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Available online at www.sciencedirect.com

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Immunohistochemical markers in lymphoid malignancies: Protein correlates of molecular alterations Caleb Ho, MD, Scott J. Rodig, MD, PhDn Department of Pathology, Brigham and Women ’s Hospital, Boston, Massachusetts

article info

abstra ct

Keywords:

Histomorphology, immunohistochemistry (IHC), and genetics are essential tools for the

Immunohistochemistry

evaluation and classification of lymphoid malignancies. Advances in diagnostic techniques

Hematologic malignancy

include the development of immunohistochemical assays that can serve as surrogates for

Protein expression

genetic tests. We review the performance of a select subset of assays that detect the aberrant

Genetic alteration

expression of onco-proteins secondary to chromosomal translocations (MYC; BCL2), somatic mutations (BRAF V600E; NOTCH1), and gene copy number gains (CD274 (encoding PD-L1); PDCD1LG2 (encoding PD-L2)) in fixed tissue biopsy sections. We discuss the limitations of IHC, but also its primary advantage over genetics; specifically, its ability to assess the final, common phenotypic consequences of a multitude of genetic and non-genetic events that influence protein expression. The information provided by IHC and genetic testing are thus intimately related; surgical pathologists will increasingly need to interpret and integrate the results of both to provide a comprehensive assessment of tumor biology and guide therapy. & 2015 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction Our understanding of the classification and underlying pathogenesis of lymphomas has improved tremendously in the past few decades, as recurrent genetic alterations have been described in a large number of lymphoproliferative disorders. In some cases, these alterations are known to be oncogenic drivers, while others have unclear functions. The cumulative effect is to promote tumorigenesis through the aberrant expression and function of proteins that affect critical intracellular signaling pathways, growth factors or their receptors, and the cellular microenvironment. Genetic testing is increasingly being used in conjunction with immunohistochemistry (IHC) to facilitate tumor classification in

routine diagnostics. Common tests include tumor cell karyotype to establish the identity, number, and structural rearrangements of chromosomes by morphology, fluorescence in situ hybridization (FISH) of labeled DNA probes to tumor cell nuclei to screen for specific chromosomal translocations or genetic copy gains or loss, and next-generation sequencing of nucleic acids from unfractionated tumors to survey for hundreds or thousands of somatic mutations. Genetic testing is appealing because the results are easily quantifiable and come from an analyte (DNA) that is extremely stable. IHC uses antibodies to detect the expression of specific proteins in tissue sections and, in contrast to genetic testing, the analytes are labile. The results of IHC can be seriously affected by pre-analytical variables, such as the chemicals

Conflict of interest and financial disclosures: Dr. Rodig has patent application submitted on the use of PD-L1 immunohistochemical stain for diagnostic work. Dr. Rodig also received research support from Ventana/Roche; and Bristol-Myers Squibb. n Correspondence to: Department of Pathology, Brigham and Women ’s Hospital, 75 Francis St, Boston, Massachusetts 02115. E-mail address: [email protected] (S.J. Rodig). http://dx.doi.org/10.1053/j.semdp.2015.02.016 0740-2570/& 2015 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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used for tissue fixation, the duration of tissue fixation, and the protocols used for tissue processing. In addition, the results of IHC are reliant upon the performance of specific antibodies on specific tissue types. Given these factors, optimal staining protocols must be determined empirically for every individual test. In addition, substantial experience in recognizing the staining patterns resulting from any of the hundreds of tests available to diagnostic pathologists is critical for accurate interpretations. Despite these limitations, IHC remains an essential tool in diagnostic laboratories for several reasons. First, pathology laboratories have had decades of experience with IHC, which can be performed routinely, economically, and quickly. Second, IHC captures essential phenotypic information that cannot be determined by genetic analysis alone, such as a tumor ’s cell of origin. Third, IHC preserves the histomorphological features of a tumor, which facilitates the interpretation of the results. Fourth, IHC can detect disrupted protein expression due to a variety of genetic, epigenetic, translational, post-translational, and microenvironmental aberrations rather than focusing on specific genetic changes. This article will focus on a few examples of how recent scientific discoveries have improved our understanding of IHC findings in correlation with the underlying genetic lesions in various lymphoid neoplasms, and hence our abilities to utilize IHC results in detecting or screening for these lesions.

MYC The MYC (c-MYC) gene on chromosome 8 at band q24 codes for a multifunctional nuclear protein that acts as a transcriptional activator or repressor, which plays a critical role in cell growth, cell cycle progression, apoptosis, angiogenesis, and cellular transformation.1 A balanced translocation involving the MYC gene is the hallmark feature of Burkitt lymphoma (BL), the first lymphoma in which a recurrent chromosomal abnormality was described.2,3 Most commonly, the translocation juxtaposes MYC with the immunoglobulin heavy chain gene (IGH) enhancer on chromosome 14q32 (approximately 80% of cases) or, less frequently, the immunoglobulin kappa gene (IGK) on chromosome 2p12 or the lambda light chain gene (IGL) on chromosome 22q11.4 MYC translocations involving non-immunoglobulin genes, such as PAX5 (chromosome 9p13) and BCL6 (chromosome 3q27), have also been described.5–7 Each of these translocations leads to constitutive expression of the MYC transcript and protein in the malignant cells. Traditionally, BL is diagnosed through recognition of stereotypical histomorphological features, coexpression of antigens associated with mature, germinal center B-cells, and genetic studies demonstrating a chromosomal rearrangement involving 8q24. The detection of the MYC oncoprotein in tumor cells by IHC would seem an obvious way to confirm a diagnosis of BL in the absence of genetic testing. Yet, early studies on MYC IHC yielded conflicting results, possibly due to the lack of antibodies amenable to detecting the protein in fixed tissues.8,9 More recently, however, it was found that a novel monoclonal rabbit anti-human MYC antibody (Y69 clone, Epitomics Inc.) was useful for detecting MYC in

