Molecular Genetics in the Diagnosis and Biology of Lymphoid Neoplasms

2017 Society for Hematopathology/European Association for Haematopathology Workshop Report

Megan S. Lim, MD, PhD; Nathanael G. Bailey, MD; Rebecca L. King, MD; Miguel Piris, MD


Am J Clin Pathol. 2019;152(3):277-301. 

In This Article

Aggressive B-cell Lymphomas

Genetic Testing in the Diagnostic Evaluation of Aggressive B-cell Lymphomas

DLBCL, not otherwise specified (NOS) represents 30% to 40% of all cases of non-Hodgkin lymphoma. From a histopathologic standpoint, identification of DLBCL is relatively straightforward. Innumerable studies over the past several decades have revealed genetic diversity and complexity in DLBCL, which have improved our understanding of the biology of the disease, in turn altering classification schemes to better reflect prognostic and predictive information. The 2017 WHO update included a number of changes that impact the diagnostic evaluation of DLBCL and served as a focus for discussion in this session Table 2.

Cell of Origin

Gene expression profiling (GEP) studies, initially performed on fresh tissue specimens, reveal three distinct cell-of-origin subgroups of DLBCL with mRNA expression profiles similar to their normal-cell counterparts: germinal center B-cell-like (GCB), ABC, and a third "unclassifiable" group.[17] The clinical significance lies in its ability to predict prognosis, with GCB subtype cases having more favorable prognosis in most studies.[12,17] As illustrated by virtually all of our submitted DLBCL cases, Hans algorithm immunohistochemistry has become the most widely used surrogate for determining cell-of-origin in DLBCL in the clinical setting.[18] This algorithm uses a panel of three stains (CD10, BCL6, MUM1), to approximate the GEP categories, classifying DLBCL as GCB or non-GCB. Although not widely available in the clinical setting yet, GEP assays using NanoString technology have now been developed for formalin-fixed, paraffin-embedded tissue and may represent the next wave of molecular testing to be routinely applied to DLBCLs.[19,20]

High-grade B-cell Lymphoma With MYC and BCL2 and/or BCL6 Rearrangements

While poor prognosis of MYC rearrangements in DLBCL has been documented for many years,[21,22] the updated WHO 2017 classification includes a new diagnostic entity, high-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements. This new entity has made testing for MYC by fluorescence in situ hybridization (FISH) essential in cases of DLBCL NOS.[12] While some cases of this entity, often referred to as "double-hit" (DHL) or "triple-hit" (if all three genes are rearranged) lymphomas have cytology resembling Burkitt lymphoma (BL), up to 50% have morphology indistinguishable from standard DLBCL. Clinically, these DHL cases have inferior prognosis and are often treated more aggressively, supporting their classification as a distinct entity.[23–27]

Two cases of DHLs were submitted to the workshop (cases 160 and 206). Case 206 showed large-cell cytology. Case 160 showed high-grade Burkitt-like cytology with a predominance of medium-sized cells. The latter case was reported to have a GCB-cell phenotype, and the patient had a history of FL. Case 160 demonstrated lack of correlation between MYC immunohistochemistry and MYC translocations identified by FISH: because 10% to 26% of cases with MYC rearrangements may be negative (below the 40% cutoff) for MYC by immunohistochemistry, it is currently felt to be essential to test all cases with DLBCL morphology by FISH.[28,29] Of note, so-called double-expresser DLBCL cases in which MYC and BCL2 are both positive by immunohistochemistry, are distinct from DHL and this immunophenotype lacks predictive value for true DHL. Although expression of MYC and BCL2 by immunohistochemistry may also hold prognostic value, it was stressed that protein expression is not sensitive or specific enough to be used as a screening for which DLBCL cases to FISH.[30–32] Additionally, although the majority of MYC/BCL2 DHL cases are of the GCB phenotype, a subset of MYC/BCL6 DHL cases have a non-GCB phenotype.[31] Thus, proposals to screen only GCB DLBCL with MYC FISH should be undertaken with the knowledge that some DHL cases will be missed.

