Emerging Immunohistochemical Biomarkers for Myeloid Neoplasms

Isocitrate dehydrogenase (IDH) is an enzyme located in the cytoplasmic and mitochondrial matrix of the cell that participates in the tricarboxylic acid cycle. It has 3 different isoforms. IDH1 is present in the cytoplasm and peroxisome, whereas IDH2 is present in the mitochondria. They are structurally similar and catalyze a reversible reaction between isocitrate and 2-oxoglutarate. IDH3 is structurally different and catalyzes an irreversible reaction in the mitochondria.¹ 

Mutations in IDH1 and IDH2 cause a gain of function resulting in accumulation of (R)-2-hydroxyglutarate (HG). Various studies have shown that (R)-2HG is an oncometabolite that suppresses 5-methyl cytosine hydroxylases and histone lysine demethylases, leading to hypermethylation.^2,3  (R)-2HG can also cause leukemogenesis by cytokine-independent proliferation and blockage of differentiation in the hematopoietic cells.³ 

IDH mutations were initially described in glioblastomas when Parsons et al⁴  demonstrated IDH1R132H mutations exclusively in secondary glioblastomas compared with primary. Subsequently, mutations in IDH2 were also demonstrated in secondary glioblastomas.⁵  Since their initial description, IDH mutations have been most commonly described in AML, MDS, chondrosarcoma, cholangiocarcinoma, and angioimmunoblastic T-cell lymphoma, among other tumors.^1,3 

IDH mutations are seen in approximately 15% to 20% of AML, 25% to 30% of normal karyotype AML, and 5% to 7% of MDS. These mutations are mainly seen in adults. IDH2 mutations are slightly more frequent than IDH1 mutations in myeloid neoplasms.^1,3,6,7  In AML, IDH2 mutations are seen in 8% to 19% of patients, whereas IDH1 mutations are seen in 7% to 14% of cases and are usually mutually exclusive.⁶  In MDS, IDH2 mutations account for 4% to 5% of cases, and IDH1 accounts for 2% to 3% of cases.^3,7,8  The IDH mutations involve arginine residues located in the active enzymatic site of IDH1 (R132) and IDH2 (R140 and R172). IDH2R140Q followed by IDH1R132H are the most common mutations. Other rare mutations include IDH2R140W/L, IDH2R172K/G/M/Q, and IDH1R132C/S/L/G/P.^1,3,6–8  In MDS, IDH2 mutations are more commonly seen in RAEB subtype and are associated with serine/arginine-rich-splicing-factor-2 (SRSF2) mutations.⁷  In AML and MDS, patients with IDH mutations tend to have higher blast count in the bone marrow and a higher platelet count.^3,8,9  The impact of mutated IDH on prognosis is still not clear. The recent literature suggests that IDH1 is associated with poor prognosis, whereas IDH2 and in particular IDH2R172 mutations are associated with relatively favorable prognosis.¹⁰  There is also a suggestion that the prognostic impact may be linked to comutation or cytogenetic abnormality, with better prognosis in cases with NPM1 mutations compared with those associated with fms-like tyrosine kinase 3 (FLT3) mutations.^7,10  Even with MDS, the prognostic significance of mutant IDH is being studied, but it is possibly associated with poorer overall survival.¹¹ 

