Received: 15 August 2023 | Accepted: 25 September 2023 DOI: 10.1111/bjh.19142 O R I G I N A L P A P E R IGJ and SPATS2L immunohistochemistry sensitively and specifically identify BCR::ABL1+ and BCR::ABL1-like B-acute lymphoblastic leukaemia Catherine K. Gestrich1 | Shanelle J. De Lancy1 | Adam Kresak1 | Howard Meyerson1 | Irina Pateva2 | Akua K. Yalley3 | Christopher Ryder1 | Shashirekha Shetty1 | Jacob Bledsoe4 | Erika M. Moore5 | Kwadwo A. Oduro Jr1 1Department of Pathology, University Hospitals Cleveland Medical Center, Summary University Hospitals Rainbow Babies and Therapeutic management and prognostication for patients with B-acute lympho- Children's Hospital and Case Western Reserve University, Cleveland, Ohio, USA blastic leukaemia (B-ALL) require appropriate disease subclassification. BCR::ABL1- 2Division of Hematology and Oncology, like B-ALL is unique in that it is defined by a gene expression profile similar to Department of Pediatrics, University BCR::ABL1+ B-ALL rather than a unifying recurrent translocation. Current molecu- Hospitals Rainbow Babies and Children's lar/cytogenetic techniques to identify this subtype are expensive, not widely acces- Hospital and Case Western Reserve University, Cleveland, Ohio, USA sible, have long turnaround times and/or require an adequate liquid biopsy. We have 3Department of Medical Laboratory Sciences, studied a total of 118 B-ALL cases from three institutions in two laboratories to iden- School of Biomedical and Allied Health tify surrogates for BCR::ABL1+/like B-ALL. We report that immunoglobulin joining Sciences, University of Ghana, Accra, Ghana chain (IGJ) and spermatogenesis associated serine-rich 2-like (SPATS2L) immuno- 4Department of Pathology, Boston Children histochemistry (IHC) sensitively and specifically identify BCR::ABL1+/like B-ALL. Hospital, Boston, Massachusetts, USA 5 IGJ IHC positivity has a sensitivity of 83%, a specificity of 95%, a positive predictive Department of Pathology, University of Pittsburgh Medical Center, Pittsburgh, value (PPV) of 89% and a negative predictive value (NPV) of 90%. SPATS2L stain- Pennsylvania, USA ing has similar sensitivity and NPV but lower specificity (85%) and PPV (70%). The presence of either IGJ or SPATS2L staining augments the sensitivity (93%) and NPV Correspondence Kwadwo A. Oduro Jr, Department of (95%). While these findings would need to be validated in larger studies, they suggest Pathology, University Hospitals Cleveland that IGJ and/or SPATS2L IHC may be utilized in identifying BCR::ABL1-like B-ALL Medical Center, 11100 Euclid Avenue, or in selecting B-ALL cases for confirmatory molecular/genetic testing, particularly Cleveland, OH 44106, USA. Email: kwadwo.oduro@uhhospitals.org; in resource-limited settings. kaoduroj@gmail.com K E Y W O R D S Present address B-ALL, BCR::ABL1, BCR::ABL1-like, IGJ, SPATS2L Catherine K. Gestrich, Department of Pathology, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania, USA I N TRODUC TION as hyperdiploidy, hypodiploidy, t(9;22) with BCR::ABL1, t(12;21) with ETV6::RUNX1, t(1;19) with TCF3::PBX1, B-acute lymphoblastic leukaemia (B-ALL) is the most com- KMT2A rearranged (KMT2A-R), t(5;14) with IGH::IL3 and mon malignancy in the paediatric population globally.1 intrachromosomal amplification of RUNX1 on chromosome In the United States, B-ALL comprises about 25% of pae- 21 (iAMP21). Newer subtypes recognized in the 2017 World diatric cancers, and about 40% of total B-ALL cases occur Health Organization (WHO) classification and the 2022 in adults. Classification of B-ALL is largely based on the proposed WHO and International Consensus Classification identification of recurrent cytogenetic abnormalities such (ICC) classifications2–5 are less amenable to routine This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. © 2023 The Authors. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd. Br J Haematol. 