Since the date of the workshop, several studies have revealed that the genomic complexity of DLBCLs is beyond what our current classification reflects. Comprehensive genomic analysis evaluating somatic copy number alterations, recurrent mutations, and structural variants reveals up to six genomic subgroups of DLBCL that do not precisely correspond to our current ABC vs GCB or DHL vs non-DHL paradigm.[33] Additionally, RNA sequencing studies have uncovered a genomic signature common to DHL which can be seen in a subset of non-DHL cases as well.[34] Results of these studies are likely to change our diagnostic evaluation of such tumors in the future.

MYD88 Mutations in DLBCL

Although not recognized under one unifying 2017 WHO category, MYD88 L256P mutations are common in certain subgroups of DLBCL. Among an unselected group of DLBCLs, MYD88 mutations were described in 22% of all cases and correlated with older age, higher serum lactate dehydrogenase (LDH), and frequent extranodal involvement.[35] These cases are most often non-GCB subtype by the Hans algorithm, in keeping with its role in upregulation of the NF-κB and JAK-STAT pathways, which represent the major signaling pathways active in ABC DLBCL.[36] This study, as well as others, has suggested a poor prognosis for MYD88-mutated cases.[35,36] Other studies of specific DLBCL subgroups have identified a high frequency of MYD88 mutations in primary testicular DLBCL, primary central nervous system DLBCL, and primary cutaneous DLBCL, leg type, the latter of which is a distinct entity within the 2017 WHO classification.[35,37–39] The co-occurrence of MYD88 L265P mutation and CD79B mutation is highly predictive of response to BTK inhibitor, ibrutinib.[40] Case 191 was that of a MYD88- mutated DLBCL, presenting with features suggestive of primary cutaneous DLBCL, leg type. This case highlights the unique extranodal predominance of MYD88-mutated cases. The session moderators emphasized that while testing for MYD88 is not necessary in all cases of DLBCL, it should be considered in these specific subtypes, as it may guide therapeutic and prognostic discussions.

Large B-cell Lymphoma With IRF4 Rearrangements

This entity was introduced as a diagnostic category in the 2017 WHO classification, and two representative cases (cases 172 and 341) were submitted to the workshop. This is an uncommon entity, which may present with any combination of follicular and/or diffuse growth patterns.[41] Case 341 Image 5 illustrated the partially diffuse and partially follicular growth pattern and characteristic medium-to-large sized cells described in this entity. These cases virtually always express BCL6 and MUM1/IRF4 (reflecting the IRF4 translocation), while CD10 is expressed in approximately two-thirds of cases.[12] IRF4 rearrangements are identified by FISH, and usually have an IG gene partner.[41] Clinically they most often present in the head and neck area, especially in the Waldeyer ring (tonsils).[41] Although the median age is only 12 years, they can occur in older adults such as the 63-year-old male presented in case 341. Most patients present with localized disease, and prognosis is overall favorable when patients are treated with rituximab and combination chemotherapy. However, prognosis is likely less favorable than in pediatric-type FL, a diagnosis with which there may be clinicopathologic overlap.[41,42] Therefore, testing for IRF4 rearrangements by FISH should be considered especially relevant in the workup of diffuse or FLs in the pediatric population, especially when presenting in the head and neck area.

Image 5.

Case 341. Large B cell lymphoma with IRF4 rearrangement. A, H&E stained sections of a typical case show a partially diffuse and partially nodular growth of atypical lymphocytes. B, The infiltrate is composed of large irregular cells. C, Tumor cells express CD20. D, They show a germinal center phenotype with expression of CD10. E, Expression of BCL6. F, The neoplastic lymphoid cells also show MUM1 expression. (A, ×100; B-F, ×400).