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formalin-fixed, paraffin-embedded (FFPE) tissue sections.10 In an initial study of MYC IHC using BL cases, most (15/17 cases) with confirmed MYC translocations, it was found that the tumor cells comprising the majority of the cases (88%) demonstrated intense nuclear staining for the oncoprotein, while the remaining cases showed equal intensity of nuclear and cytoplasmic staining. In contrast, almost all cases (18/19 cases; 95%) of diffuse large B-cell lymphoma (DLBCL) without MYC rearrangement (MYC–DLBCL) lacked nuclear staining of the tumor cells for MYC.10 This initial study also included a small number of DLBCL with MYC rearrangement (MYCþDLBCL) and B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and BL (BCL-U). MYC IHC showed intense nuclear staining for the oncoprotein in these cases as well. Overall, nuclear staining for MYC was found to be 96% sensitive and 90% specific for MYC rearrangement, with high positive and negative predictive values (0.92 and 0.95, respectively) and a high inter-observer concordance in classifying the MYC staining pattern between two pathologists (kappa statistic ¼ 0.90). The results suggested that IHC effectively captures the dysregulated expression of the MYC oncoprotein in BL, the prototypical tumor harboring a MYC translocation. MYC translocations occur in additional lymphoid malignancies, including plasmablastic lymphoma, transformed follicular lymphoma, blastoid mantle cell lymphoma, and de novo DLBCL, albeit at much lower frequencies than observed for BL.11–13 Approximately 10–15% of patients with de novo DLBCL are MYCþ, and a number of studies have demonstrated that these patients have inferior 5-year progressionfree survival and overall survival rates, higher rate of central nervous system relapse, as well as poor responses to CHOP or R-CHOP chemotherapy compared to patients with de novo MYC– DLBCL when treated with standard immunochemotherapy.14–17 MYC IHC has been tested on genetically annotated DLBCLs in several studies. It was found that high nuclear MYC staining by IHC (450% of tumor cells) was detected not only in all MYCþ DLBCLs, but also in a subset of MYC– DLBCLs.18 Both MYCþ and MYC– DLBCLs with high MYC protein were found to exhibit high MYC transcript and MYC target gene expression by gene set enrichment analysis (GSEA). Moreover, the transcriptional signature of DLBCLs with high MYC protein expression resembled that of the so-called molecular Burkitt lymphoma (mBL) reported previously.14,19 Finally, DLBCL cases with high MYC protein expression were shown to have poorer overall survival after R-CHOP chemotherapy compared to DLBCLs with low MYC protein expression.18 The mechanisms responsible for MYC dysregulation in MYC– DLBCL with high MYC protein are under active investigation but are likely multi-factorial and include MYC copy number gain, dysregulated micro-RNAs that govern MYC transcript abundance, and mutations in MYC itself.1,18,20 Fig. 1 shows examples of two cases of DLBCL with high nuclear MYC staining by IHC. One showed MYC gene rearrangement as demonstrated by FISH study, while the other one did not show MYC gene rearrangement. Several subsequent studies have validated the negative prognostic significance of MYC overexpression in patients with DLBCL treated with R-CHOP, especially in the context of

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Fig. 1 – MYC IHC in two diffuse large B-cell lymphomas (DLBCL). (A) DLBCL case with high MYC protein expression and demonstrated MYC gene rearrangement by FISH using break-apart probes (inset). (B) DLBCL case with high MYC protein expression, but no MYC gene rearrangement detected by FISH using break-apart probes (inset).

concurrent BCL2 overexpression.21–25 Thus, MYC IHC appears more sensitive than conventional karyotyping and FISH in detecting MYC-driven DLBCLs, as up to 50% of DLBCLs with high MYC protein and high MYC biological activity lack a MYC translocation. However, the challenge for pathologists incorporating MYC IHC into routine practice remains the employment of rigorously standardized staining and scoring protocols to ensure reproducible results on a daily basis.