2023;00:1–11. wileyonlinelibrary.com/journal/bjh | 1 2 | IMMUNOHISTOCHEMISTRY FOR BCR::ABL1-LIKE B-ALL cytogenetic testing for their identification. Considering the with putative roles in ribosome biogenesis and response to important role of B-ALL classification in risk stratification oxidative stress29 and no established function in the hae- and guiding up front or subsequent risk-adapted or targeted matopoietic system. IGJ IHC is utilized as a reliable marker management decisions,6–8 techniques to identify these new for neoplastic cells of nodular lymphocyte-predominant B-ALL subtypes in a rapid and cost-effective manner would Hodgkin lymphoma (NLPHL).30–32 Although SPATS2L an- be clinically significant. tibodies are commercially available, there is no current diag- BCR::ABL1-like B-ALL has a gene expression (transcrip- nostic use for SPATS2L IHC. tome) profile similar to BCR::ABL1+ B-ALL but lacking We demonstrate in this report that IHC for IGJ and the BCR::ABL1 translocation.9,10 Subsequent studies have SPATS2L sensitively and specifically identifies BCR::ABL1+/ demonstrated that about 90% of BCR::ABL1-like B-ALL like B-ALL. cases harbour other genetic alterations resulting in activa- tion of kinase and cytokine receptor signalling, most com- monly rearrangements involving CRLF2 and fewer cases M ATER I A L S A N D M ETHODS with rearrangements involving ABL1 (non-BCR partner), EPOR and other genes.11 BCR::ABL1+ and BCR::ABL1-like Patient selection B-ALL are considered high-risk B-ALL subtypes and to- gether comprise about 10%–20% of paediatric and about Patients diagnosed with de novo B-ALL at University 50% of adult B-ALL.9,12,13 Classification of a new B-ALL into Hospitals (UH; 100 cases) and with available bone marrow one of these subtypes allows identification of patients who (BM) biopsies in formalin fixed paraffin embedded (FFPE) may benefit from targeted kinase inhibitor therapy either as blocks were identified as previously described.18 Additional a part of an established standard of care (for BCR::ABL1+ cases from Boston Children Hospital (BCH; 9 cases) and the B-ALL) or emerging treatments in the context of a clinical University of Pittsburgh Medical Center (UPMC; 9 cases), trial (for BCR::ABL1-like B-ALL).14,15 BCR::ABL1 identifica- most BCR::ABL1-like, were also included. In all but seven tion by karyotype, fluorescence in situ hybridization (FISH) cases, the initial diagnostic BM biopsy was utilized. For or PCR is routine in many diagnostic laboratories. However, seven cases, the initial biopsy was not available or was in- identification of BCR::ABL1-like B-ALL is less straightfor- adequate, and a relapse/refractory disease sample was used ward. DNA- or RNA-based techniques such as FISH, RNA instead. The study was performed in accordance with re- sequencing and gene expression arrays to identify either spective Institutional Review Board approvals. structural abnormalities prevalent in BCR::ABL1-like B-ALL or to identify its defining gene expression programme have been utilized.16,17 However, these techniques are expensive, B-ALL diagnosis and classification not widely accessible, may have long turn-around times, rely on an adequate marrow aspirate sample or the presence of B-ALL diagnosis and classification were made by board- circulating blasts, may be susceptible to RNA stability con- certified hematopathologists based on morphological, im- cerns and/or lack of correlative spatial information. munophenotypic and molecular/genetic characteristics as Immunohistochemistry (IHC) is a rapid, widely available described previously.18 Targeted archer-based RNA next and relatively low cost means of evaluating protein expres- generation sequencing (NGS), DNA NGS and/or single nu- sion on tissue sections and thus could circumvent or miti- cleotide polymorphism microarray (SNP array) to identify gate many of the challenges in identifying BCR::ABL1-like BCR::ABL1-like translocations or reveal gene copy number B-ALL if sensitive/specific markers are identified. We hy- variants/mutations was performed in a subset of patients. pothesized that genes upregulated at the protein level may Although all CRLF2-rearranged (CRLF2-R) cases evaluated serve as useful IHC markers and have recently shown that showed CRLF2 overexpression by flow cytometry or at the MUC4 IHC is specific for BCR::ABL1+/like B-ALL, albeit mRNA level, overexpression of CRLF2 alone without a cor- not very sensitive.18 In the current study, we have tested pro- roborating genetic abnormality was not used to designate a tein expression of IGJ and SPATS2L, both previously shown case as BCR::ABL1-like since overexpression can rarely be to be upregulated at the mRNA level in BCR::ABL1+/like observed in non-CRLF2-R/non-BCR::ABL1-like B-ALL.33,34 B-ALL relative to other subtypes,10,19,20 which is incorpo- Of the 118 B-ALL cases evaluated, 95 were classifiable rated in currently utilized or reported targeted low density into categories conforming to WHO 2017, WHO 2022 array (LDA) or quantitative real-time PCR assays for identi- and ICC 2022 criteria after standard cytogenetics and fying BCR::ABL1-like B-ALL.21–26 BCR::ABL1-like testing. Sophisticated DNA/RNA sequenc- The IGJ expression in B-ALL is paradoxical for two rea- ing, transcriptome evaluation and specialized FISH testing sons: First, IGJ is not appreciably expressed in normal com- to identify molecular/genetic abnormalities defining newly mitted B lineage precursors, and second, the known function described definitive and provisional rare B-ALL subtypes of IGJ protein is in mature B cells/plasma cells, where it con- in the most recently proposed WHO 2022 & ICC 2022 clas- catenates IgA and IgM monomers into multimeric mature sifications,3–5 many of which are cryptic to routine testing, forms and is involved in mucosal transport of these secreted were not performed for most cases. Also, since BCR::ABL1 immunoglobulins.27,28 SPATS2L is an understudied gene FISH was performed on total bone marrow cells and not on 13652141, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/bjh.19142 by University of Ghana - Accra, Wiley Online Library on [01/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License GESTRICH et al. | 3 separately sorted myeloid and lymphoid cells, BCR::ABL1+ or one board-certified haematopathologist and one to two B-ALL were not further subtyped into those with ‘lymphoid pathologists in training as diffusely positive, partially posi- only’ versus ‘multilineage’ involvement (ICC 20223,4). The 23 tive, or negative. The intensity of staining was also recorded patients who could not be classified into a specific B-ALL as weak or strong. Staining was considered diffuse when type were designated ‘Not Further Specified’ (NFS). The over 50% of the blasts were stained. Most negative cases NFS terminology is employed to avoid confusion with the showed no staining. Few cases with very rare cells (less than WHO 2022/ICC 2022 B-ALL, NOS category, since such des- 10%) showing weak staining were also scored as negative (i.e. ignation depends on the exclusion of other defined types of partial staining was 10%–50% of blasts). B-ALL, which in turn depends on the degree of comprehen- Additional Materials and Methods are provided in a sive molecular testing performed. Supplementary document. Immunohistochemistry R E SU LTS IHC for IGJ and SPATS2L were performed at UH on BM Sensitive and specific identification of FFPE sections as previously described.18 For IGJ, a mouse BCR::ABL1+/like B-ALL by IGJ IHC monoclonal antibody (clone OTI3B3; Invitrogen; 1:3200 dilution) was used as the primary antibody. Reactive tonsil To determine whether BCR::ABL1+/like express IGJ protein or lymph nodes involved by NLPHL30 were used for anti- and explore the utility of IGJ IHC in identifying these B-ALL body validation and as technical controls. IGJ IHC at UPMC subtypes, we stained BM biopsy samples from 116 cases from was performed as previously described (Clone 3B3, Lifespan three institutions and across a wide age range (1–82 years), Biosciences, 1:3000 dilution).30 For SPATS2L, a rabbit poly- including 64 paediatrics (<18 years), 20 adolescent-young clonal antibody (Invitrogen; 1:250 dilution) was used as a adults (AYA; 18–39 years) and 32 older adults (>39 years). The primary. Hippocampus sections were used for antibody vali- cohort was comprised of 23 BCR::ABL1-like, 17 BCR::ABL+ dation and for technical controls. and 75 non-BCR::ABL1+/like (including 21 hyperdiploid, For IGJ, cellular staining of lymphoblasts was distinguish- 13 ETV6::RUNX1, 5 TCF3::PBX1, 4 hypodiploid, 7 KMT2A- able from occasional background staining (possibly attrib- rearranged, 2 iAMP21, 1 t(5;14) and 23 NFS). We also stained utable to serum immunoglobulins as occurs frequently in 11 normal bone marrow cases. IGJ IHC was positive in 20/23 immunoglobulin heavy or light chain IHC) and scored in a (87%) of BCR::ABL1-like, 13/17 (76%) BCR::ABL1+ B-ALL blinded manner by two board-certified haematopathologists and 4/75 (5%) non-BCR::ABL1+/like (Figure  1, Table  1). F I G U R E 1 Variable expression of IGJ protein in B-ALL. Representative images of the IgJ IHC. (A) Negative, 400×, UH-077. A rare positive plasma cell is present. (B) Partial positive, 400×, UH-005. (C) Diffuse weak positive, 40×, UH-025. (D) Diffuse weak positive, 400×, UH-025. (E) Diffuse strong positive, 40×, UH-012. (F) Diffuse strong positive, 400×, UH-012. 