Sanger sequencing, next-generation sequencing, and polymerase chain reaction (PCR)–based mutation tests are usually used for mutation analysis. IDH1/2 mutation has also been shown to be a good marker for detecting minimal residual disease.³  Analyzing IDH mutation by detecting mutant protein by IHC can be valuable because it is likely to be more cost-efficient, have a lower turnaround time, and also be applicable to formalin-fixed, paraffin-embedded specimens. We found 3 studies analyzing IDH1R132H mutation by IHC using monoclonal antibodies in myeloid neoplasms like AML-not otherwise specified, AML-myelodysplasia–related changes, therapy-related AML, MPN-blast phase, MDS, MDS/MPN, and MPN, but none analyzing IDH2 mutations by IHC.^12–14  IHC has been shown to be 100% specific and sensitive in pretreatment samples of myeloid neoplasms with IDH1R132H mutations (Figure 1, A and B). No cross-reactivity with other IDH1 mutations has been seen.¹⁴  It has also been shown to be more sensitive than molecular tests, particularly in posttreatment samples, and could complement flow cytometry in analyzing minimal residual disease (MRD; Figure 1, C through F).^13,14  The antibody localizes to cytoplasm and can range from weak to strong in intensity. The antibody uniformly stains the neoplastic blasts in AML with no or minimal differentiation. Staining of mature and immature neoplastic cells in AML with differentiation and MPN-blast phase can be seen. IHC also stains the mature cells in MDS, MPN, and MPN/MDS. The staining is usually diffuse and interstitial in distribution but can be multifocal or unifocal and paratrabecular, and rarely perivascular, in distribution.¹⁴  The megakaryocytes may stain with weak intensity. This is likely a true staining of neoplastic cells because negative megakaryocytes may be seen within the same positive cases and no nonspecific staining has been observed in wild-type and non-IDH1R132H mutant cases. Other marrow elements, like erythroid, lymphocytes, osteoblasts, and endothelial cells, do not show any staining. Focal nonspecific nuclear staining may be seen.¹⁴ 

Kurt et al¹⁴  found discrepancy in molecular and IHC results in low–tumor burden samples, like posttreatment AML and MDS cases. In their study, the criterion for positive cases was any staining other than <1% weak staining.¹⁴  Their analysis showed discordant results in 8 cases. All of these cases were negative by molecular methods but positive by IHC, out of a total of 20 PCR-negative cases from patients with known mutation. These 8 cases showed positivity ranging from 1% to 25% (median, 8%) of bone marrow cells. The authors suggested that the discrepancy could be due to sampling error or due to limitations of molecular testing, like low tumor burden and sensitivity.¹⁴  Andrulis et al¹³  also found that 2 cases of MDS with IDH positivity in 1% to 5% of cells turned out to be negative by sequencing.

Kurt et al¹⁴  also compared MRD by flow cytometry and IDHR132H staining and found that in 9 cases with MRD detected by flow cytometry, IDHR132H was positive in maturing and mature myeloid cells. In 5 bone marrow specimens with complete morphologic and flow immunophenotypic remission, they found residual mutant clones by sequencing. By IHC, these positive cells were localized in mature myeloid cells. This discordance was probably because flow cytometric gating strategies are focused on blast detection, whereas the residual mutant cells could be mature. The clinical significance of this residual mutation in mature cells is unknown and probably requires a large-scale clinical study for better understanding.¹⁴ 

Since the discovery of these mutations, a few targeted inhibitors of IDH have been developed and have gained US Food and Drug Administration (FDA) approval.¹⁰  These drugs inhibit the (R)-2HG oncometabolite and advance the maturation of myeloid cells. Ivosidenib, an IDH1 inhibitor, has been approved by the FDA for relapsed/refractory AML, where it has shown complete response/complete response with partial hematologic response in 30% and an overall response rate in 42% of patients. It is also FDA approved for newly diagnosed AML in older and chemotherapy-ineligible populations. Enasidenib, an IDH2 inhibitor, has been approved by the FDA for relapsed/refractory AML, and it shows approximately a complete response in 20%, a complete response with incomplete hematologic recovery in 10%, and overall response in 40% of cases. Patients with IDH1/2 mutation also respond well to the BCL-2 inhibitor venetoclax with hypomethylating agents like azacitidine; this combination is now standard of care treatment for older patients who are ineligible for intensive chemotherapy. Because patients with IDH mutations respond well to BCL-2 and IDH inhibitors, their sequential and combined use is under trial.¹⁰ 

Thus, IHC for IDH1R132H is a highly sensitive and specific marker. Although molecular tests are still necessary for initial diagnostic workup, IHC may be more sensitive in the posttreatment biopsy and provide a cheaper and faster alternative to molecular tests to assess residual disease and monitor relapse in an already known mutant leukemia. Because of the arrival of IDH-specific inhibitors, it may also be helpful in monitoring treatment response.

Erythroblast transformation–specific regulated gene (ERG) is a member of the E26-transformation–specific sequence family of transcription factors encoded on chromosome 21 and plays an important role in normal and aberrant hematopoiesis, and particularly in megakaryopoiesis.^15–17  In hematopoiesis, it helps maintain hematopoietic stem cells, prevent their differentiation, and maintain peripheral platelet counts.^18,19  It also has a substantial role in cartilage formation and angiogenesis, possibly via the VE-cadherin-wnt/β-catenin pathway.^17,20 

The precise oncogenic mechanism involving ERG is not known, but it has been strongly linked to various malignancies and is involved in translocations like TMPRSS2-ERG in prostatic adenocarcinoma, EWS-ERG in Ewing sarcoma, and TLS/FUS-ERG in AML.¹⁵  These fusion proteins are weaker activators of transcription than the normal protein.²¹  High expression of ERG has been reported in cytogenetically normal AML in adults and in pediatric AML with normal and abnormal karyotype.^22,23  ERG also directly induces the expression of proto-oncogene serine/threonine-protein (PIM1) and indirectly activates rat sarcoma virus (RAS), which may be involved in the development of a small subset of AML.¹⁶  Overexpression of ERG in trisomy 21 and its interaction with a mutated GATA-binding factor 1 (GATA1) has been suspected to cause acute leukemia in patients with Down syndrome (DS).¹⁵ 

ERG IHC shows nuclear staining and has traditionally been used as a specific and highly sensitive marker in the diagnosis of benign and malignant vascular and lymphatic lesions.²⁴  In recent years, studies have described the utility of ERG IHC in myeloid neoplasms.^17,20,25  ERG antibody localizes to the nucleus and stains mature and immature myeloid cells in a normal marrow; the intensity of staining wanes as the cell matures (Figure 2, A and B). The erythroid cells do not show any staining, whereas the megakaryocytes show absent to faint staining.^17,20  In one study, lymphocytes in the lymph nodes showed weak to moderate staining by rabbit monoclonal antibody but were negative by mouse monoclonal antibody, whereas in other studies lymphocytes were negative by rabbit monoclonal antibody in bone marrow, lymph node, spleen, and thymus.^17,20,26  Hematogones may show mild to moderate staining, whereas indolent T-lymphoblastic proliferations have been reported to be negative.¹⁷  The lower intensity of hematogone staining, along with the scarcity of hematogone populations in myeloid sarcomas, render this an unlikely confounding. In AML and myeloid sarcomas, ERG IHC shows a sensitivity of approximately 90%.^17,20,24  IHC is also highly sensitive in CD34- and CD117-negative AML/myeloid sarcoma cases.¹⁷  It can be used to differentiate cutaneous myeloid sarcomas from reactive leukocytic infiltrate. ERG staining is negative to weak in reactive leukocytic infiltrate compared with neoplastic cells of myeloid sarcoma.²⁵  ERG shows a high concordance with myeloperoxidase staining and has an edge over myeloperoxidase in being a nuclear stain with less background staining (Figure 2, C through F).²⁰  Although megakaryocytes and erythroid cells do not show ERG staining, the neoplastic cells in pure erythroid leukemia and acute megakaryoblastic leukemia show strong nuclear staining.¹⁷  Weak to moderate ERG staining may be seen in almost all cases of T-lymphoblastic leukemia, B-lymphoblastic leukemia, and systemic mastocytosis.¹⁷  Blastic plasmacytoid dendritic cell neoplasm and blastoid mantle cell lymphoma are negative.¹⁷  Plasma cell myeloma shows strong and diffuse staining.²⁷  Lymphomas have usually been reported to be negative for ERG, although a few weak-positive follicular lymphomas and diffuse large B-cell lymphoma cases are reported in the literature.^17,24,28  In the past, ERG overexpression by reverse transcription–PCR (RT-PCR) and microarray analysis has been shown to be an independent prognostic marker with poor response to chemotherapy and reduced survival in cytogenetically normal AML, but there are no studies correlating ERG expression by PCR and IHC.^22,29 