BCL2 BCL2 (B-cell leukemia–lymphoma-2), located on chromosome region 18q21, codes for a protein that is expressed normally by resting T and B cells, but not by normal germinal center cells or cortical thymocytes, and regulates cell death by functioning as an important anti-apoptotic protein.26–27 It was originally cloned from follicular lymphomas cell lines with t(14;18)(q32;q21), which juxtaposes the immunoglobulin heavy chain gene IGH promoter with BCL2, and is now known to be characteristic of up to 90% of follicular lymphomas (FLs). BCL2 translocations have also been observed in a subset of DLBCLs,28–32 but they were not observed in Hodgkin and other types of non-Hodgkin lymphomas.31 This translocation is also absent in many grade 3B FLs, which are likely more closely related to DLBCL.33 The translocation results in a hybrid transcript consisting of the 50 half of the BCL2 mRNA fused to an IGH mRNA, which continues to encode a normal but overexpressed BCL2 protein, as the breakpoint is located outside the BCL2 coding region.34 Overexpression of BCL2 confers survival advantage by preventing apoptosis, as demonstrated by an in vitro experiment on growth factordeprived hematopoietic cell lines.35 Although t(14;18)(q32;q21) is a characteristic of FL, in practice, the diagnosis of FL is usually made by a combination of cellular morphology (typically a combination of centrocytes and centroblasts), architectural pattern (typically at least partially follicular), IHC positivity for antigens associated with mature, germinal center B cells, and the detection

of aberrant BCL2 protein expression by the malignant cells.36 There are several caveats to consider applying BCL2 IHC as a surrogate for genetic testing. First, BCL2 expression is more frequent among grades 1–2 FL cases (85–97%) compared to grade 3 cases (50–75%), consistent with the distribution of t(14;18) in these tumors.37,38 Second, BCL2 is also expressed by the malignant cells of various other low-grade B-cell lymphomas, such as mantle cell lymphoma and marginal zone lymphoma, although the characteristic t(14;18) is absent.36 Third, and most importantly, up to 10% of FLs are negative for BCL2 by IHC using the conventional monoclonal antibody raised against residues 41–54 of the BCL2 protein.39 In a study by Schraders et al., grades 1, 2, and 3A FLs that were negative or showed heterogenous staining for BCL2 IHC with the standard antibody were stained with alternative antibodies raised against residues 1–201 as well as against the whole BCL2 protein. Five of these 18 cases were positive for BCL2 using the alternative antibodies, and these cases showed t(14;18) by FISH analysis. Of the remaining FL cases, one case had t(14;18) but was negative for BCL2 using all the antibodies tested. Twelve cases lacked t(14;18) and were negative for BCL2 by IHC using the standard and alternative antibodies. PCR and sequencing analysis on the cases showing BCL2 positivity with the alternative antibodies, which all harbor t(14;18), demonstrated somatic BCL2 mutations in a subset of cases, resulting in amino acid replacements within the epitope recognized by the conventional antibody.39 To provide further proof of the usefulness of alternative antibodies raised against different residues of the BCL2 protein, in a study by Masir et al., only about 50% of the 33 cases of t(14;18)-positive FL cases were strongly positive for BCL2 by IHC using a widely used monoclonal antibody BCL2/124 targeting residues 41–54. 20% of the cases were negative for BCL2 with the conventional antibody but were positive using an alternative antibody (clone E17 targeting residues 61–76).27 Sequencing of the genomic region encoding the epitope recognized by the two different BCL2 antibodies showed that some cases harbored BCL2 mutations resulting in amino acid

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replacements within the epitope recognized by the conventional BCL2/124 antibody, just like in the study by Schrader et al. The remaining 30% of the cases showed heterogeneous positivity for BCL2, using both the BCL2/124 and the clone E17 antibodies.27 In summary, BCL2 IHC is commonly used as a surrogate for genetic testing in cases of suspected FL. IHC is not infallible, as BCL2 negativity can be observed in FL cases with t(14;18) due to mutations within the epitope recognized by the anti-BCL2 antibody. The use of multiple antibodies targeting different epitopes can largely overcome this problem. Fig. 2 shows an example of follicular lymphoma involving the bone marrow, wherein the tumor cells are negative by IHC using a conventional BCL2 antibody clone 124 and positive using an alternative BCL2 antibody clone E17. In aggressive B-cell lymphomas, BCL2 translocations have been primarily evaluated in the context of co-existent MYC translocations, where the presence of both identifies a subset of tumors with dismal prognosis. A Canadian study on B-cell non-Hodgkin lymphomas with concurrent translocations involving BCL2 and MYC, including cases of de novo DLBCL; DLBCL transformed from FL or chronic lymphocytic leukemia; and B-cell lymphoma unclassifiable (BCL-U) showed that close to 60% died within 6 months of diagnosis, and only 11% remained in remission in 5 years.40 The median survival remained short despite the addition of Rituximab to the CHOP therapy, or the use of high-dose chemotherapy with stem cell transplantation.40 The inferior prognosis of socalled “genetic double-hit lymphomas” (MYC and BCL2 translocations) has been confirmed in additional series.41 BCL2 is expressed in a greater percentage of aggressive Bcell lymphomas than those that harbor the t(14;18), and its expression provides prognostic information. An early study found that high BCL2 protein expression had an adverse effect on overall survival within the activated B-cell (ABC) group of DLBCL, but not within the germinal center B-cell-like (GCB) group, although the study did not take into consideration the presence of MYC gene translocation or MYC protein expression in these cases, factors that have been associated with poor prognosis.42 Subsequent studies have focused on the synergistic effects of BCL2 and MYC overexpression in