13652141, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/bjh.19142 by University of Ghana - Accra, Wiley Online Library on [01/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 4 | IMMUNOHISTOCHEMISTRY FOR BCR::ABL1-LIKE B-ALL T A B L E 1 Summary of IgJ IHC results by B-ALL subtype. CD10−, CD15+ phenotype of most KMT2A-R B-ALL37 and Total # Diffuse Partial instead having a mature immunophenotype (Tdt-, CD34−, Subtype of cases IgJ+ staining staining CD20+, dim surface lambda light chain) as described in a 38–41 BCR::ABL1-like 23 20 (87%) 13 7 minority of KMT2A-R B-ALL cases. No distinguishing BCR::ABL1+ 17 13 (76%) 10 3 features were seen between the IGJ+ and the respective IGJ− ETV6::RUNX1 and NFS cases. We also did not identify any Hyperdiploidy 21 0 0 0 unifying features in IGJ+ non-BCR::ABL1+/like cases or be- ETV6::RUNX1 13 2 (15%) 0 2 tween these cases and the BCR::ABL1+/like cases. TCF3::PBX1 5 0 0 0 In summary, IGJ protein expression was significantly Hypodiploidy 4 0 0 0 associated with BCR::ABL1+/like B-ALL, identifying these KMT2A-rearranged 7 1 (14%) 0 1 subtypes with an 83% sensitivity, 95% specificity, 89% PPV iAMP21 2 0 0 0 and 90% NPV (p < 0.0001). For cases that were unclassifiable t(5;14) 1 0 0 0 after routine cytogenetic testing, the PPV was even better (19/20 IGJ IHC-positive cases were BCR::ABL1-like; PPV NFS 23 1 (4%) 0 1 of 95%), indicating IGJ IHC could provide added value to Abbreviations: B-ALL, B-acute lymphoblastic leukaemia; NFS, not further routine cytogenetics for the identification of BCR::ABL1-like specified. cases. Importantly, the data also indicates that CRLF2 rear- rangement is neither necessary nor sufficient for IGJ protein Diffuse positive staining was only present in the BCR::ABL1- expression. like (13/20) and BCR::ABL1+ (10/13) groups. All the control normal bone marrows were negative. The BCR::ABL1-like cases (Table  2) were mostly SPATS2L IHC augments the sensitivity of CRLF2-R, and IGJ staining was independent of CRLF2 identifying BCR::ABL1+/like B-ALL partner—IGJ+ in 8/9 IGH::CRLF2 (7 diffuse, 1 partial stain- ing) and 8/10 P2RY8::CRLF2 (4 diffuse, 4 partial staining). We tested SPATS2L protein expression by IHC in 104 B-ALL Importantly, there were three non-CRLF2-R BCR::ABL1-like cases from UH and BCH (Figure 2, Table 4). SPATS2L was cases—one with a JAK2 rearrangement (BCH-9), one with positive in 14/17 (82%) BCR::ABL1+ B-ALL and 12/15 (80%) an IGH::EPOR rearrangement (UPMC-4) and one with an BCR::ABL1-like B-ALL, including both non-CRLF2-R cases ETV6::ABL1 rearrangement (BCH-10)—and they were all tested (BCH-9 with JAK2-R and BCH-10 with ETV6::ABL1). IGJ+. All three BCR::ABL1-like cases, which were IGJ− (UH- SPATS2L IHC was positive in 11/72 (15%) non-BCR::ABL1- 006, BCH3, BCH4) and CRLF2-R, also had a CDKN2A loss, like cases, which included 6 NFS, 2 ETV6::RUNX1, 2 hy- but CDKN2A loss was also seen in a subset (4/14, 29%) of podiploidy, 1 KMT2A-R. Most of the SPATS2L-positive IGJ+ BCR::ABL1-like cases. For BCR::ABL1+ (Table  3), all cases (34/37) showed diffuse staining. Diffuse staining in 5/5 (100%) cases with a major breakpoint were diffuse IGJ+, BCR::ABL1+/like cases predominantly (21/23 cases) showed a while staining in the 10 cases with a minor breakpoint was strong intensity, while diffuse staining in non-BCR::ABL1+/ more variable (4 diffuse IGJ+, 3 partial IGJ+, 3 IGJ−; p = 0.04). like cases (6/11 cases) was mostly weak (p = 0.007). None Nevertheless, the prevalent IGJ+ result in BCR::ABL1+ B-ALL of the B-ALL cases with hyperdiploidy (0/19), TCF3::PBX1 further indicates that IGJ+ is not restricted to CRLF2-R or (0/5), iAMP21 (0/2) or t(5;14) (0/1) were SPATS2L+. No JAK–STAT-activated B-ALL, despite the few non-CRLF2-R SPATS2L staining was seen in all three normal bone mar- BCR::ABL1-like cases in our cohort. No consistent distin- rows evaluated. guishing