Other tumors that express ERG are prostatic adenocarcinoma in 45.4% of cases, Ewing sarcoma in 7% of cases, meningioma in 40% of cases, chondrosarcoma in approximately 80% of cases, and EWSR1-SMAD3⁺ fibroblastic tumor in 100% of cases.^24,28,30,31  In prostatic adenocarcinoma it is frequently positive in high-grade prostatic adenocarcinoma and small cell carcinoma. Approximately 25% of prostatic small cell carcinomas express ERG by IHC.^24,32  Cytoplasmic staining has been reported in gastrointestinal stromal tumor and ductal carcinoma of the breast, and membranous staining has been reported in papillary thyroid carcinoma.²⁴ 

Thus, ERG appears to be a sensitive but not specific marker for myeloid neoplasms. It can be used in a panel of antibodies in cases of undifferentiated neoplasm at extramedullary sites.¹⁷  ERG IHC is also helpful in differentiating myeloid sarcomas from its mimics, such as blastic plasmacytoid dendritic cell neoplasm, blastoid variant of mantle cell lymphoma, and reactive leukocytic infiltrate. In the case of the last differential diagnosis, it is more useful than myeloperoxidase. Potential pitfalls include small cell carcinoma of prostate, Ewing sarcoma, T-lymphoblastic leukemia, and B-lymphoblastic leukemia, which may have a morphology similar to AML and are also positive for ERG. ERG, along with other markers, like CD34, CD31, and FLi1, overlaps between myeloid and vascular neoplasms, and therefore a wide panel of IHC may be necessary in the context of undifferentiated histology.^17,20,25 

Blast quantitation in acute monocytic leukemias has been a significant problem because of the lack of markers, like CD34, that can differentiate between early precursors and their mature counterparts. Although residual disease detection can be performed by a multitude of cutting-edge technologies, including next-generation sequencing, assuming there are level 1 or 2 mutations, and “different from normal” flow cytometry, these studies can be riddled with confounders, such as clonal hematopoiesis.^33,34  Blast counting of aspirates has remained the gold standard for all subtypes of AML, but in monocytic leukemias it is difficult to come to consensus among pathologists.^35,36 

Recently discovered, IHC for interferon regulatory factor 8 (IRF8) shows promise as a reliable marker for monoblasts in bone marrow core biopsies.³⁷  The authors of that study surveyed the gene expression profiles of monocyte progenitors, which led to the validation of IRF8 in 90 cases of acute monocytic leukemia, including posttherapy staging bone marrows. They found very a high correlation (R = 0.95) between the percentage of cells staining for the marker and aspirate blast count. The marker was negative in reactive marrows showing monocytosis as well as in other subtypes of AML (without monocytic differentiation). A smaller cohort of chronic myelomonocytic leukemias were also tested and showed good correlation between aspirate blast count and staining result (R = 0.86; Figure 3). Although IRF8 also stains B cells and hematogones, the staining intensity was weaker and only rarely led to count discrepancies in this initial study.

IRF8 is a lineage-determining transcription factor that is necessary for the production of monocyte and dendritic cell progenitors. It regulates interferon-stimulated genes and also the expression of a number of genes in apoptosis.^38,39  In mice, knockout of IRF8 leads to spontaneous myeloproliferative disorders that resemble chronic myeloid leukemia, with proliferation of the granulocytic lineage.⁴⁰  Even more recently, investigators performed clustered regularly interspaced short palindromic repeats (CRISPR) screening that identified an IRF8-myocyte enhancer factor 2D (MEF2D) transcriptional circuit as an AML-biased dependency.⁴¹  They showed that a chromatin “reader” zinc finger MYND-type containing 8 (ZMYND8) directly activates IRF8 through lineage-specific enhancers. Many of the cell lines used were AMLs with monocytic differentiation. Another study showed that IRF8 is an AML-specific susceptibility factor and that high IRF8 expression is associated with poorer patient prognoses.⁴²  These findings suggest a rapidly increasing understanding of the role of IRF8 in myeloid neoplasms in the near future.