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predicting patient outcomes in high-grade B-cell lymphomas. A study by Johnson et al. found that among a cohort of DLBCL patients treated with R-CHOP, poor overall and progressionfree survival were only seen in the group with both BCL2 and MYC positivity by IHC (defined as Z50% and Z40% tumor cells showing positivity, respectively). The negative prognostic impact was significant for the MYC/BCL2 positive cases by IHC, regardless of the BCL2 translocation status, and persisted even after adjusting for the International Prognostic Index (IPI) score and cell-of-origin (COO) gene signatures (ABC vs. GCB groups).22 Similarly, a study by Hu et al.24 found that high BCL2/MYC coexpression, as detected by IHC (defined as Z70% and Z40% tumor cells showing positivity, respectively), was associated with significantly worse overall and progression-free survival rates, after adjusting for the IPI scores and other clinico-pathological parameters. Meanwhile, neither MYC nor BCL2 expression alone significantly impacted survival rates. BCL2/MYC-positive cases correlated significantly with DLBCL of ABC type, a group associated with inferior outcome, as determined by a combination of GEP and IHC data. After excluding all BCL2/MYC-positive cases within the DLBCL ABC group, the remaining cases have similar prognoses to the cases of GCB type.24 Finally, a study by Green et al.21 also found that high expression of both MYC and BCL2 in DLBCL, as detected by IHC using the same cutoff as the study by Hu et al., was significantly associated with lower complete treatment response rate and shorter survival, independent of the IPI score. Again, neither MYC nor BCL2 gene rearrangement alone significantly impacted prognosis. The findings in these and other similar studies led to the proposal of various scoring systems that utilized the expression of MYC, BCL2, and BCL6, as detected by IHC, in stratifying DLBCL patients into prognostic subgroups. The study by Green et al.21 proposed a double-hit score that ranged from 0 to 2, using IHC results for MYC and BCL2. On the other hand, a study by Horn et al.23 found that a scoring system with a total score of 0–3, by assigning a risk score of 1 to each of the adverse features (high MYC, high BCL2, and low BCL6 expression), can predict overall survival and event-free survival independent of the IPI score. In summary, genetic studies

Fig. 2 – BCL2 IHC in a follicular lymphoma involving the bone marrow. (A) BCL2 IHC using the conventional antibody clone 124 is negative in the tumor cells. (B) BCL2 IHC using the alternative antibody clone E17 is positive in the tumor cells.

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can identify a group of DLBCL with concurrent MYC and BCL2 translocations, so-called “double-hit lymphomas,” which is a group associated with poor treatment response and survival. However, detection of protein overexpression of MYC and BCL2 by IHC identifies a larger group of DLBCL cases, so-called “double-hit score lymphomas” that also have a poor prognosis, many of which are not driven by MYC or BCL2 gene translocations and are therefore not captured through routine genetic studies.

BRAF V600E BRAF, also known as v-Raf murine sarcoma viral oncogene homolog B, codes for a serine threonine kinase that is part of the RAF/MEK/ERK cascade, also known as the ERK/MAP kinase pathway. It is a signaling pathway that mediates cellular responses to growth signals, cell differentiation, and survival.43–45 The most common somatic mutation in BRAF among human tumors leads to the substitution of glutamic acid for valine at codon 600 (V600E), which has often been found in melanomas46,47 and papillary thyroid cancers.48,49 The amino acid substitution results in the activation of BRAF,46,49 which in turn, leads to abnormal activation of the ERK/MAP kinase pathway to promote tumor growth.46–48 BRAF inhibitors have shown efficacy in the treatment of solid tumors with the BRAF V600E mutation.50,51 During whole-exome sequencing in search of mutations associated with hairy cell leukemia (HCL), the BRAF V600E mutation was found in the peripheral blood samples or marrow biopsy of all 48 cases studied.52 This mutation was not found in the matched non-neoplastic cells from the same patients or patients with other types of B-cell lymphomas/ leukemias that can be difficult to distinguish from HCL, including splenic marginal zone lymphoma (SMZL) and hairy cell leukemia-variant (HCL-v). Furthermore, MEK and ERK, downstream kinase targets of BRAF, were shown to be phosphorylated (activated) in the HCL samples, and the degree of phosphorylation decreased with a BRAF inhibitor.52 Thus, exome sequencing revealed a molecular hallmark for HCL and suggested a novel therapy for this disease. Given that BRAF V600E is a common mutation observed in several neoplasms, a novel monoclonal antibody (clone VE1) recognizing the mutated protein was generated, validated for IHC53 and subsequently applied to HCL.54 BRAF V600E-specific IHC demonstrated cytoplasmic positivity in all 32 HCL bone marrows, which were mostly confirmed to have the specific mutation by direct DNA sequencing, except two cases with low tumor burden (o15%), thought to be below the detection threshold of heterozygous mutation by Sanger sequencing. The mutation-specific IHC was also positive in one case of CLL/SLL with confirmed BRAF V600E mutation, but negative in a variety of other B-cell neoplasms.54 Thus the BRAF V600Especific IHC is useful for establishing a diagnosis of HCL and to exclude the diagnoses of SMZL and HCL-v. Fig. 3 shows a case of hairy cell leukemia involving the bone marrow with positivity for BRAF V600E-specific IHC, which was confirmed to harbor the mutation by pyrosequencing. Besides HCL, the BRAF V600E mutation is also found in close to 60% of Langerhans cell histiocytosis (LCH), a rare disease with no previously reported recurrent genomic abnormalities.55 Early