features were seen in the BCR::ABL1+/like cases Although IGJ and SPATS2L stain results were mostly con- negative for IGJ IHC. cordant (83% concordance, p < 0.0001), there was some dis- Intriguingly, four non-BCR::ABL1+/like cases were cordant staining (Figure 3). Specifically, 6/31 BCR::ABL1+/ also IGJ+, all with partial staining. These included two like (2 IGJ+SPATS2L−, 4 IGJ−SPATS2L+) and 11/72 non- ETV6::RUNX1 cases (UH-047 & BCH-2), one KMT2A-R BCR::ABL1+/like (2 IGJ+SPATS2L−, 9 IGJ−SPATS2L+) cases case (UH-072) and one NFS case (UH-100). BCR::ABL1- showed discordant staining. Concordant cases included like testing in two of these cases revealed a concomitant BCR::ABL1+/like cases (UH-006, UH-011, BCH-4) negative P2RY8::CRLF2 in BCH-2, which has been reported before,35,36 for both IGJ and SPATS2L and 2 non-BCR::ABL1+/like cases and no rearrangements in UH-100. BCR::ABL1-like testing (UH-047 and UH-100) positive for both stains. The other performed on 24 other non-BCR::ABL1-like cases showed two IGJ+ non-BCR::ABL1+/like cases (BCH-2 and UH-072) three additional cases with a co-occurring P2RY8::CRLF2 were SPATS2L negative. All non-BCR::ABL1-like cases with rearrangement—BCH-6 (hyperdiploidy), UPMC-7 (hyper- a concomitant P2RY8::CRLF2 rearrangement evaluated— diploidy) and UH-076 (iAMP21)—all of which were IGJ−. UH-076 (iAMP21), BCH-2 (ETV6::RUNX1) and BCH-6 (hy- Therefore, co-occurring CRLF2-R or other BCR::ABL1-like perdiploidy)—were negative for SPATS2L. genetic abnormality does not appear to explain the few non- Overall, IGJ and SPATS2L IHC have comparable sensitiv- BCR::ABL1+/like cases with IGJ+ staining. UH-072 differed ity (81%–83%) and negative predictive value (91%) for identi- from the other KMT2A-R cases in lacking the characteristic fying BCR::ABL1+/like cases. However, IGJ has far superior 13652141, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/bjh.19142 by University of Ghana - Accra, Wiley Online Library on [01/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License GESTRICH et al. | 5 T A B L E 2 BCR::ABL1-like cases. ICC 2022 Age BCR::ABL1-like Positive BCR::ABL1-like genetic/molecular CRLF2 Case # (years) class testing overexpression? Karyotype Downs? Additional positive genetics findings UH-001 49 JAK–STAT CRLF2-R (FISH) Y (flow) 46,XY,del(6)(q13q23),add(19)(q13.4) N CRLF2 gain (FISH) [12]/46,XY[10] UH-002 80 JAK–STAT CRLF2-R (FISH) Y (flow) CF N None UH-003 44 JAK–STAT Loss of 5′ (FISH) Y (flow) 46,XY[20] N None UH-004 41 JAK–STAT CRLF2-R (FISH), JAK2 L681_I682insGQD Y (flow, mRNA) CF N Non-productive KMT2A-R (FISH). (NGS) IKZF1 loss (NGS). UH-005 37 JAK–STAT CRLF2-R (FISH) Y (flow) 46,Y,t(X;20)(p22;q13.3),del(1) N None (q32q42),i(7)q10)[15]/46,XY[5] UH-006 31 JAK–STAT CRLF2-R (FISH) Y (flow) 47,XY,+21c[25] Y CRLF2 gain and CDKN2A loss (FISH) UH-007 19 JAK–STAT Loss of 5′ (FISH), Xp22.3 del (SNP array) Y (flow) 47,XY,+21c[8]/48,idem,+X[12] Y IKZF1 and CDKN2A losses (SNP array) UH-008 4 JAK–STAT P2RY8-CRLF2+ (PCR), JAK2 p.R683S Y (flow, mRNA) 46,XX,dic(9;20)(p13.2;q11.2),+21[5]/47, N CDKN2A loss (FISH) (NGS), Xp22.3 del (SNP array), LDA+ idem,+20[3]/46,XX[2] UH-009 2 JAK–STAT Loss of 5′ (FISH), P2RY8-CRLF2+ (PCR), Y (flow) 46,XX,dic(9;20)(p13.2;q11.21),+21[18] N CDKN2A loss (FISH) JAK2 p.R683G (NGS), Xp22.3 del (SNP /46,XX[2] array), LDA+ UPMC-1 2 JAK–STAT Loss of 5′ (FISH) N/A 46,XX,i(9)(q10)[7]/46,XX[1] N PAX5 loss, ABL1 gain and sublconal IGH-R (FISH) UPMC-2 52 JAK–STAT CRLF2-R (FISH) N/A 47,XX,+21[9]/46,XX[1] N IGH-R (FISH) UPMC-3 16 JAK–STAT CRLF2-R (FISH), JAK2 p.R683S (NGS) N/A 46,XY,del(6)(q15q24),del(12) N IGH-R and ETV6 loss (FISH). EBF1, (p12p13),add(15)(q24) ETV6, and BTG1 losses (SNP array) [8]/46,XY[12] UPMC-4 22 JAK–STAT EPOR-IGH (NGS) N/A 46,XY,der(19)t(1;19)(q21;p13)[10] N PBX1 gain and TCF3 loss (FISH). IKZF1 loss (SNP array). No PBX1::TCF3 by FISH. UPMC-5 4 JAK–STAT Loss of 5′ (FISH), Xp22.3 del (SNP array) N/A 48,XY,+X,+21[5]/46,XY[15] N IKZF1, PAX5 and ETV6 losses (SNP array) UPMC-6 12 JAK–STAT CRLF2-R (FISH) N/A 47,XY,+21c[20] Y EBF1, SETD2 and FBXW7 losses (SNP array) UPMC-8 2 JAK–STAT Loss of 5′ (FISH), Xp22.3 del (SNP array) N/A 49,XY,+X,+17,+21[3]/46,XY[18] N PAX5 loss (SNP array) UPMC-9 12 JAK–STAT CRLF2-R (FISH) N/A 49,XY,+X,der(1)t(1;8) N IKZF1 loss (SNP array) (q44;q11.2),del(3)(p11.2),ins(4;3) (p14;p24.3p26.3),+7,del(8) (p12p23.2),+del(8)(p12p23.2) [cp8]/46,XY[4] BCH-1 5 JAK–STAT P2RY8-CRLF2 (PCR), LDA+ N/A 46,XY[11] N None (Continues) 13652141, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/bjh.19142 by University of Ghana - Accra, Wiley Online Library on [01/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 6 | IMMUNOHISTOCHEMISTRY FOR BCR::ABL1-LIKE B-ALL T A B L E 2 (Continued) ICC 2022 Age BCR::ABL1-like Positive BCR::ABL1-like genetic/molecular CRLF2 Case # (years) class testing overexpression? Karyotype Downs? Additional positive genetics findings BCH-3 5 JAK–STAT P2RY8-CRLF2+ (NGS), CRLF2 p.F232C Y (flow) 46,XY,del(9)(p21)[2]/46,sl,del(11)(q23) N CDKN2A and KMT2A losses (FISH). [1]/46,XY[19] IKZF1, CDKN2B, ATM losses (NGS). BCH-4 3 JAK–STAT P2RY8-CRLF2 (NGS) Y (flow); 4% of 46,XY[20] N CDKN2A loss (FISH). CDKN2B blasts loss (NGS). PAX5::ZCCHC7, ZCCHC7::PAX5 (NGS) BCH-7 15 JAK–STAT P2RY8-CRLF2 (PCR), LDA+ Y (flow) 46,X,-X,add(2)(p21),- N RUNX1 gain (FISH). CREBBP loss 9,add(16)(p13.3),add(21) and SH2B3 c.519_520insGCCCG (q21),+2mar[6]/46,XX[14] p.P173fs* (NGS) BCH-9 6 JAK–STAT JAK2-R (FISH) N (flow) 46,XX[20] N IKZF1 loss (NGS) BCH-10 5 ABL ETV6::ABL1 (unknown), LDA+ N (flow) 46,XY[20] N IKZF1 loss and ABL1 gain (NGS) BCH-11 2 JAK–STAT JAK2 p.R683G (NGS), LDA+ Y (flow) 45,XX,add(9)(p13),-20,der(21) N CDKN2A loss (FISH). JAK2 loss t(20;21)(p11.2;p13) [5]/46,sl,+X[3]/46,XX[12] Note: CRLF2 FISH was performed using break-apart probes. “CRLF2-R” in the table indicates separation of the 5′ and 3′ FISH probes which typically correlates with IGH::CRLF2. “Loss of 5′” in the table indicates loss of the 5′ FISH probe, which is typically due to an intrachromosomal deletion event resulting in P2RY8::CRLF2. For BCH11, No CRLF2 FISH, PCR or RNA sequencing was performed to directly identify the CRLF2-R. Therefore whether this case is IGH::CRLF2 or P2RY8::CRLF2 is unknown. No assessment for CDKN2A loss by FISH or SNP microarray performed for UH-001, UH-002, UPMC-1, UPMC-2 and BCH-7. Abbreviations: CF, culture failure; -R, rearranged; N/A, not available. 13652141, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/bjh.19142 by University of Ghana - Accra, Wiley Online Library on [01/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License GESTRICH et al. | 7 T A B L E 3 BCR::ABL1+ cases. BCR-ABL1 Case # Karyotype Additional FISH Breakpoint UH-010 CF BCR::ABL1, subclonal −17 and +17 Minor UH-011 47,XX,+4,t(9;22)(q34;q11.2)[9]/47,idem,del(1)(q42),der(7)t(7;21) BCR::ABL1, RUNX1 gain Minor (q22;q11.2)[14]/46,XX[2] UH-012 45,XX,-7,t(9;22)(q34;q11.2)[11]/46,XX,idem,+6[2]/46,XX[2] BCR::ABL1 Neither UH-013 46,XX,t(9;22)(p24;q11)[6]/46,idem,-11,+mar[9]/46,XX[5] BCR::ABL1 Minor UH-014 45,XX,del(7)(p15),t(9;22)(q34;q11.2),-16[11]/46,XX[9] BCR::ABL1 Minor UH-015 46,XX,t(9;22)(q34;q11.2)[18]/46,XX[2] BCR::ABL1 Minor UH-016 CF BCR::ABL1 Minor UH-017 46, XX[5] BCR::ABL1 Neither UH-018 N/A BCR::ABL1 Major UH-019 46,XX,t(9;22)(q34;q11.2)[11]/48,idem,-der(9)t(9;22)(q34;q11.2),- BCR::ABL1 Major 14,+22,+der(22)t(9;22)(q34;q11.2),+der(22)t(9;22)(q34;q11.2),+mar[9] UH-020 46,XY,t(9;22)(q34;q11.2)[4]/46,XY[16] BCR::ABL1 Major UH-021 46,XX,t(9;22)(q34;q11.2)[20] BCR::ABL1 Minor UH-022 45,XX,-7,dup(8)(q24.1q13),t(9;22)(q34;q11.2)[14]/46,XX[11] BCR::ABL1, subclonal C-MYC gain Major UH-023 46,XY,t(9;22)(q34;q11.2)[1]/46,idem,t(9;14)(p21;q13),del(12)(p11.2) BCR::ABL, subclonal ETV6 loss and Major [14]/49,idem,+8,+17,+21[3]/46,XY[2] KMT2A gain UH-024 45,XY,-3,der(7;9)t(7;9)(q10;q10)t(9;22)(q34;q11.2),+mar[5]/46,XY[6] BCR::ABL1, CDKN2A loss, Minor subclonal +7 UH-025 48,XX,t(9;22)(q34;q11.2),+19,+mar[3]/46,XX[22] BCR::ABL1 Minor UH-026 CF BCR::ABL1 +4 Minor specificity (95%) and PPV (89%). Combining both stains im- (70%) than IGJ (89%). Also, in contrast to IGJ and SPATS2L proved performance for detection of BCR::ABL1+/like cases; IHC, sensitivity for MUC4 IHC is only about 30%, even for example, the presence of either IGJ or SPATS2L staining though it has 100% specificity.18 In unpublished work, we improved sensitivity (from 83% to 93%) and NPV (from 90% have failed to detect some mRNA-overexpressed genes at the to 95%) (Table 5). protein level by IHC. These differences justify empiric eval- uations to identify optimal IHC markers for