GATA1 is a transcription factor of the GATA family encoded on the X chromosome that binds to (A/T)GATA(A/G) motif in the promoter or enhancer of erythroid genes.⁴³  GATA1 plays an important role in the specific gene expression of megakaryocytes, and its loss induces apoptosis in erythroid precursors and proliferation of megakaryocytes without terminal differentiation.⁴⁴  Dysregulated GATA1 is associated with the development of Diamond-Blackfan anemia, MDS, thrombocytopenia, or leukemia.^45,46 GATA1 mutations can be inherited or acquired. The inherited ones are seen in Diamond-Blackfan anemia, whereas the acquired ones are seen in transient abnormal myelopoiesis (TAM) and myeloid leukemia associated with DS (ML-DS).⁴⁴ 

GATA1 translation due to 2 different start codons in exon 2 and exon 3 produces a full-length GATA1f and a functionally deficient shorter variant that lacks N-terminal transactivation domain GATA1s.⁴⁵  The known GATA1 mutations in DS result in formation of only GATA1s due to mutation in the start codon or as a result of the splicing of exon 2.⁴⁶  Approximately 30% of newborns with DS harbor GATA1 mutations. Approximately 10% of these then develop TAM.⁴⁷  A total of 20% of TAMs may progress to ML-DS, which phenotypically resembles acute megakaryoblastic leukemia.⁴⁷  The cause of frequent GATA1 mutations in DS is not known and is a topic of active research. A triple-hit model of ML-DS has been suggested in which trisomy 21 is the first hit, followed by GATA1 mutation causing TAM, and a final hit that involves additional mutations in genes such as myeloproliferative leukemia (MPL), Janus kinase 2 (JAK2), stromal antigen 2 (STAG2), enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2), and additional sex combs-like 1 (ASXL1) that may lead to ML-DS.⁴⁴ 

Some studies have also shown low GATA1 expression in BCR-ABL⁻ MPNs that are associated with mutations involving JAK2, MPL, and calreticulin (CALR). These mutations cause increased activity of JAK/STAT signaling, which inhibits ribosomal proteins, resulting in decreased translation of GATA1.⁴⁸ 

The utility of GATA1 IHC in formalin-fixed, paraffin-embedded samples has been explored recently. It is a highly sensitive and specific marker for erythroid and megakaryocytic lineage.⁴⁹  The staining localizes to the nucleus. In a normal bone marrow, it stains the megakaryocytes with a uniform strong intensity. The intensity is variable in the erythroid lineage, with strong staining in erythroid precursors, that is reduced with erythroid maturation. GATA1 IHC is negative in mature red blood cells and platelets.⁴⁹  Staining of mast cells and eosinophils may be seen.⁴⁹  In acute leukemias, the blasts of pure erythroleukemia and acute megakaryoblastic leukemia are positive for GATA1 in 100% of cases. The blasts of other acute myeloid and lymphoid leukemias are negative.⁴⁹  GATA1 IHC can also be used to differentiate between TAM and ML-DS and other myeloid neoplasms, including AKML in non-DS patients.⁵⁰  The megakaryocytes in TAM and ML-DS are negative for GATA1 antibody clones that are raised against an amino acid at the N-terminal (mAb D52H6) but are positive for a clone raised against an amino acid downstream in exon 3 (mAb D24E4) in 100% of cases. This differential staining dependent on the clone is attributable to truncating mutations of GATA1 in TAM and ML-DS. Because these mutations are not seen in other pediatric myeloid neoplasms and adult acute megakaryoblastic leukemia, they stain with both clones of GATA1.⁵⁰ 