Fig. 3 – BRAF V600E-specific IHC in hairy cell leukemia involving the bone marrow. BRAF V600E-specific IHC shows diffuse cytoplasmic positivity in the tumor cells. The presence of the mutation was confirmed by pyrosequencing of tumor DNA isolated from paraffin-embedded sections. studies suggest that mutation-specific IHC will be useful for the diagnostic evaluation of LCH as well.56,57 The extreme specificity of mutation-specific IHC is also its limitation. A more recent study described a rare subtype of HCL lacking a mutation in BRAF but with an activating mutation in MAP2K1, which codes for MEK1, a direct effector of BRAF.58 These HCLs express the IGHV4-34 immunoglobulin variable heavy chain, and as a group, have higher disease burden, worse response to treatment, and shorter survival compared to typical HCL.58,59 These cases would not be expected to be captured by BRAF V600E-specific IHC. However, one can speculate that IHC tests targeting downstream signaling molecules in the ERK/MAP kinase pathway, such as phospho-ERK (pERK), may capture lymphomas with dysregulated ERK/MAPK signaling in general.60,61 As a conclusion, the BRAF V600E mutation is the most common mutation detected in HCL, and this specific mutation can be detected by IHC. In contrary to IHC for MYC and BCL2, both of which aim to detect the final result of protein overexpression by a variety of mechanisms, IHC for BRAF V600E aims to detect an altered protein produced by a specific genetic mutation. In this case, the advantages of IHC over genetic studies are the former method ’s faster turnaround time, lower cost, and possibly higher sensitivity, when compared to PCRbased mutation detection from FFPE tissue. Usage of pERK IHC could potentially detect HCL cases with a broader range of genetic alterations, as some of these cases can have mutations other than BRAF V600E that cause dysregulation of the ERK/MAP kinase pathway. However, additional studies will be needed to validate its sensitivity and specificity in HCL and HCL-v cases.

NOTCH1 NOTCH1, located on chromosome 9, encodes a heterodimeric transmembrane protein that acts as a ligand-activated transcription factor. Notch1 plays a key role in determining cell fate, including the differentiation of lymphoid precursors into

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T cells.62–65 After ligand binding to Notch1, a series of proteolytic cleavages eventually lead to the cleavage of the Notch receptor’s transmembrane domain, liberating the Notch intracellular domain (NICD), allowing its translocation to the nucleus with transcriptional activation of multiple target genes, including MYC and NF-κB.64–67 NOTCH1 can function as either a tumor suppressor gene, or as an oncogene, depending on the cellular context.68 The chromosomal translocation t(7;9)(q34;q34.3) was recognized in rare cases of T-cell acute lymphoblastic leukemia/lymphoma (T-ALL) and, upon cloning, was found to fuse the intracellular domain of NOTCH1 to the beta T-cell receptor locus.69 The subsequent introduction of active NOTCH1 alleles into mice resulted in the development of T-cell neoplasms, consistent with an oncogenic role for the truncated protein.70 However, chromosomal translocation is a rare mechanism of pathogenesis in T-ALL.62,69 Weng et al.71 described up to 50–60% of T-ALL cases contained activating NOTCH1 mutations. These mutations can affect the heterodimerization (HD) domain (most common), the proline glutamate serine threonine (PEST) domain, or both.62,71–73 Mutations in the HD domain enhance cleavage of Notch1 to NICD, in some cases in a ligand-independent manner, while mutations in the PEST domain reduce the degradation of NICD.64,71 The final common result is constitutive Notch1 signaling. As one might suspect, mutations in other genes that participate in the Notch pathway can also cause activation of the Notch pathway and are associated with tumorigenesis. By specifically looking at T-ALL cell lines and patient samples with constitutive expression of NICD but lacked C-terminal PEST domain truncating mutations in NOTCH1, a study identified either missense mutations or homozygous deletions of the gene FBXW7 (FBW7), which codes for a ubiquitin ligase that is critical in the degradation of NICD and is dependent on binding to the PEST domain of Notch1.67 The identified mutations affect residues within the substratebinding domain of FBXW7, impairing FBXW7–NICD interactions and hence NICD ubiquitination. Due to the resulting stabilizing effects on NICD, these FBXW7 mutations also render the tumor cells resistant to gamma secretase inhibitors (GSI), a class of drug that usually decreases the level of NICD by blocking the activation of Notch to NICD.67 NOTCH1 PEST domain mutations and FBXW7 mutations appeared to be mutually exclusive in this study and another study,74 but they were found to rarely coexist in a subsequent study,73 suggesting that FBXW7 mutations represent an alternative pathway of Notch deregulation and pathogenesis for T-ALL. Besides playing an important role in the pathogenesis of TALL, recurrent NOTCH1 mutations have also been identified in chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) in several studies,65,75,76 including a recent study that detected a recurrent frameshift mutation (P2514fs) in a subset of the 91 CLL patients during whole-exome and -genome sequencing of matched leukemic cells and normal tissue.77 The mutation rate was significantly higher than the background rate, suggesting NOTCH1 as a driver mutation in CLL. This particular frameshift mutation is thought to result in a premature stop codon resulting in a protein with a truncated C-terminal PEST domain, leading to impaired degradation and accumulation of the active isoform of

