Commonly used immunophenotypic and genetic markers in the diagnosis of NK- and T-cell neoplasms
| Marker . | Diagnostic utility . | Interpretation and comments . |
|---|---|---|
| Antigens assessed by immunohistochemistry (IHC) or flow cytometry (FC) | ||
| Pan-T-cell antigens: CD2, CD3, CD5, CD7, CD43 (IHC, FC) | Identification T-cell (or NK) populations. Aberrant loss of T-cell antigen expression is a phenotypic marker favoring neoplastic over benign lymphoproliferation. | NK and T cells are distinguished by the expression of surface CD3 (assessed on fresh cells by FC), positive in T cells and negative in NK cells, whereas both T and NK cells express cytoplasmic CD3 (assessed by IHC on routinely processed tissues); in addition, CD5 is constitutively absent in NK cells (and in TCRγδ+ T cells) Extensive loss of T-cell antigens and possibly «null» immunophenotype is characteristic of ALK+ and to a lesser extent ALK− ALCL. CD7 loss is typical of ATLL. |
| CD4 and CD8 (IHC, FC) | Characterization of T-cell proliferations according to the CD4+CD8− or CD4−CD8+ lineages. | Double negative expression may indicate immature lymphoid cells, TCRγδ+ or NK cells, or aberrant phenotype in mature TCRαβ+ T cells, found in ALPS or lymphomas. Double positive expression may indicate immature lymphoid cells, or aberrant phenotype in mature TCRαβ+ T cells, usually indicative of malignancy. |
| TCRβ and TCRδ chains (IHC, FC) | Identification of TCRαβ+ vs TCRγδ+ T cells. | Most nodal PTCLs derive from TCRαβ+ T cells. PTCL may show TCR downregulation, or silencing of TCR expression. |
| TRBC1 (IHC, FC) | Identification of clonal T-cell population by monotypic expression pattern. | Only applicable if surface CD3 and TCRαβ expression is retained. |
| CD30 (ICH, FC) | Activation marker, not lineage-specific. Strong uniform expression in ALCLs, heterogeneous expression in a proportion of cases in many TCL entities, usually in a subset of the cells. | Only scattered cells may be positive in small-cell and histiocyte-rich patterns of ALK+ ALCL. CD30 expression in bystander Reed–Sternberg-like cells in TFHL and PTCL-NOS. May be targetable by antibody–drug conjugates. |
| CD15 (IHC, FC) | Glycan determinant, marker of myeloid cells. May be positive in the neoplastic cells in ALCL and a subset of PTCL-NOS. | Coexpression of CD30 and CD15 in bystander HRS B cells in various PTCLs. |
| ALK (IHC) | Demonstration of ALK expression is essential for the diagnosis of ALK+ ALCL. IHC is an excellent surrogate to ALK gene fusion detection by genetic assays. | Variable patterns of subcellular staining according to the partner gene of ALK in the fusion. Nuclear and cytoplasmic expression is characteristic of NPM1::ALK, which is the most common fusion. |
| Cytotoxic molecules: TIA-1, perforin, granzyme B (IHC) | Identification of a nonactivated (TIA1+ alone) or activated (perforin+ or granzyme B+) cytotoxic phenotype, characteristically seen in ALCL and a subset of PTCL-NOS. | Many reactive cytotoxic cells may be present in noncytotoxic TCL (eg, TFHL-AI). |
| TFH cell markers: PD1, ICOS, CXCL13, CD10, BCL6 (most commonly used), CD57, CXCR5, SAP, MAF, CD200 (IHC, FC for cell surface markers) | TFH immunophenotype is defined by the expression of at least 2 and ideally 3 TFH markers in CD4+ cells. | None of the TFH markers are in isolation sensitive or specific for the TFH phenotype. Intensity of expression should be similar to that of reactive TFH cells, in a significant proportion of the presumed neoplastic cells. |
| EBV (ISH, IHC) | Detection mandatory to diagnose EBV-associated lymphomas. