–Down syndrome (DS) cases. Strong nuclear reactivity is detected erythroid and megakaryocytic elements in normal marrow (A, ×100) and neoplastic blasts of acute megakaryocytic leukemia non-DS (B, ×100) in a similar pattern as mAb D52H6. However, in contrast to mAb D52H6, the neoplastic megakaryocytes (arrows) in transient abnormal myelopoiesis (C, ×100) and myeloid leukemia associated with Down syndrome (ML-DS) (D, ×100), as well as neoplastic blasts in ML-DS (E, ×100), retained robust nuclear reactivity, consistent with expression of truncated GATA1 proteins. Image 3 View largeDownload slide Detection of truncated and full-length forms of GATA1 (using mAb D24E4) in myeloid proliferative disorders associated with Down syndrome (MPD-DS) and non–Down syndrome (DS) cases. Strong nuclear reactivity is detected erythroid and megakaryocytic elements in normal marrow (A, ×100) and neoplastic blasts of acute megakaryocytic leukemia non-DS (B, ×100) in a similar pattern as mAb D52H6. However, in contrast to mAb D52H6, the neoplastic megakaryocytes (arrows) in transient abnormal myelopoiesis (C, ×100) and myeloid leukemia associated with Down syndrome (ML-DS) (D, ×100), as well as neoplastic blasts in ML-DS (E, ×100), retained robust nuclear reactivity, consistent with expression of truncated GATA1 proteins. Utility of GATA1f/CD61 Double Immuno histochemical Study GATA1f-negative megakaryocytes in MPD-DS can be easily discerned morphologically in single-marker GATA1f immunohistochemical studies, as described above. This approach potentially underestimates the extent of involvement as some of the dysplastic small hypolobated GATA1f-negative megakaryocytes can be difficult to recognize. This problem is highlighted by two cases of TAM, in which heterogeneous populations of GATA1f-positive and GATA1f-negative megakaryocytes were present in a patchy distribution Image 4E. To address this concern, we explored the approach of using double markers—CD61, a megakaryocytic marker, in combination with GATA1f immunostain on all cases of TAM, ML-DS, NL non-DS, and NL-DS. In cases with GATA1f expression, costaining of CD61 and GATA1f in megakaryocytes are seen Image 4A and Image 4B . In cases with extensive involvement, we found that CD61/GATA1f double staining performed similar to single GATA1f staining Image 4C and Image 4D . However, in cases with patchy involvement, the CD61/GATA1f double staining highlighted many of the GATA1f negative small hypolobated megakaryocytes that were difficult to discern using the GATA1f single immunostain (Image 4E). Image 4 View largeDownload slide Examples of double immunostain for full-length GATA1 (GATA1f) (brown) and megakaryocytic marker, CD61 (red), highlight the specificity of the GATA1f immunostain. GATA1f reactivity in CD61-positive megakaryocytes is retained in marrow cores with maturing trilineage hematopoiesis from patients without Down syndrome (A, x100) and marrow cores with maturing trilineage hematopoiesis from patients with Down syndrome (B, ×100) and is lost in transient abnormal myelopoiesis (TAM) (C, ×100) and myeloid leukemia associated with Down syndrome (D, ×100). The double immunostain is also useful in detecting patchy involvement, as illustrated in this case of TAM (E, ×100). Image 4 View largeDownload slide Examples of double immunostain for full-length GATA1 (GATA1f) (brown) and megakaryocytic marker, CD61 (red), highlight the specificity of the GATA1f immunostain. GATA1f reactivity in CD61-positive megakaryocytes is retained in marrow cores with maturing trilineage hematopoiesis from patients without Down syndrome (A, x100) and marrow cores with maturing trilineage hematopoiesis from patients with Down syndrome (B, ×100) and is lost in transient abnormal myelopoiesis (TAM) (C, ×100) and myeloid leukemia associated with Down syndrome (D, ×100). The double immunostain is also useful in detecting