BCR::ABL1-like and other newer subtypes of B-ALL. Mechanistic studies DISCUSSION would also be important to determine the contribution of these genes to the biology of BCR::ABL1+/like B-ALL. The fields of diagnostic pathology and oncology are in dire One limitation of the study is the fact that most BCR::ABL1- need of facile, accessible and inexpensive methods of iden- like cases included in the study were CRLF2-R, by far the tifying BCR::ABL1-like B-ALL, a high-risk B-ALL subtype most prevalent genetic abnormality in BCR::ABL1-like with the potential for emerging targeted therapy. We report B-ALL. This raises the question of whether IGJ & SPATS2L here for the first time IHC markers with high sensitivity and IHC are simply recognizing CRLF2-R B-ALL, which will specificity for identifying BCR::ABL1+/like B-ALL. diminish their utility since CRLF2 flow cytometry already The IHC results corroborate prior mRNA-based gene provides a robust method for identifying CRLF2-R B-ALL. expression data that have demonstrated preferential up- However, we do not think this is the case because, first, regulation of IGJ and SPATS2L genes in BCR::ABL1+/like IGJ & SPATS2L IHC stains BCR::ABL1+ B-ALL (which B-ALL and the incorporation of their overexpression in lack CRLF2 rearrangement) with a similar frequency as LDA/qRT-PCR screeners for BCR::ABL1-like B-ALL.10,19–24 BCR::ABL1-like B-ALL, and second, all three non-CRLF2-R This is noteworthy because many upregulated genes from BCR::ABL1-like cases (1 with JAK2-R, 1 with EPOR-R and transcriptome analyses either have limited known function 1 with ETV6::ABL1) were also IHC positive. These results (e.g. SPATS2L), have previously known function/proper- contrast with MUC4 IHC, which appeared to show prefer- ties that would not predict protein expression in haemato- ential expression in B-ALLs with ABL1 class rearrangement poietic cells (e.g. MUC4), or in immature precursors (e.g. versus those with CRLF2-R and other JAK–STAT pathway IGJ). Preferential upregulation at the mRNA level does not activation.18,42 Our findings suggest that IGJ and SPATS2L always translate into a diagnostically useful IHC test for would be useful in identifying both JAK–STAT-activated BCR::ABL1+/like identification, however. For instance, even and ABL1-rearranged classes of BCR::ABL1-like B-ALL rec- between IGJ and SPATS2L, SPATS2L had a much lower PPV ognized by the ICC 2022 classification,3,4 although direct 13652141, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/bjh.19142 by University of Ghana - Accra, Wiley Online Library on [01/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 8 | IMMUNOHISTOCHEMISTRY FOR BCR::ABL1-LIKE B-ALL F I G U R E 2 Variable expression of SPATS2L protein in B-ALL. Representative images of SPATS2L IHC. (A) Negative, 200×, UH-032. (B) Partial positive, 200×, UH-008. (C) Diffuse strong positive, 200×, UH-001. (D) Diffuse weak positive, 200×, UH-064. T A B L E 4 Summary of SPATS2L IHC results by B-ALL subtype. Subtype Total # of cases SPATS2L+ Diffuse staining Partial staining BCR::ABL1-like 15 12 (80%) 9 3 BCR::ABL1+ 17 14 (82%) 14 0 Hyperdiploidy 19 0 0 0 ETV6::RUNX1 12 2 (17%) 2 0 TCF3::PBX1 5 0 0 0 Hypodiploidy 4 2 (50%) 2 0 KMT2A-rearranged 7 1 (14%) 1 0 iAMP21 2 0 0 0 t(5;14) 1 0 0 0 NFS 22 6 (27%) 6 0 Abbreviations: B-ALL, B-acute lymphoblastic leukaemia; NFS, not further specified. testing of the rare ABL1-class, non-CRLF2-R JAK–STAT other class-defining genetic abnormalities. CRLF2 rear- class and BCR::ABL1-like NOS in larger cohorts would need rangements occur in about 20%–25% of iAMP21 B-ALL to be performed in the future for validation. and at a lower frequency in B-ALL with hyperdiploidy, Although genetic identification of rearrangements in ETV6::RUNX1 and the novel subtypes of B-ALL with tyrosine kinase receptor genes has proven invaluable in PAX5alt or ETV6::RUNX1-like gene expression.7,35,43–45 In the identification of BCR::ABL1-like B-ALL,11,21 one of many of these cases, the CRLF2-R is thought to be sub- the challenges in interpretation is the occasional co-oc- clonal, not a driver of disease biology or clinical behaviour, currence of CRLF2-R (especially P2RY8::CRLF2) with and may be lost at relapse.36,46 Furthermore, CRLF2 13652141, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/bjh.19142 