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

Mature T-cell Lymphomas/Leukemias

T-cell lymphomas/leukemias represent a heterogeneous group of rare malignancies with overlapping clinical, immunologic, and histologic features. Genetic aberrations in mature T-cell lymphomas/leukemias have not been well studied due to the rarity of these neoplasms. Traditionally, the determination of T-cell receptor (TCR) gene rearrangement by Southern blot analysis and subsequently polymerase chain reaction (PCR) has been helpful in the assessment of clonal T-cell populations. However, results of TCR PCR dictate cautious interpretation of the information in establishing a diagnosis of T-cell lymphoma and more importantly do not contribute to subclassification of this highly heterogeneous group of neoplasms. Cytogenetics and FISH studies may be of great utility in certain clinical contexts, for example the identification of a disease-specific cytogenetic abnormality such as inv(14) in T-prolymphocytic leukemia (T-PLL) or iso(7q) in hepatosplenic T-cell lymphoma (HSTL). Additionally, FISH studies may reveal genetic alterations that have prognostic value such as DUSP22 rearrangements in ALK-negative ALCL. GEP studies and NGS technologies have led to the identification of genetic alterations in many T-cell lymphoma subtypes and contributed to improved understanding of the pathogenetic mechanisms. These studies have led to better delineation of diagnostic categories and provided novel insights into T-cell lymphoma biology. The discussion below summarizes the cases submitted to the workshop which serve to highlight the role of molecular genetics and, more specifically, recurrent genetic features that are characteristically seen in distinct and overlapping subtypes of mature T-cell lymphomas/leukemias Table 4.

Anaplastic Large Cell Lymphoma

ALCL represent a group of T-cell lymphomas with pleomorphic nuclear features that express strong CD30. ALK-positive ALCL are relatively well characterized. They occur primarily in children or young adults and may involve lymph nodes and extranodal sites with an overall excellent prognosis. The presence of rearrangements involving ALK1 and its fusion to over 21 distinct partner genes can be easily detected by routine immunohistochemistry for ALK protein. ALK-negative ALCL is considered a definitive entity in the 2017 WHO classification and includes several cytogenetic subtypes.[12] Their cytologic features are similar to those of ALK-positive ALCL; however, the tumor cells exhibit more pleomorphic features. Immunophenotypic features are similar to those of ALK-positive ALCL, except for absence of ALK protein expression. Rearrangements of DUSP22, TP63, and TYK2 are present in ALK-negative ALCL. Those with DUSP22 rearrangements are more likely to exhibit so-called doughnut-like cells.[72] Interestingly, ALK-negative ALCLs with DUSP22 rearrangements have better clinical outcome relative to TP63 rearranged cases and those lacking DUSP22, TP63, or TYK2 rearrangements.[73] Importantly, DUSP22 and TYK2 rearrangements are also seen in cases of lymphomatoid papulosis and cutaneous ALCL,[74] suggesting that there may be biologic overlap between these entities.

Two cases of ALK-positive ALCL (case 153 and 132) were submitted to the workshop. Case 132 described a t(X;2) MSN-ALK rearrangement in a 2-year-old girl presenting with extensive lung masses and pleural implants. Notably, while tumor cells demonstrated ALK protein in a membranous staining pattern, no definitive T-cell markers were expressed, with only weak expression of CD43 and CD4. Importantly, CD30 was essentially negative while keratin AE1/AE3 was weakly positive. Karyotype of the pleural effusion demonstrated (X;2)(q13;p23), as was originally described in this entity,[75] while FISH detected an ALK rearrangement. This fusion represents one of the numerous variant gene fusions involving ALK.[76] This case highlights the contribution of immunohistochemistry, karyotype, and FISH in determining the status of ALK-positive lymphomas. Case 153 represented a small-cell variant of ALK-positive ALCL that demonstrated a leukemic presentation in a 24-year-old woman. FISH analysis using break-apart probes for chromosome 2 demonstrated a translocation involving ALK, while PCR analysis of TCR gene rearrangement demonstrated four different clonal peaks. These cases serve to underscore the important contribution of genetic assessment of the ALK gene rearrangement in tumors that have unusual immunophenotypic features or clinical presentation that may have made the diagnosis challenging.