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Notch1—NICD.65,76,77 The result is an activation of the Notch pathway, as demonstrated by gene expression analysis of differentially expressed genes in NOTCH1-mutated and unmutated CLL cases.76 NOTCH1 mutation was also associated with unmutated immunoglobulin heavy chain (IGHV), the presence of trisomy 12, higher rate of transformation into DLBCL, more advanced clinical stage, and shorter overall survival.65,76,77 Additionally, mutations in FBXW7 were also detected in some cases of CLL,77 further implicating the Notch1 pathway in the pathogenesis of this common mature B-cell malignancy. Just as in the other examples mentioned above, NOTCH1 mutations resulting in an accumulation of activated Notch1 protein (NICD1) can be detected by IHC. IHC is especially helping in screening for activating NOTCH1 mutations, as genetic screening is difficult, considering the large size of the NOTCH1 gene and the variety of genetic alterations described so far.68 Kluk et al. made use of a rabbit monoclonal antibody specific for a neo-epitope at the amino terminus of NICD1, resulting from the proteolysis of Notch1 by a gamma secretase. The study showed either diffuse nuclear or no positivity by IHC in FFPE tissue sections of T-ALL cases, thought to correspond to the NOTCH-mutated and unmutated cases, respectively. Staining for CLL/SLL cases was more variable, with close to 90% of cases showing positivity in at least 10% of tumor cells, but 33% of cases showing positivity in 450% of tumor cells, and 15% of cases being positive in 480% of tumor cells. Deep sequencing on frozen tissue identified NOTCH1 activating mutation in 17% of the cases, most commonly the frameshift mutation in codon 2514 mentioned above. Although there was a correlation between the percentage of mutated reads by sequencing, and the percentage of positive tumor nuclei by NICD1 IHC among the NOTCH1-mutated cases (p o 0.0005), some CLL/SLL cases with wild-type NOTCH1 alleles showed significant positivity for NICD1 staining. It was thought that among these wild-type cases, microenvironmental factors, such as the presence of Notch ligands, mediated Notch1 activation. This hypothesis was supported by the observation that NICD1 IHC staining was much weaker in tumor cells found in perinodal soft tissue when compared to tumor cells within the adjacent nodal tissue.68 Fig. 4 shows two cases of CLL/SLL, one with a high fraction of tumor cells being positive for NICD1 IHC staining and the other with almost no tumor cells being positive. The NOTCH1 p2514fs mutation status for both cases was confirmed by pyrosequencing. In the study by Kluk et al.,68 NICD1 staining was also positive in some cases of various types of peripheral Tcell lymphomas, such as angioimmunoblastic lymphoma, but the role of NOTCH1 activating mutations in this lymphoma group is still unclear. Overall, the presence of activating NOTCH1 mutations, as well as other mutations resulting in the activation of the Notch pathway, can be detected by IHC for NICD1, the activated form of the Notch1 protein. IHC is particularly useful in this situation, as genetic mutation analysis is difficult due to the large size of the NOTCH1 gene and the variety of known genetic alterations in the NOTCH1 gene as well as related genes such as FBXW7. For T-ALL cases, the correlation between NICD1 positivity by IHC and activating NOTCH1 mutations appears to be more straightforward,

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Fig. 4 – NICD1 IHC in two chronic lymphocytic leukemia/small lymphocytic lymphomas (CLL/SLL). (A) CLL/SLL case with a high amount of tumor cell NICD1 staining, which was confirmed to harbor the NOTCH1 P2514fs frameshift mutation by pyrosequencing. (B) CLL/SLL case with a very low amount of tumor cell NICD1 staining, which was confirmed not to harbor the NOTCH1 P2514fs frameshift mutation by pyrosequencing.

while for CLL, it appears to be more complicated, with an apparently high negative predictive value but a less robust positive predictive value. Regardless of this, the use of NICD1 IHC on T-ALL and CLL patients can be helpful in selecting patients for drug trials involving Notch pathway inhibitors,71 as well as identifying the subset of CLL patients with more rapid disease progression and poor prognosis, who may require more frequent monitoring and aggressive treatments.

PD-1 ligands Programmed death-1 (PD-1) receptors are inducibly expressed on activated T cells, and function to limit T-cell-mediated immune responses. Normal antigen-presenting cells, dendritic cells, and macrophages can express PD-1 ligands, which upon binding to PD-1 receptors, leads to the attenuation of T-cell receptor (TCR) signaling, which is essential in maintaining immune tolerance and balanced responses to eliminate pathogens and tumor cells.78 PD-1 ligand 1 (PD-L1) is constitutively expressed in a subset of cells of the hematopoietic lineage and can be upregulated in solid tumors and certain hematologic malignancies,79,80 while PD-1 ligand 2 (PD-L2) is usually inducibly expressed on dendritic cells, macrophages, and mast cells.79 PD-L1 can also competitively bind to CD80 (B7-1), the ligand for CD28, a constitutively expressed receptor on T cells that provide co-stimulatory signal for T-cell activation and cytokine production.79 The result is a temporary and reversible suppression of T-cell activation and proliferation.81 Furthermore, PD-L1 plays an essential role in the induction and maintenance of induced regulatory T (iT reg) cell functioning, including the suppression of effector T cells, inhibition of immune-mediated tissue damage, and peripheral immune tolerance.82 Due to its role in suppressing T-cell activation and effector T cells, one can see why the PD-1 pathway can be utilized by tumors to reduce or evade the host antitumor immune responses.