Myeloid neoplasms that can show loss of GATA1 staining in megakaryocytes are BCR-ABL⁻ MPNs. The decrease in staining is more marked in overt primary myelofibrosis, and to a lesser degree in prefibrotic primary myelofibrosis, polycythemia vera, and essential thrombocythemia. The intensity of staining in primary myelofibrosis decreases as the severity of fibrosis increases. Decreased GATA1 expression in BCR-ABL⁻ MPN is due to JAK mutations causing decreased expression of GATA1.^48,51,52 

Although GATA1 cannot distinguish between erythroid and megakaryocytic blasts, it has an advantage of being a nuclear stain with high sensitivity and specificity and can be used as a dual IHC marker with CD61 and CD71. Loss of GATA1 can help in diagnosing GATA1 mutation–associated TAM and ML-DS, especially with atypical presentations or due to insufficient clinical/genetic history, and distinguishing it from other pediatric leukemias. There are studies evaluating MRD by GATA1 using PCR; these findings may be applicable to IHC in the future. Reduced expression of GATA1 in BCR‐ABL⁻ MPNs is a subject of active research as a predictive marker for new future therapies.^48–50 

Nucleophosmin (NPM1) is a phosphoprotein that is encoded on chromosome 5 and found in abundance in nucleoli.^53,54  It shuttles between nucleoli and cytoplasm, acting as a molecular chaperone, and helps in executing multiple crucial cellular functions, like transport of preribosomal particles, ribosome biogenesis, response to stress, genomic stability, control of cellular ploidy, DNA repair, histone chaperoning, centrosome duplication, and chromatin condensation.^53–55  NPM1 controls the activity of tumor suppressors like alternative reading frame (ARF) and p53 and can act as an oncogene when its overexpression leads to development of a solid neoplasm. In hematolymphoid neoplasms, it typically acts as a tumor suppressor gene if there is a mutation, translocation or deletion.⁵⁵ 

As an oncogene, NPM1's role in ribosome biogenesis and protein synthesis plays a key role in tumorigenesis. Mutations in other genes like MYC can cause overexpression of NPM1 in tumorigenesis.⁵⁴  NPM1 functions as an antiapoptotic protein by enhancing DNA repair, resisting apoptosis induced by hypoxia and ultraviolet radiation. As a tumor suppressor it complexes with ARF and stabilizes it and inhibits mouse double minute 2 homolog (MDM2) to stabilize p53.^53,54,56  ARF is a protein that can cause cell cycle arrest and induce apoptosis. The complex also prevents the shuttling of NPM to cytoplasm and prevents ribosome assembly and cell proliferation.^53,54  Cells with reduced NPM protein also show reduced ARF levels.^54,56  NPM1 arrests the cell cycle by the p53-dependent pathway under stressful conditions.⁵⁴  It also maintains genomic stability and chromosomal ploidy by enhancing DNA repair in multiple ways, including by enhancing the activity of DNA repair kinases ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and RAD3-related (ATR).^54,56 

NPM1-mutated AML was introduced as a separate diagnostic entity in the World Health Organization 2017 edition but is not considered to be a leukemia-defining mutation at the time of this publication and still needs to fulfill the criteria of 20% blast count in bone marrow.⁵⁵  The mutations are seen in approximately 30% of all AML and 50% to 60% of normal karyotype AML.^53,55  The somatic mutations are almost always in exon 12 and are usually insertions of 4 bp next to Trp288.⁵⁵  The most common mutation, also known as type A mutation, occurs due to duplication of TCTG at Trp288.⁵⁵  These leukemias often show a characteristic “cuplike” nuclear invagination on bone marrow smears and frequently show dysplasia in multiple lineages.⁵⁶  By immunophenotyping, the blasts are very commonly negative for CD34.⁵⁶  Patients with AML with NPM1 mutations have a favorable prognosis unless it is associated with FLT3-ITD mutations. NPM1 mutations have been identified in MDS and MDS/MPN in <5% of cases and are associated with an aggressive course, with frequent and rapid progression to AML.⁵⁵  Because it is separate diagnostic entity, NPM mutations are frequently tested for in AML by one of the methods like sequencing and/or RT-PCR.