patchy involvement, as illustrated in this case of TAM (E, ×100). Discussion The most striking finding in this study is the lack of GATA1f reactivity in megakaryocytes in all cases of MPD-DS, which is in contrast with the robust reactivity in erythroid elements. Our understanding of the pathogenesis of MPD-DS began with the discovery of the founder GATA1 mutations, which force the expression of GATA1s while prohibiting the production of GATA1f.13,16,24 The interplay of the GATA1f loss of function, the GATA1s gain of function, and the effect of trisomy 21 has just begun to unravel through a number of studies using transgenic mouse and patient-derived induced pluripotent stem cell models. In these experimental models, trisomy 21 alone can result in varying degrees of aberrant hematopoiesis with a skewing toward erythromegakaryocytic differentiation, providing the backdrop for MPD-DS.25,26 The lack of GATA1f severely disrupts erythroid development and results in aberrant megakaryocytic differentiation, while the overexpression of GATA1s, which appears to preferentially drive genes for megakaryocytic differentiation, further exacerbates aberrant megakaryocytic proliferation.7,9-11,25,26 Therefore, our observations of retained GATA1f reactivity in erythroid elements and the lack of GATA1f reactivity in megakaryocytes are consistent with our current understanding of MPD-DS pathogenesis. Given that GATA1 is encoded on the X-chromosome, erythroid progenitors giving rise to GATA1f-positive erythroid elements must carry a functional wild-type GATA1 gene. As substantial numbers of GATA1f-positive erythroid elements are observed in all cases of MPD-DS, one would expect that these progenitors with wild-type GATA1 also would give rise to GATA1f-positive megakaryocytes even in samples with extensive involvement. Indeed, rare GATA1f-positive megakaryocytes are almost always found using the CD61/GATA1f double stain. Truncating GATA1 mutations have also been described in two other settings. First, a single case of adult acute megakaryocytic leukemia not associated with DS was reported to harbor a 20-nucleotide insertion in exon 2 of GATA1, resulting in a premature stop codon.27 In this case, the neoplastic megakaryocytic progenitors are expected to lack GATA1f reactivity, similar to MPD-DS. However, we have previously observed robust GATA1f expression in neoplastic megakaryoblasts in all cases of adult megakaryocytic leukemia tested at our institutions using the same monoclonal antibody.15 This discrepancy likely reflects the rare occurrence of truncating GATA1 mutations in adult acute megakaryocytic leukemia. Second, rare cases of Diamond-Blackfan anemia with an X-linked recessive pattern of inheritance were reported to harbor mutations involving the exon 2 donor splice site, leading to the deletion of exon 2 in GATA1 messenger RNA and the sole production of GATA1s.17,19 One would expect a lack of GATA1f reactivity in both erythroid and megakaryocytic elements. Both scenarios may represent additional applications for the immunohistochemical detection of GATA1f. We believe that this method can be useful in the diagnostic study of MPD-DS. In our experience, MPD-DS often can be diagnosed based on the appropriate clinicopathologic correlation, given its relatively unique clinical presentation. However, in challenging cases with atypical presentations, demonstration of GATA1 mutations becomes pertinent in the diagnostic workup of MPD-DS. Currently, targeted sequencing (either based on next-generation sequencing technology or Sanger’s method) remains to be the only way to demonstrate GATA1 mutations. These sequencing methods require nondecalcified samples and, in most practices, are send-out tests. Therefore, our immunohistochemistry-based method has several advantages. First, our method is compatible with decalcified bone marrow core biopsy specimens, from which extraction of high-quality DNA is difficult. Second, our method can be implemented using existing immunohistochemistry setups, allowing for on-site testing and more rapid turnaround. Third, this is only method available where one can observe the loss of full-length GATA1 at the single cell level, allowing correlation for lineage specificity. Therefore, we believe that our method for detecting truncated GATA1 proteins can be useful to practicing pathologists in the diagnostic workup of MPD-DS. In summary, this study demonstrates that the lack of GATA1f reactivity in megakaryocytes is a sensitive and specific immunohistochemical marker for truncating GATA1 mutations in the diagnosis of DS-related myeloid proliferative disorder. In most cases, GATA1f-negative megakaryocytes can be readily discerned using single-color immunohistochemistry in conjunction with morphologic studies. However, in cases with partial involvement, combination with CD61 can further increase the sensitivity of detection. This work was supported in part by National Institutes of Health–Supported Training Grant Award T32 (HL007627 to W.Y.L.). Acknowledgments: We thank Jon Aster, MD, PhD, and Li Chai, MD (Brigham and Women’s Hospital, Boston, MA), for providing cell lines and materials necessary for Western blot analysis. We also thank Alyson Campbell for expert technical assistance. References 1. Gruber TA, Downing JR. The biology of pediatric acute megakaryoblastic leukemia. Blood . 2015; 126: 943- 949. Google Scholar CrossRef Search ADS PubMed  2. Roy A, Roberts I, Norton A et al.   Acute megakaryoblastic leukaemia (AMKL) and transient myeloproliferative disorder (TMD) in Down syndrome: a multi-step model of myeloid leukaemogenesis. Br J Haematol . 2009; 147: 3- 12. Google Scholar CrossRef Search ADS PubMed  3. Arber DA, Orazi A, Hasserjian R et al.   The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia. Blood . 2016; 127: 2391- 2405. Google Scholar CrossRef Search ADS PubMed  4. Langebrake C, Creutzig U, Reinhardt D. Immunophenotype of Down syndrome acute myeloid leukemia and transient myeloproliferative disease differs significantly from other diseases with morphologically identical or similar blasts. Klin Padiatr . 2005; 217: 126- 134. Google Scholar CrossRef Search ADS PubMed  5. Karandikar NJ, Aquino DB, McKenna RW et al.   Transient myeloproliferative disorder and acute myeloid leukemia in Down syndrome: an immunophenotypic analysis. Am J Clin Pathol . 2001; 116: 204- 210. Google Scholar CrossRef Search ADS PubMed  6. Bombery M, Vergilio JA. Transient abnormal myelopoiesis in neonates: GATA get the diagnosis. Arch Pathol Lab Med . 2014; 138: 1302- 1306. Google Scholar CrossRef Search ADS PubMed  7. Alford KA, Reinhardt K, Garnett C et al.  ; International Myeloid Leukemia–Down Syndrome Study Group. Analysis of GATA1 mutations in Down syndrome transient myeloproliferative disorder and myeloid leukemia. Blood . 2011; 118: 2222- 2238. Google Scholar CrossRef Search ADS PubMed  8. Cantor AB, Orkin SH. Transcriptional regulation of erythropoiesis: an affair involving multiple partners. Oncogene . 2002; 21: 3368- 3376. Google Scholar CrossRef Search ADS PubMed  9. Byrska-Bishop M, VanDorn D, Campbell AE et al.   Pluripotent stem cells reveal erythroid-specific activities of the GATA1 N-terminus. J Clin Invest . 2015; 125: 993- 1005. Google Scholar CrossRef Search ADS PubMed  10. Li Z, Godinho FJ, Klusmann JH et al.   Developmental stage-selective effect of somatically mutated leukemogenic transcription factor gata1. Nat Genet . 2005; 37: 613- 619. Google Scholar CrossRef Search ADS PubMed  11. Birger Y, Goldberg L, Chlon TM et al.   Perturbation of fetal hematopoiesis in a mouse model of Down syndrome’s transient myeloproliferative disorder. Blood . 2013; 122: 988- 998. Google Scholar CrossRef Search ADS PubMed  12. Yoshida K, Toki T, Okuno Y et al.   