Two cases of ALK-negative ALCLs with DUSP22 rearrangement (cases 277 and 270) were submitted to the workshop. While ALK fusions can be easily detected by either routine immunohistochemistry or FISH that are available in most clinical cytogenetic laboratories, DUSP22 and TYK2 rearrangements require FISH testing and are currently not available in many clinical cytogenetic laboratories. Case 270 illustrated an ALK-negative ALCL from an 84-year-old male presenting with skin lesions and regional lymphadenopathy. FISH analysis of the DUSP22/IRF4 locus (6p25.3) with a laboratory-developed break-apart probe showed a biallelic DUSP22 rearrangement, with no intact copy of the 6p25.3 locus. Case 277 described a comprehensive molecular profiling study of a DUSP22 rearranged ALK-negative ALCL where FISH demonstrated a DUSP22-IRF4 (6p25.3) gene rearrangement. Hybridization-capture based targeted NGS of 400 genes showed genetic mutations in CARD11, GRIN2A, and PIK3R1. The significance of these variants is unknown although they have been implicated in both lymphomas as well as other cancers.

Angioimmunoblastic T-cell Lymphoma and Other Nodal Lymphomas Derived From T-follicular Helper Cells

There are three subtypes of T-cell lymphomas that are thought to be derived from T follicular helper cells: angioimmunoblastic T-cell lymphoma (AITL), follicular T-cell lymphoma, and nodal peripheral T-cell lymphoma (PTCL) with T follicular helper phenotype.[77,78] All occur predominantly in adults and present with generalized lymphadenopathy, hepatosplenomegaly, skin rash, and constitutional symptoms. The tumor cells express markers characteristic of T-follicular helper phenotype: CD3, CD4, CD10, CXCL13, ICOS (CD278), and CD279. Large Epstein-Barr virus encoded RNA (EBER)-positive B immunoblasts are frequently present and can give rise to subsequent DLBCL.

Follicular T-cell lymphoma is a rare nodal form of PTCL that exhibits T follicular helper phenotype. Clinical presentation is similar to that of AITL; however, some patients have localized disease without B symptoms. They exhibit a predominantly follicular growth pattern without the characteristic features of AITL. The neoplastic cells are intermediate in size and appear monotonous, with abundant pale cytoplasm. In some cases the pattern resembles FL while in others it can resemble progressive transformation of germinal centers. Nodal PTCL with T follicular helper phenotype is a provisional entity that typically demonstrates a diffuse pattern of infiltration without the prominent polymorphic inflammatory background, vascular proliferation, or expansion of follicular dendritic cell meshwork seen in AITL. Genomic studies of AITL and other T-cell lymphomas of follicular T-cell derivation have shown frequent mutations in three genes involved in the regulation of DNA methylation and hydroxymethylation. Mutations in TET2 (80%), DNMT3A (20%-30%), and IDH2 (20%-30%) are characteristically observed in AITL. Additionally, RHOA G17V mutations are found in 50% to 70% of AITL patients.[79] From a diagnostic standpoint, TET2 family genes are mutated in a variety of hematologic malignancies including most T-cell lymphoma types, cutaneous T-cell lymphoma and Sézary syndrome, ALK-negative ALCL, and adult T-cell leukemia/lymphoma and thus are not disease specific; however, TET2 mutations are most prevalent in AITL.

Case 377 represents a patient with AITL associated with TET2, DNMT3A, and RHOA G17V mutations documented on three sequential biopsies over a 20-year period. The patient initially presented at the age of 36, with the second relapse occurring 17 years later. Mutational analysis focused on recurrent genetic events in AITL and other nodal T-follicular helper PTCL demonstrates that TET2 and DNMT3A, and RHOA G17V variants were found in all three samples. While the IDH2 R172S mutation was present in the first two biopsies and not in the third, numerous neoplastic T cells with T follicular helper phenotype demonstrated EBV in only the second relapse (third biopsy). Clonal relationship was demonstrated by TCR γ rearrangement in the initial and relapse samples. The findings suggest that a "lymphoma-initiating cell," which preceded TCR rearrangements and harbored TET2, DNMT3A, and possibly RHOA mutations, may survive over a long period in quiescent form. IDH2 R172 mutations and EBV infections may represent secondary events that promote lymphoma progression. This case also illustrates the pathologic spectrum of T-follicular helper PTCLs.