The significance of the PD-1/PD-ligand signaling axis in inhibiting antitumor immune responses has become apparent with recent clinical trials. In a multicenter phase 1 drug trial with an anti-PD-L1 antibody, which blocks the interaction of PD-L1 with PD-1 on different types of advanced cancers, partial or complete response was seen in a subset of melanomas, renal cell carcinomas, non-small-cell lung cancer, and ovarian cancers with sustained tumor regression and disease stabilization at least 1 year after treatment.80 Another phase 1 trial published at the same time investigated instead the use of anti-PD-1 antibody, which blocks the interaction of PD-1 with its ligands, PD-L1 and PD-L2, in treating various advanced malignancies.83 Partial or complete response was again seen in a subset of melanomas, renal cell carcinomas, and non-small-cell lung cancer, with durable treatment response with at least 1 year of follow-up. Just as in the anti-PD-L1 antibody trial, most treatment-related adverse events were low grade, with some potential immune-related adverse events. These data suggest that immunotherapy targeting PD-1 and PD-L1 will be increasingly integrated into the treatment of selected solid tumors. Analyses of lymphoma cell lines and primary tissues have revealed a genetic basis for PD-ligand expression. Using lasercapture microdissection to purify Reed–Sternberg cells seen in classical Hodgkin lymphomas (CHL), and high-resolution array comparative genomic hybridization (aCGH) analysis, Steidl et al.84 identified various recurrent chromosomal abnormalities associated with CHL, including gains of chromosome 9p, with 9p24.1–24.3 being one of the regions with the most frequent chromosomal gains. Amplification of the 9p24.1 locus in CHL was further characterized by Green et al.81 using high-density single nucleotide polymorphism (HD SNP) arrays with paired transcriptional profiles of CHL cell lines, identifying CD274 (encoding PD-L1), PDCD1LG2 (encoding PD-L2), and Janus kinase 2 (JAK2), all located in close proximity to each other at 9p24.1, as the target genes of amplification. Chromosome 9p24.1 amplification was

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confirmed by allele-specific PCR using DNA isolated from micro-dissected Reed–Sternberg cells in primary CHL.81 As predicted, increasing copy number of CD274 and PDCD1LG2 encoded at 9p24.1 correlated with increased PDligand expression. However, Green et al. also showed that JAK2 copy gain also contributed to this effect. The CD274 promoter, and to a lesser extent the PDCD1LG2 promoter, contains an interferon (IFN)-stimulated regulatory element/ IFN-regulatory factor 1 (ISRE/IRF1) module that is responsive to JAK/STAT signaling. Amplification of the JAK2 locus on 9p24.1 results in an increase in JAK2 transcript, phosphorylated (active) JAK2 and STAT1, and an induction of PD-L1 in CHL cell lines in a manner sensitive to JAK2 inhibition.81 Thus 9p24.1 copy gain results in PD-ligand overexpression by multiple, reinforcing mechanisms. Perhaps not surprisingly, 9p24.1 copy gain is not restricted to CHL. Primary mediastinal large B-cell lymphoma (PMBL) is an entity with shared clinical and molecular genetic features with CHL.85,86 Consistent with this, a subset of PMBL also has 9p24.1 copy gain and PMBL cell lines express the PD-ligands.81 A subset also overexpresses PD-L1 or PD-L2 due to chromosomal translocations involving CD274 or PDCD1LG2, respectively. Twa et al.87 used a break-apart FISH assay to show that 20% of 125 cases of PMBL have chromosomal translocations, a frequency much higher than other B-cell lymphomas. Using qRT-PCR, PD-L1 and PD-L2 transcripts were shown to be significantly higher in translocation-positive cases compared to cases with neutral copy numbers at 9p24.1.87 Finally, within classical Hodgkin lymphomas, chromosome 9p24.1 copy gain was specifically restricted to the EBV-negative, nodular sclerosis subtype (NSHL). In EBV-positive tumors, an alternative mechanism for PD-L1 expression was described. Using EBV-transformed lymphoblastoid cell lines (LBLs), Green et al.88 showed that the EBV-encoded latent membrane protein (LMP-1) promotes constitutive activation of AP-1, an enhancer element in CD274 (which encodes PD-L1), as well as JAK-STAT signaling, which induces PD-L1 expression. Due to the variety of mechanism that can drive PD-1 ligand overexpression in lymphoid neoplasms (gene copy number