ALK-positive Large B-cell Lymphoma

One case of ALK-positive large B-cell lymphoma was submitted to the workshop (case 161). This is a rare entity and typically presents in young males, although a wide age range has been reported. Diagnostically, ALK-positive DLBCL should be considered in tumors with immunoblastic or plasmablastic cytology, and which lacks staining for typical B-cell lineage antigens (CD20, PAX5, CD79A). CD138 and MUM1 are usually expressed, and CD45 is variable.[12] These tumors often have sinusoidal infiltration, similar to anaplastic large cell lymphoma (ALCL). Distinction from ALCL is critical, and typically is easily done with a CD30 stain, which should be strong and diffuse in ALCL and is only weak or patchy in ALK-positive large B-cell lymphoma, if expressed at all. Other entities in the differential diagnosis typically include plasmablastic lymphoma and HHV-8–positive large B-cell lymphomas. ALK is positive by immunohistochemistry, usually in a cytoplasmic granular pattern indicating the CLTC-ALK fusion.[12] ALK FISH can be performed but is usually not necessary, as immunohistochemistry is both sensitive and specific.

Primary Mediastinal Large B-cell Lymphoma

One case of primary mediastinal large B cell lymphoma (PMBL) was submitted to the workshop (case 378). PMBL represents a subset of DLBCL presenting typically in young females, arising in the mediastinum. These cases show some distinctive morphologic and phenotypic features such as fine fibrosis, clear-cell cytology, and expression of CD30 and CD23. However, none of these features is sensitive or specific for PMBL. Although not widely available for clinical use, molecular genetic studies reveal them to be a biologically distinct subgroup of DLBCL, with GEP similarities to classic Hodgkin lymphoma.[43] As in the submitted case, clinicopathologic distinction from DLBCL, NOS involving the mediastinum can be challenging, and this distinction may alter therapeutic decisions.[44] Immunohistochemical algorithms using markers such as MAL, PD-L2, CD30, CD23, TNFAIP2, C-REL, and TRAF1, which are more frequently expressed in PMBL, have been proposed to distinguish these lymphomas from DLBCL, NOS. MAL and PD-L2 appear to have high sensitivity and specificity for PMBL in some studies. However, in current practice, diagnosis of PMBL still requires clinicopathologic correlation.[45–48] As such, molecular methods to aid in the diagnosis of PMBL have been sought. The submitters of case 378 performed a 500-gene next-generation sequencing (NGS) panel on their case and demonstrated somatic mutations in TNFAIP3, SOCS1, and STAT6, components of the NF-κB and JAK-STAT pathways known to show constitutive activation in PMBL.[49] Also identified was a pathogenic intrachromosomal structural rearrangement involving PDCD1LG. Structural rearrangements of PDL1 (CD274), CIITA, and PDL2 (PDCD1LG2) are recurrent in PMBL.[48,50] Finally, amplification of chromosome 9p, including the JAK2, CD274, and PDCD1LG2 genes, was noted, a finding reported in up to 75% of PMBL cases and uncommon in other DLBCL subtypes.[51,52] Molecular features have also helped in identifying lymphomas outside the mediastinum that show GEP signatures similar to PMBLs, suggesting that there may be cases of extramediastinal PMBL.[53] As GEP becomes more widely available on paraffin-embedded material, this represents another rapidly evolving area where assays are likely to soon emerge for clinical use.[54]

Burkitt Lymphoma

Burkitt lymphoma (BL) is an aggressive B-cell neoplasm, which in Western countries presents most often as an extranodal mass in children or young adults (sporadic type), as well as in immunosuppressed hosts (immunodeficiency associated). The endemic type is seen in children in equatorial Africa or patients who have emigrated from this region. All three types present with similar histologic findings, with diffuse growth of medium-sized, monomorphic cells with blastic chromatin and multiple small nucleoli. The tingible-body macrophages that impart the characteristic "starry-sky" appearance are a reflection of the extremely high rate of cell turnover in this tumor. Clinically and morphologically, the main differential diagnosis is typically with other high-grade B-cell lymphomas, such as high-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 translocations (DHL), high-grade B-cell lymphoma, not otherwise specified (HGBCL, NOS), and sometimes B-lymphoblastic leukemia/lymphoma (B-ALL). BL shows MYC rearrangements in close to 100% of cases, most often with an immunoglobulin gene partner. BCL2 and BCL6 rearrangements are absent, by definition.