Recent studies indicate that TET2, DNMT3A mutations may be found not only in the neoplastic T cells of AITL but also in CD34-derived progenitor cells, suggesting the notion that TET2 and DNMT3A mutations occur in a hematopoietic progenitor cell, while RHOA and IDH2 mutations are restricted to T cells. These observations suggest a multistep oncogenic hypothesis in which TET2 inactivation and DNMT3A mutations lead to a variety of hematologic disorders, including T-cell lymphoma, with low penetrance.[80] On the other hand, TET2 inactivation in conjunction with RHOA G17V may lead to AITL-like lymphoproliferations with high penetrance.[81]

T-large Granular Lymphocytic Leukemia

T-large granular lymphocytic (T-LGL) leukemias are clonal diseases characterized by persistent increase in the number of CD3+, CD8+ cytotoxic T cells. T-LGLs are often associated with autoimmune disorders and immune-mediated cytopenias. STAT3 mutations are commonly observed in LGL leukemias of either T or NK cells.[82,83]

Three cases of T-LGL leukemia (cases 111, 179, and 190) were submitted to the workshop. Case 190 represents a rare T-LGL leukemia in a child (13 years old), with typical clinical presentation of neutropenia, anemia, and splenomegaly. Karyotype was normal and TCR PCR demonstrated a clonal T-cell population. Sanger sequencing demonstrated a STAT3 N647I (p.Asn647Ile, c.1940A>T) mutation in exon 21 (SH2 domain). Molecular findings helped in establishing the clonality and differentiating the LGL proliferation from a benign cause. LGL leukemia is thought to be causally associated with activating mutations in STAT3, but this mutation has not yet been reported in pediatric T-LGL leukemia. Case 179 illustrated the contribution of STAT3 mutation (STAT3 K658R; p.Lys658Arg, c.1974A>G) that was observed in a Down syndrome patient who developed aggressive NK-cell leukemia after a diagnosis of indolent T-cell LGL leukemia, which was made over 21 years prior. STAT3 mutations are a unifying feature of T-LGL and NK-LGL leukemia, currently classified as distinct entities but with overlapping clinicopathologic features. The findings may support the notion of a continuum of malignant differentiation in this disease, or evolution from a common precursor into two distinct neoplasms. In this case, the evidence of a pathogenic somatic mutation in STAT3 provided confirmation of a malignant LGL-type leukemia.[84]

Case 23 described an interesting case of T-LGL leukemia that was shown to harbor STAT3, DNMT3A, and TET2 mutations in additional to a distinct population of CD33+ cells with DNMT3A p.R882S (22% variant allele frequency [VAF]) mutation. Targeted high-throughput sequencing of whole peripheral blood demonstrated the presence of DNMT3A p.R882S (4% VAF) and p.E907del (38% VAF) mutations, a STAT3 p.Y640F mutation (40% VAF), and a TET2 p.E1144K (40% VAF) mutation. Flow cytometric sorting of CD3+ and CD33+ peripheral blood leukocyte subsets demonstrated the CD3+ T cells to exclusively harbor DNMT3A p.E907del, STAT3 p.Y640F, and TET2 p.E1144K mutations, whereas the DNMT3A p.R882S mutation was only detected in the CD33+ myeloid cells. This case expands the mutational spectrum of T-LGL leukemia to include DNMT3A and TET2 mutations and, in addition, it demonstrates the presence of a molecularly distinct clonal hematopoiesis of indeterminate potential (CHIP). Finally, it highlights the importance of careful morphologic and immunophenotypic examination in conjunction with molecular analysis to distinguish CHIP and myelodysplastic syndromes from LGL leukemias, both of which may cause cytopenias.

Hepatosplenic T-cell Lymphoma

Two cases of HSTL were submitted to the workshop. Mutations in STAT3 and STAT5B are seen in about 9% and 31% of HSTL, respectively.[85] Case 114 was that of a 26-year-old man who presented with severe weight loss, fever, night sweats, skin rash, and marked splenomegaly without adenopathy. A diagnosis of HSTL was made based on the bone marrow findings showing neoplastic tumor cells in a typical intrasinusoidal pattern, comprising 30% of the cellularity. Bone marrow karyotype demonstrated 47XY,r(7)i(7)(q10)dup(7)(q11.2q36),+8[4]/46,XY[16]. Cytogenetic analysis revealed trisomy 8 and a ring chromosome 7, which appeared to be a duplicated 7q arm. Ring chromosome 7 is detected in the vast majority of HSTLs. Detection of ring chromosome 7/isochromosome 7q10 in this case played a significant role in establishing a diagnosis of HSTL in the bone marrow biopsy in the absence of a liver or splenic biopsy. Case 331 was that of an aggressive hepatosplenic T-cell lymphoma with isochromosome 7q10, t(4;8)(q21;q13), losses of multiple chromosomes, and tetraploidy identified by cytogenetics and FISH analyses. PCR showed a clonal TCR β gene rearrangement. Targeted NGS detected three pathogenic alterations: TET2 c532G>T p.E178* at 28% VAF, KRAS c35G>C p.G12A at 23% VAF, and TP53 c524 G>A p.R175H at 47% VAF. KRAS and TP53 mutations can be observed in NK/T-type lymphomas but are pres-ent in a variety of other lymphomas. While TET2 mutation occurs in AITL and in PTCL, NOS, it is unusual in NK/T-lymphoma/leukemia.

T-prolymphocytic Leukemia

T-PLL is an aggressive mature T-cell leukemia. Case 327 demonstrates an unusual clinical presentation of a patient with T-PLL with indolent presentation and with TCL1A rearrangement. FISH study performed on a blood sample showed 29% nuclei with rearrangement involving the TCL1A (14q32) locus, which may indicate an inv(14) or t(14;14). Additional FISH study demonstrated a 17p deletion in 64% of nuclei, a deletion of a chromosome 11 centromere with retention of both copies of ATM (11q22.3), monosomy 13 in 60% of nuclei, and a 6q deletion in 21% of nuclei. Mutation of TP53 G334V was seen with VAF of 55%. Besides the inv(14) or t(14;14), additional chromosomal abnormalities and TP53 mutations are also frequently seen in T-PLL. Considering the atypical indolent presentation (seen only in 10%-15% of cases), a diagnosis of T-PLL would have been challenging based solely on morphologic and immunophenotypic features. Given that the classic T-PLL with aggressive clinical behavior is associated with recurrent mutations in the JAK1/3-STAT5B pathway,[86] it may be important to assess for these mutations to identify those with indolent or aggressive behavior.

Other T-cell Lymphoproliferative Disorders

Three cases of other T-cell lymphoproliferative disorders (LPDs; cases 114, 115, and 326) were submitted to the workshop. Hydroa vacciniforme-like lymphoproliferative disorders (HV-LPD) are EBV-positive T- or NK-cell lymphoproliferative disorders of childhood, mainly affecting children from Central and South America and Asia. These disorders may represent a spectrum of diseases. Thus, the term HV-LPD encompasses both diseases. Case 326 serves to illustrate a HV-LPD in an 8-year-old boy who presented with chronic relapsing papulovesicular eruptions with hemorrhagic crusted erosions over ears and lower lip. Skin biopsy revealed hydroa vacciniforme-like lymphoma with low-level bone marrow involvement. Clonal TCR gene rearrangements were observed in the skin. The atypical lymphocytic population in the skin biopsy was positive for CD3, TIA1, TCR gamma, and subset CD30, and was negative for CD4, CD8, CD56, and TCR β. The majority of the lymphocytes were positive for EBER by in situ hybridization. A sparse infiltrate of EBER-positive cells, which were also positive for CD3 and TIA1, was also identified on marrow core biopsy. A clonal TCR-γ gene rearrangement was present in the skin biopsy, consistent with a clonal T-cell process. In this case, the TCR gene rearrangements helped to establish the clonal nature of the disease.

Other new provisional entities added to the 2017 WHO classification, including breast-implant ALCL, indolent T-cell lymphoproliferative disorder of the gastrointestinal tract, monomorphic epitheliotropic intestinal T-cell lymphoma, and primary cutaneous acral CD8+ T-cell lymphoma were not submitted to the workshop.