ALK-positive large B-cell lymphoma (ALK + LBCL) with aberrant CD3 expression

ALK-positive ( +) large B cell lymphoma (ALK + LBCL) is a rare distinct subtype of diffuse large B cell lymphoma presenting with high stage and aggressive behavior. Although B cell markers such as CD20, CD19, and CD22 are generally negative, plasmacytic markers including CD138, CD38, and MUM1 are positive. T cell markers are negative with rare exceptions. We report an unusual case of ALK1 + LBCL in a 58-year-old man with partial expression of CD3 without other T cell antigen expression. The tissue was evaluated with flow cytometry, immunohistochemistry, fluorescent in situ hybridization, and gene rearrangement studies. Gene rearrangement studies for IGH and TCR gamma were performed. Flow cytometry did not demonstrate any abnormal lymphoid populations. Tissue sectioning shows a malignant plasmacytic large cell neoplasm which expresses CD45 but is negative for CD20, CD79a, and PAX5. Plasmacytic markers CD138 and MUM1 are positive with kappa light chain restriction. Strong granular cytoplasmic expression of ALK is present. FISH showing disrupted ALK supports the diagnosis while MYC, BCL6, and BCL2 are intact. Gene rearrangement studies show coexisting IGH and TCR gamma clones; however, the TCR peak was present within a polyclonal background suggesting the disputed cells are likely only a subset of the T cell population. ALK + LBCL can present with an ambiguous immunophenotype, which warrants the use of multiple B cell, T cell, and plasmacytic antibodies. CD3 expression in this entity is rare and of uncertain clinical significance, but warrants further study.

The ALK protein is a receptor tyrosine kinase, encoded by the ALK gene on chromosome 2p23 [9].Aberrant kinase activity associated with ALK can be seen with gene rearrangement, amplification, and point mutations of the ALK gene [4]; however, fusion proteins with ALK are seen most commonly in ALK + LBCL.ALK is rearranged in ALK + LBCL, most commonly CLTC::ALK, but other translocations have been reported involving the ALK gene [4-9, 11, 15, 16].Multiple signaling pathways are stimulated in response to ALK, including Ras/ERK, JAK/STAT, PI3k/Ark, and PLCγ pathways [4].Activation of STAT3 and STAT5 is reported in ALK + LBL [4] with STAT3 expression reported in some cases [17].

Case history
A 58-year-old man presented with cervical adenopathy.The specimen was noted to be involved by a large cell neoplasm, which was favored to be hematopoietic, and referred to our institution.There is no other available past medical history or follow-up.Flow cytometry from an outside laboratory at the time of excision showed "no monotypic B-cell, phenotypically aberrant T-cell or blast cell population with identified."

T cell receptor (TCR) and immunoglobulin heavy (IGH) chain rearrangement
PCR-based detection for clonal TCR gamma and IGH was performed.Extracted DNA from the paraffin-embedded tissue using a modified version of QIAGEN QIAmp DNA purification protocol and the PCR-based BIOMED-2 assay (Invivoscribe, San Diego, CA) was performed.The manufacturer's instructions were strictly followed in interpreting this assay.TCR beta was not performed.
Fluorescence in situ hybridization (FISH) reveals intact MYC, BCL6 genes, and negative IGH::BCL2.ALK is disrupted (Fig. 2D).Clonal IGH and TCR gamma chain gene rearrangements are present (Fig. 2A-C).The TCR gamma shows a prominent peak in an otherwise polyclonal background.