One case of BL was submitted to the workshop (case 287) in which the differential diagnosis between BL and B-ALL was challenging based on morphologic and phenotypic features. This patient was a 72-year-old man who presented with lymphadenopathy and elevated LDH. Bone marrow aspirate was performed first in the diagnostic evaluation, which revealed a high-grade lymphoma expressing CD19, CD20, and CD10, as well as dim CD45 and terminal deoxynucleotidyl transferase (TdT), and lacking surface immunoglobulin. Epstein-Barr virus (EBV)-ISH, however, was positive strongly supporting a mature B-cell process rather than B-ALL. FISH studies revealed a IGH/MYC translocation, without concurrent BCL2 or BCL6 translocations, supporting a diagnosis of BL. Finally, the authors performed additional NGS, which revealed an ID3 mutation, seen in up to 70% of sporadic BL cases.[12] Although rapid diagnosis of BL, especially in the pediatric setting, may be necessary before FISH results are available, confirmation of MYC rearrangement by FISH is recommended to be routinely performed in the setting of a new diagnosis of BL. Assessment for BCL2 and BCL6 rearrangements is also critical, in order to fully exclude a DHL or triple-hit lymphoma, which may show very similar morphologic and phenotypic features to BL.

Burkitt-like Lymphoma With 11q Aberration

Burkitt-like lymphoma without MYC rearrangement is rare; many cases are likely due to technical limitations within the clinical laboratory. Although a relatively recently described entity, four cases of Burkitt-like lymphoma with 11q aberration were submitted to the workshop (cases 43, 58, 137, and 356). This entity represents cases that show typical histologic and clinical features of BL, as well as similar GEP and microRNA profiles but lack the IGH/MYC rearrangements seen in more than 90% of BL. Instead, complex but recurrent gains and losses of chromosome 11q are found, specifically gains at 11q23.2–23.3, and terminal losses of 11q24.1.[55]

The submitted cases, including case 137 Image 6, demonstrated the similarities to standard BL, but all lacked MYC rearrangements by FISH. Oncoscan microarray analysis, which utilizes both single nucleotide polymorphism and array-comparative genomic hybridization, is currently the most widely utilized modality for detecting the 11q aberrations. Case 137 included the results of an 11q-directed FISH assay[56] but acknowledged that, if negative, a microarray would be necessary to evaluate the 11q region for the characteristic gains/losses.

Image 6.

Case 137. Burkitt-like lymphoma with 11q aberration. A, H&E stained sections show medium-size cells with blastic chromatin and abundant tingible body macrophages. Tumor cells are positive for CD20 (B), CD10 (C), and are negative for BCL2-like typical Burkitt lymphoma (D). (A-D, ×400). E, They are positive for MYC by immunohistochemistry. F, Ki-67 shows a proliferation index of essentially 100%.

Biologic Insights

Low-Grade Lymphoid Neoplasms With Genetic Events Associated With Aggressive Biology. Several cases submitted to the workshop exhibited morphologic features typical of a low-grade B-cell lymphoma but harbored genetic abnormalities associated with an aggressive B-cell lymphoma Table 3. As FISH testing for translocations of MYC, BCL2, and BCL6 become more routine, it is perhaps not surprising that genetic double-hits are identified in what appears morphologically to be low-grade lymphomas. Two cases submitted to the workshop documented the presence of both MYC and BCL2 translocations in FL (cases 129 and 358) Image 7. Both cases had grade 1 to 2 morphology and diffuse growth patterns. These cases are in these respects dissimilar to the two largest series of double-hit FL published to date, in which most cases had at least some regions of grade 3 FL, and where a follicular growth pattern was described.[57,58] While in one series double-hit FL patients were thought to have an aggressive disease course warranting more intensive therapy than conventional FL,[57] this conclusion was not supported by the other series.[58] Ultimately, too few cases have been studied for firm conclusions regarding the clinical implications of double-hit genetics in morphologically low-grade lymphoma, though the finding would obviously cause clinical and diagnostic consternation.

Image 7.