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gain, chromosomal translocation, JAK/STAT signaling, and EBV-driven induction), the use of IHC would be expected to have an advantage over targeted genetic testing. Chen et al.89 validated IHC for PD-L1 to confirm protein expression in Reed–Sternberg cells of CHL and malignant B-cells of PMBL. That study also showed that PD-L1 expression was fairly common among EBV-positive malignancies including EBVpositive DLBCL (DLBCL of the elderly and immunodeficiencyrelated DLBCL), EBV-positive post-transplant lymphoproliferative disorders (PTLD), plasmablastic lymphoma, extranodal NK/T-cell lymphoma, and nasopharyngeal carcinoma.88,89 In addition, PD-L1 was identified in a subset of EBV-negative PTLD, HHV8-associated primary effusion lymphoma, and Tcell/histiocyte-rich large B-cell lymphoma.89 On the other hand, positivity for PD-L1 was uncommon among nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL); DLBCL, not otherwise specified (NOS); Burkitt lymphoma; and HHV8-associated Kaposi sarcoma. An unanticipated result of these studies was the detection of robust PD-L1 expression by non-neoplastic cells, primarily macrophages, within the tumor microenvironment of many PD-L1-positive malignancies.89 Indeed, for tumors such as CHL, in which non-neoplastic inflammatory cells far exceed the malignant Reed–Sternberg cells, the majority of PD-L1 protein in the cellular microenvironment is attributable to tumorassociated macrophages (TAMs) as demonstrated by double staining for CD68 and PD-L1.89 The mechanism of PD-L1 induction in TAMs remains unclear, but cytokine-mediated JAK/STAT signaling likely contributes. Moreover, it is not clear whether PD-L1 positivity in TAMs is associated with tumor aggressiveness and prognosis. However, several studies have described an association between increased TAMs in CHL and shorter disease-free and overall survival rates.90,91 More recently, Shi et al.92 validated IHC for PD-L2 and demonstrated that over 70% of PMBL cases, and only 3% of DLBCL cases, showed membranous positivity by IHC in at least 20% of tumor cells. The majority of PMBL cases showed 3þ (strong) staining in the majority of the tumor cells, while the one case of DLBCL that was positive showed staining in

Fig. 5 – PD-L2 IHC in two primary mediastinal (thymic) large B-cell lymphomas (PMBL). (A) PMBL showing positive membrane staining for PD-L2. This case also demonstrated PDCD1LG2 copy number gain by qPCR. (B) PMBL showing no membrane staining for PD-L2. This case lacked PDCD1LG2 copy gain by qPCR.

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only approximately 20% of tumor cells. Using quantitative PCR, 75% of PMBLs with frozen and paraffin-embedded tissues available showed amplification of the PDCD1LG2 gene, which were all positive for PD-L2 by IHC. High copy number gain of PDCD1LG2 was associated with intense membranous positivity for PD-L2 by IHC. On the contrary, none of the DLBCLs showed gain of copy number of PDCD1LG2.92 Fig. 5 shows examples of PMBL with or without PD-L2 expression by IHC that are in accordance with genetic analyses. Immunohistochemical testing for PD-ligand expression may prove important for the selection of patients who are likely to respond to targeted immunotherapy. In the phase I trial of a PD-1 inhibitor in patients with solid tumors, a subset of patients (36%) who had PD-L1-positive tumors had objective response to the anti-PD-1 drug, while none of the patients who had PD-L1-negative tumors had objective response.83 A trial of an anti-PD-1 antibody (Nivolumab) in 23 patients with relapsed/refractory Hodgkin lymphomas suggests a high response rate (17% with complete response and 70% with partial response) among patients with Hodgkin lymphoma positive for PD-ligand expression.93 As a summary, the use of IHC for PD-L1 and PD-L2 can be helpful for diagnostic purposes, such as in the case of differentiating PMBL from DLBCL, and may yet prove predictive of tumor responses to anti-PD-1 or anti-PD-L1 therapies. The use of IHC has many advantages in the case of PD-1 ligands, as the variety of mechanisms causing PD-L1 and PD-L2 overexpression (chromosomal copy gain, translocation, cytokine-mediated, and EBV-driven induction) creates difficulties in detection when relying upon genetics studies alone. IHC also captures PD-ligand expression by non-neoplastic cells within the tumor microenvironment, which may contribute to the overall immune evasion strategy of a tumor.

Conclusions The examples above show that IHC can be an economical and reliable method of detecting underlying genetic aberrations in lymphoid neoplasms. It can be performed routinely in many pathology laboratories and requires less instrumentation and personnel expertise necessary for genetic analyses such as karyotyping, FISH, and PCR mutation studies. In the cases of MYC, BCL2, Notch1, and PD-1 ligands, not only can IHC capture dysregulated protein expression due to known and specific genetic lesions, it can also serve as a common final read-out for other known and unknown genetic lesions, epigenetic modifications, or microenvironmental regulation that result in dysregulated protein expression. Thus, IHC captures information that has diagnostic, prognostic, and therapeutic implications, which may not be detected by genetic analyses alone. Moreover, the use of IHC also allows the concurrent evaluation of staining pattern, tissue architecture, and cellular morphology. However, from the examples above, it is also apparent that the selection of appropriate and specific antibodies, a rigorous validation process, use of relevant controls, and standards for comparisons are all crucial for performing IHC. It is also important to understand that certain genetic mutations, as in the case of BCL2, can result in false negativity due to the loss of a

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recognizable epitope by the antibody. Genetic analysis also has the advantage of a digital read-out, which may be important from the perspectives of treatment decisionmaking and disease monitoring. In the future, pathologists will be required to integrate the complementary data from multiple diagnostic modalities including IHC and genetics to classify lymphoid malignancies with greater precision.

Acknowledgments The authors would like to thank Drs. Paola Dal Cin, Elizabeth Morgan, Michael Kluk, as well as Cynthia McLaughlin for providing representative clinical cases discussed in this review.

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