The landscape of somatic mutations in Down syndrome–related myeloid disorders. Nat Genet . 2013; 45: 1293- 1299. Google Scholar CrossRef Search ADS PubMed  13. Roberts I, Izraeli S. Haematopoietic development and leukaemia in Down syndrome. Br J Haematol . 2014; 167: 587- 599. Google Scholar CrossRef Search ADS PubMed  14. Pine SR, Guo Q, Yin C et al.   GATA1 as a new target to detect minimal residual disease in both transient leukemia and megakaryoblastic leukemia of Down syndrome. Leuk Res . 2005; 29: 1353- 1356. Google Scholar CrossRef Search ADS PubMed  15. Lee WY, Weinberg OK, Pinkus GS. GATA1 is a sensitive and specific nuclear marker for erythroid and megakaryocytic lineages. Am J Clin Pathol . 2017; 147: 420- 426. Google Scholar CrossRef Search ADS PubMed  16. Wechsler J, Greene M, McDevitt MA et al.   Acquired mutations in GATA1 in the megakaryoblastic leukemia of Down syndrome. Nat Genet . 2002; 32: 148- 152. Google Scholar CrossRef Search ADS PubMed  17. Hollanda LM, Lima CS, Cunha AF et al.   An inherited mutation leading to production of only the short isoform of GATA-1 is associated with impaired erythropoiesis. Nat Genet . 2006; 38: 807- 812. Google Scholar CrossRef Search ADS PubMed  18. Cabelof DC, Patel HV, Chen Q et al.   Mutational spectrum at GATA1 provides insights into mutagenesis and leukemogenesis in Down syndrome. Blood . 2009; 114: 2753- 2763. Google Scholar CrossRef Search ADS PubMed  19. Sankaran VG, Ghazvinian R, Do R et al.   Exome sequencing identifies GATA1 mutations resulting in Diamond-Blackfan anemia. J Clin Invest . 2012; 122: 2439- 2443. Google Scholar CrossRef Search ADS PubMed  20. Nikolaev SI, Santoni F, Vannier A et al.   Exome sequencing identifies putative drivers of progression of transient myeloproliferative disorder to AMKL in infants with Down syndrome. Blood . 2013; 122: 554- 561. Google Scholar CrossRef Search ADS PubMed  21. Thiollier C, Lopez CK, Gerby B et al.   Characterization of novel genomic alterations and therapeutic approaches using acute megakaryoblastic leukemia xenograft models. J Exp Med . 2012; 209: 2017- 2031. Google Scholar CrossRef Search ADS PubMed  22. Roberts I, Alford K, Hall G et al.  ; Oxford-Imperial Down Syndrome Cohort Study Group. GATA1-mutant clones are frequent and often unsuspected in babies with Down syndrome: identification of a population at risk of leukemia. Blood . 2013; 122: 3908- 3917. Google Scholar CrossRef Search ADS PubMed  23. Toki T, Kanezaki R, Kobayashi E et al.   Naturally occurring oncogenic GATA1 mutants with internal deletions in transient abnormal myelopoiesis in Down syndrome. Blood . 2013; 121: 3181- 3184. Google Scholar CrossRef Search ADS PubMed  24. Bhatnagar N, Nizery L, Tunstall O et al.   Transient abnormal myelopoiesis and AML in Down syndrome: an update. Curr Hematol Malig Rep . 2016; 11: 333- 341. Google Scholar CrossRef Search ADS PubMed  25. Banno K, Omori S, Hirata K et al.   Systematic cellular disease models reveal synergistic interaction of trisomy 21 and GATA1 mutations in hematopoietic abnormalities. Cell Rep . 2016; 15: 1228- 1241. Google Scholar CrossRef Search ADS PubMed  26. Kazuki Y, Yakura Y, Abe S et al.   Down syndrome–associated haematopoiesis abnormalities created by chromosome transfer and genome editing technologies. Sci Rep . 2014; 4: 6136. Google Scholar CrossRef Search ADS PubMed  27. Harigae H, Xu G, Sugawara T et al.   The gata1 mutation in an adult patient with acute megakaryoblastic leukemia not accompanying Down syndrome. Blood . 2004; 103: 3242- 3243. Google Scholar CrossRef Search ADS PubMed  © American Society for Clinical Pathology, 2018. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

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American Journal of Clinical PathologyOxford University Press

Published: Apr 1, 2018

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