Case 358. Low-grade follicular lymphoma with MYC and BCL2 rearrangement. A, H&E stained sections show a low-grade follicular lymphoma. B, There is also focal diffuse growth pattern. The cells are small with slightly irregular nuclei with occasional centroblasts. C, The cells have fairly low MYC protein expression. D, Fluorescence in situ hybridization studies demonstrated the presence of both IGH/BCL2 rearrangement (D) and MYC rearrangement (E). (A, ×100; B and C, ×400; D and E, ×1,000).

Case 36 emphasized the utility of FISH testing to clarify karyotype results in lymphoma. Case 36 was a CLL/small lymphocytic lymphoma (SLL) with numerous prolymphocytes and a karyotype containing trisomy 12 and t(8;14)(q24;q32). This karyotype suggested the presence of IGH/MYC translocation, a rare event in CLL that has been associated with increased prolymphocytes and an aggressive clinical course.[59,60] However, FISH testing performed on the case demonstrated intact MYC and IGH loci, and the finding had been present 13 years prior without aggressive clinical behavior.

Genetic Events Associated With Transformation. While historically any large-cell lymphoma in a patient with a previously diagnosed low-grade lymphoma may have been considered to represent transformation, molecular tools allow study of the biologic relationship of lymphomas in greater detail (Table 3). The phenomenon of transformation has been best studied in CLL and FL. In patients with CLL, around 20% of subsequent large cell lymphomas are clonally unrelated to the underlying CLL. This has important clinical implications, as the prognosis for patients with an unrelated large B-cell lymphoma is similar to that of de novo DLBCL and far superior to the prognosis of a clonally related large-cell transformation (Richter transformation [RT]).[61] Whenever possible, clonality testing to assess for clonal identity should be performed on both the prior CLL and large-cell lymphoma in CLL patients diagnosed with what appears to be RT. The genetic background of RT also differs from that of de novo DLBCL, with frequent MYC translocations/amplifications, TP53 alterations, CDKN2A deletions, and/or NOTCH1 mutations.

In addition to overt RT, CLL may progress through a pattern of branching evolution, commonly with outgrowth of TP53- mutated subclones at disease progression following therapy.[62] Case 85 demonstrated RT with acquisition of an IGK/BCL2 rearrangement at transformation. Case 359 Image 8 illustrated both genetic CLL progression and two genetically distinct RTs. The patient had a history of CLL treated with fludarabine cyclophosphamide and rituximab, followed by bendamustine and rituximab. The initial CLL did not demonstrate typical copy number abnormalities as assessed by FISH, with no del(13q), trisomy 12, del(11q), or del(17p). Four years following diagnosis, the patient presented with an EBV-negative RT in the ileum, with a complex karyotype including t(8;22)(q24.2;q11.2), IGL/MYC rearrangement, without trisomy 12. The patient achieved remission from the RT following R-EPOCH therapy. A year later, the progressed CLL had trisomy 12 and deletion of TP53 at 17p. The patient then received ofatumumab and ibrutinib, with a clinical response. Around 2 years later, he presented with increasing lymphadenopathy, and a repeat biopsy demonstrated RT with a complex karyotype and t(8;14)(q24.2;q32), indicative of IGH/MYC rearrangement. This RT additionally contained a BTK Cys481Ser mutation, a common ibrutinib resistance mutation.[63] Subsequent clonality testing demonstrated clonal similarity between the two RTs and the progressed CLL. This case highlights current challenges of molecular-directed therapy for lymphoma: novel therapies may lead to minor subclones that frequently expand under the selective pressure of genetically targeted therapy, become dominant and manifest as therapeutically resistant relapses.

Image 8.

Case 359. Richter transformation (RT) of chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL). A, The lymph node is replaced by both CLL/SLL (left half of A and B) and areas of morphologic transformation (C) to a large B-cell lymphoma. D, MYC immunohistochemistry demonstrates low MYC expression in the CLL/SLL. E, MYC expression is high in the RT. The second RT was associated with acquisition of IGH/MYC rearrangement. (A, ×40; B and C, ×400; D and E, ×1,000).