The utility of IHC for NPM1 was described in the seminal paper⁵³  that identified NPM1 mutation in AML, and more recently it has been described to be helpful in analyzing MRD in AML.^57,58  The initial studies were done on bone marrow that was fixed in B5 or Bouin fluid, but recent studies have shown equal efficacy in formalin-fixed tissues.^53,57,59  Although there is no standard definition of positivity yet, in 1 study >3 cells with cytoplasmic staining in the entire biopsy was considered positive and 1 to 3 cells was considered borderline because the authors believed that scattered staining can be difficult to distinguish from background staining.⁵⁹  IHC has an 80% to 90% sensitivity and 100% specificity compared with sequencing and RT-PCR analysis.^53,58–60  In one study,⁵⁹  the concordance between RT-PCR and IHC was 79% compared with that between sequencing and RT-PCR, which was 60%. The discrepancy is usually reported in cases with low tumor burden.⁵⁹  There are various clones available to detect NPM1 protein. Most of the studies have used a monoclonal antibody against the N-terminal of NPM1 (mAb clone 376; pan-NPM) that detects wild-type NPM1 and almost all of the mutant NPM1 protein.^53,61  Some of the recent studies have used a polyclonal antibody (PA1-46356; mutation-specific NPM1) that detects the most common NPM1 mutant protein (type A mutation) and some of the less common mutations.⁵⁹  In type A–mutated cases, both the clones show cytoplasmic and nuclear staining of all the leukemic cells. Occasional cytoplasmic staining of megakaryocytes by mutation-specific NPM1 antibody has been observed.⁵⁹  In nonmutated cases, pan-NPM1 antibody shows cytoplasmic staining in a few blasts undergoing mitosis, whereas no staining is seen by the mutation-specific NPM antibody.^57,62,63  NPM1 IHC has been used recently to evaluate MRD. Compared with next-generation sequencing, IHC has a specificity of 100%, a sensitivity of 80%, and an accuracy of approximately 90%.^58,59  However, because only 1 study has evaluated MRD by NPM IHC, further multi-institutional correlative studies would be helpful before a conclusion can be drawn on its utility.

Recently, mutant NPM1 neoepitope has been shown to be a potential target for immunotherapy.⁵⁵  T cells with high avidity to these neoantigens are capable of killing NPM1-mutated cells. NPM1 mutation is also predictive of response to the BCL-2 inhibitor venetoclax, potentially due to its antiapoptotic role. Addition of venetoclax to conventional chemotherapy improves relapse-free survival and overall survival.⁵⁵  Menin-mixed lineage leukemia inhibitors have been tried with some success in mouse- and patient-derived xenograft (PDX) models and are now undergoing phase 1/2 trials for NPM1-mutated acute leukemias.⁵⁵ 

NPM1 IHC is therefore a cheaper and often faster alternative to molecular methods in making the diagnosis of AML with mutated NPM1. It may assist in predicting response to BCL-2 inhibition and upcoming therapies. The pan-NPM1 antibody is more sensitive and can identify the common and rare variants but also stains wild-type NPM1 protein. The mutation-specific NPM1 antibody is highly specific for type A mutation but less sensitive for NPM1 mutations in general. IHC can also measure tumor burden and analyze the architecture and cytology of the neoplastic cells and act as a complementary test to other molecular studies in low-level disease in MRD.^55,57,59  Patel et al⁵⁷  suggested an algorithm where IHC should be done as a part of workup at first remission and must be followed by molecular studies if negative.

Herein we highlight a few of the exciting immunohistochemical markers that are being studied, optimized, and validated for use in pathologic examination of myeloid neoplasms (Table). Even in the era of advancing molecular techniques, the need for accurate in situ detection of various markers on human tissue remains high. These markers are often very cost-effective and can be interpreted with extremely fast turnaround time. With greater use, the results of additional specific phenotyping can also advance our understanding of these diseases by showing the proximity and localization of cells of interest based on their expression patterns.