by University of Ghana - Accra, Wiley Online Library on [01/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License GESTRICH et al. | 9 F I G U R E 3 Heat map display of IGJ and SPATS2L IHC in individual B-ALL cases. IgJ and SPATS2L IHC results for each case evaluated in this study organized by B-ALL subtype. (A) BCR::ABL1-like. (B) BCR::ABL1+. (C) Hyperdiploidy. (D) ETV6::RUNX1. (E) TCF3::PBX1, hypodiploidy, KMT2A-R, iAMP21, t(5;14). (F) NFS (not further specified). Each row is an individual case. Columns as follows: Column 1: Case number. UH, University Hospital cases; UPMC, University of Pittsburgh Medical Center cases; BCH, Boston Children's Hospital cases. Column 2: Alternating dark grey and light grey coding are used to designate different B-ALL subtypes. In Figure 3E, there are 5 TCF3::PBX1, 4 hypodiploidy, 7 KMT2A-R, 2 iAMP21 and 1 t(5;14) in that order. Column 3: Age at diagnosis. Dark blue is older adult (>39 years old). Intermediate blue is AYA (18–39 years old). Lightest blue is paediatric (<18 years old). Column 4: IGJ IHC results. Dark green, strong diffuse positive staining. Light green, partial or weak positive staining. Red, negative staining. Black, not assessed or inadequate tissue. Column 5: SPATS2L IHC results. Dark green, strong diffuse positive staining. Light green, partial or weak positive staining. Red, negative staining. Black, not assessed or inadequate tissue. T A B L E 5 Summary of test performance of IgJ and SPATS2L IHC. Sensitivity Specificity PPV NPV p value IGJ 83% 95% 89% 90% <0.0001 SPATS2L 81% 85% 70% 91% <0.0001 IGJ or SPATS2L 93% 82% 74% 95% <0.0001 IGJ & SPATS2L 71% 97% 92% 87% <0.0001 Abbreviations: NPV, negative predictive value; PPV, positive predictive value. rearrangement without BCR::ABL1-like gene expression for IGJ and negative for SPATS2L. Future studies on ad- does occur (about 1% of paediatric and AYA B-ALL in 1 ditional cases are needed to fully understand the perfor- study47), and false positive LDA screen due to CRLF2 over- mance and significance of IGJ/SPATS2L IHC staining in expression resulting from CRLF2 genetic alterations has B-ALL cases that have co-occurring class-defining rear- been described.26 Therefore, having another assay such rangements, mutations or gene expression profiles in ad- as IGJ/SPATS2L IHC, which is not reliant on CRLF2 al- dition to CRLF2 rearrangements. terations, holds promise for guiding appropriate disease Our work highlights the undying relevance of IHC, classification. Although our cohort only included a few even in an era of technological advancement in diagnos- cases of B-ALLs with co-occurring CRLF2-R and other tic pathology, that may be applicable to newer subtypes of class-defining genetic alteration, IGJ/SPATS2L IHC was B-ALL. Current methods for BCR::ABL1-like identification different from the vast majority of other CRLF2-R cases. such as LDA screen, large-scale RNA sequencing panels Specifically, one case with iAMP21 + P2RY8::CRLF2 and or multiprobe FISH panels cost several thousand dollars two cases with hyperdiploidy + P2RY8::CRLF2 were all and are either proprietary or require sophisticated/expen- negative for both IGJ and SPATS2L IHC, while one case sive equipment. In contrast, IHC is more than an order with ETV6::RUNX1 + P2RY8::CRLF2 was partially positive of magnitude cheaper, utilizes commercially available 13652141, 0, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/bjh.19142 by University of Ghana - Accra, Wiley Online Library on [01/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 10 | IMMUNOHISTOCHEMISTRY FOR BCR::ABL1-LIKE B-ALL reagents and can be performed with minimal equipment. ORC I D Therefore, IGJ/SPATS2L IHC would likely be more amena- Kwadwo A. Oduro Jr https://orcid. ble to adoption in developing countries48,49 and should org/0000-0001-5447-3442 also prove invaluable in the United States/other developed countries where there are increasing concerns related to R E F E R E N C E S decreasing individual/institutional medical care costs, 1. Ward ZJ, Yeh JM, Bhakta N, Frazier AL, Atun R. Estimating the total the levels of insurance reimbursements or where access to incidence of global childhood cancer: a simulation-based analysis. current techniques may be limited. 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