FL is another indolent lymphoma where progression and transformation have been extensively studied. FL often arises from a "premalignant" proliferation of IGH/BCL2- rearranged cells that may or may not lead to overt FL. Case 100 demonstrated a clonal relationship between "in situ follicular neoplasia-like" colonization of the germinal centers of the peripancreatic lymph nodes and concurrent duodenal FL. The possibility that the in situ follicular neoplasia-like colonization may represent partial involvement from the concurring duodenal FL was also discussed. FL is genetically heterogenous at diagnosis, with multiple subclones that progress over time.[64] In contrast to CLL, which progresses to RT usually through a linear pattern of genetic alterations, most studied cases of transformed FL (tFL) demonstrate a branched pattern of evolution, arising from an early common progenitor that also gives rise to the FL.[65,66] Most cases of tFL exhibit morphologic features similar to conventional DLBCL, and many of the genetic attributes of tFL are similar to those present in de novo GCB DLBCL (BCL2 rearrangements, MYC alterations, and mutations of KMT2D, CREBBP, EZH2). Some differences from de novo GCB DLBCL are present: CDKN2A/B; TNFAIP3 deletions and mutations of STAT6, FAS, and ARID1A are more common in tFL than de novo DBLCL.[65,66] While most FL transform to a morphologic DLBCL, FL may more rarely transform to classic Hodgkin lymphoma or a lymphoblastic phenotype, both processes represented in the cases submitted to the workshop. Two cases of FL with classic Hodgkin lymphoma (CHL) transformation were submitted to the workshop (cases 141 and 278). These cases exhibited areas of both conventional FL and distinct areas with a T-cell–rich background and morphologically and phenotypically typical Hodgkin-Reed-Sternberg (HRS) cells. The clonal relationship between the HRS cells and FL was demonstrated through FISH testing, which showed BCL2 rearrangements in both the FL and within the HRS cells. CHL transformation of FL is very rare, and prior case reports have documented a similar relationship between the FL and HRS cells.[67,68] Other examples of unusual FL transformation were submitted (cases 227 and 373). These cases demonstrated BCL2- rearranged FLs with TdT-positive lymphoblastic transformations containing rearrangements of both BCL2 and MYC. Transformation of FL to a B-lymphoblastic lymphoma is rare, but well described. Acquisition of MYC translocation at lymphoblastic transformation, as in both cases submitted to this workshop, appears to be a typical event in these cases, present in nine of nine evaluable cases in two recent series.[69,70] The most appropriate therapy for these patients is unclear, and they exhibit an aggressive disease course.

While lymphoblastic transformation of FL is one process that may lead to a double-hit population of B-lymphoblasts, apparently de novo cases of B-ALL with both BCL2 and MYC translocations also occur, without typical recurrent fusion events of B-ALL. An otherwise morphologically and phenotypically classic case of pediatric B-ALL was submitted (case 139) with both IGH/MYC and IGH/BCL2 rearrangements, along with a relapsing disease course. While MYC- rearranged B-ALL is well described (though rare) in the pediatric population, they should be distinguishable from BL based on cytologic features.

Unexpected Genetic Findings. The clinical adoption of low-cost high-throughput sequencing allows for routine genetic characterization of lymphoid neoplasms. Such testing sometimes discloses unexpected findings (Table 3), such as the presence of BRAF Val600Glu in a case of a splenic lymphoma with morphologic and phenotypic features of SDRPL rather than HCL (case 21), or TP53 mutations in clinically indolent leukemia/lymphomas (leukemic nonnodal mantle cell lymphoma, case 86 and a gastric MALT lymphoma (case 365). The clinical importance of such discordant findings may be difficult to determine. The finding of an actionable mutation such as BRAF Val600Glu may be of therapeutic interest, but in the absence of typical morphologic and phenotypic features of HCL, the finding is not sufficient for reclassification, as BRAF mutations have very rarely been reported in non-HCL low-grade lymphomas, though most are non-Val600Glu.[62,71] Similarly, identification of a TP53 mutation in an otherwise indolent-appearing lymphoma may prompt closer clinical monitoring, but outside of the CLL/SLL setting, it is unclear whether TP53-mutated lymphomas require different therapy. Quite often, sequencing and genetic testing may find additional mutations that are of unknown significance and do not contribute to therapy or classification, and several cases submitted to the workshop were emblematic of this.