Discussion
ALK + LBCL is a distinct, rare, and aggressive type of large B cell lymphoma, which typically presents in lymph nodes, most commonly the cervical [7,8,11,15,16]; however, extranodal sites are also affected.There is a wide age range at presentation with a median of 35-38 years and a male predominance.Patients present with high stage disease in most cases with median survival of 11-24 months [4,6,8].There is no known association with immunodeficiency or viral association including Epstein Barr virus, HHV8, or HIV.Architecture of the lymph node is diffusely effaced but often shows a partial sinusoidal pattern.Cellular morphology appears immunoblastic and/or plasmablastic [7,8,11,15].Rarely large Reed-Sternberg-like cells can be present [7].There is increased mitotic activity and can be necrosis.Our case showed effaced architecture with the neoplastic cells showing a plasmablastic appearance.
Immunohistochemistry evaluation of the neoplastic cells in ALK + LBCL shows expression of plasmacytic markers including CD138, MUM1, CD38, and VS38.CD20 is often negative or weakly positive in a minority of cases.Other B cell antibodies including CD19 and CD22 are negative.PAX5 and CD79a are seen in a minority of cases.Other stains, which may be positive, include EMA, BOB-1, OCT-2, and CD45RB (may be weak).Cytoplasmic immunoglobulin most often IgA can be positive.CD10, MYC, and STAT3 can be positive.Lineage-specific T cell antigens and T cell-associated antigens are generally negative; however, CD4, CD57, and CD43 expression is reported in approximately half of cases.Immunohistochemistry in our case was compatible with ALK + LBCL, but the cells demonstrated partial CD3 expression, a finding that has been reported in only rare cases [9,12].EBV is almost uniformly negative, although rare cases have been positive [14].CD30 is negative in the majority of cases.
All cases express ALK protein or contain the ALK translocation [9].The staining pattern of ALK is most often in a cytoplasmic granular pattern; this expression pattern is the result of the lymphomagenic translocation ALK::CLTC [t(2;17) (p23;q23)] in the majority of cases.Other translocation partners include NPM1, SEC31A, SQSTM1, RANBP2 GORASP2, EML4, and IGL; these translocations can give distinct staining patterns [7].Our case shows cytoplasmic granular ALK staining.ALK by FISH was disrupted in our case; however, the partner gene cannot be determined in the FISH assay and NGS was not performed.FISH for BCL2, BCL6, and MYC were intact.
Aberrant T cell-specific markers expressed on LBCL have been reported, most notably CD5, but other markers including CD2, CD4, CD7, and CD8 have also been reported [18,19].Aberrant CD3 on LBCL is unusual but is reported sporadically [18][19][20][21][22].Some cases with CD3 expression on LBCL have been associated with EBV [13] leading to speculation that EBV may promote linage infidelity; however, EBV is not uniformly present, and our case does not express EBER.Expression of more than one T cell-specific or associated marker is seen in rare cases of LBCL.The biologic significance is unknown in cases of LCBL with aberrant CD3 due to their rarity [13,19,20].
ALK + LBCL has not been studied extensively with IGH and TCR previously, but in studies where IGH and TCR rearrangements were performed, no TCR clonal rearrangement was identified; however, no case showed CD3 expression in these studies [8,11].Coexisting IGH and TCR clones in gene rearrangement studies is not previously reported, but there are limited studies looking at TCR rearrangements in ALK + LBCL.Although our case may be considered "borderline" in some laboratories due to the apparent polyclonal background, the TCR assay was read as clonal following the manufacturer's instructions.The source of this peak may derive from the focal neoplastic cells, which express aberrant CD3 or conversely, the peak may derive background T cells reacting to the neoplastic B cells.It has also been documented that false positive TCR rearrangements can occur in B cell lymphomas and reactive processes using BIOMED-2 assays [23,24].Although theoretically TCR gamma and TCR beta assays together could be used to confirm the clone, both may sometimes be clonal in the same reactive process [23].Although the source and significance of the T cell peak in our case is uncertain, the utility of BIOMED-2 assays is well documented, and this assay is standard worldwide but caution in reading the assay should be observed.
In conclusion, we present a case of ALK + LBCL with aberrant expression of CD3 and gene rearrangement of both IGH and TCR .A panel of immunohistochemistry markers including multiple T cell, B cell, and plasmacytic markers was necessary to characterize the lymphoma, which is recommended when lineage-specific antigen expression is ambiguous.Because aberrant expression of CD3 is rare in ALK + LBCL, we performed IGH and TCR to help delineate lineage of this ALK + LBCL, testing which may be helpful particularly if aberrant T cell markers are expressed.However, as this case illustrates, gene rearrangement studies should be interpreted with caution and considered within the clinical and pathologic context.The clinical behavior of our case of ALK + LBCL is uncertain due to the rarity of aberrant CD3 in this entity and the lack of clinical followup information.The clinical behavior may not differ from conventional cases of ALK + LBCL; however, further study is necessary.
Deparaffinized tissue sections were hybridized using the manufacturer's recommendations with the following probes Vysis LSI MYC Dual Color Breakapart DNA probe, Vysis IGH/BCL2 Dual Color Fusion DNA translocation probe, Vysis LSI BCL6 (ABR) Dual Color Breakapart DNA probe, and Vysis LSI ALK Dual Color Breakapart DNA probe.

Fig. 2 A
Fig. 2 A TCR-PCR.The T cell receptor gamma-chain PCR assay demonstrates a predominant blue peak (arrow 242 bp) within a valid size range for tube A. B IGH-PCR.The immunoglobulin heavy chain gene PCR assay demonstrates a predominant black peak (arrow 279 bp) in tube B. C A predominant green peak (arrow 265 bp;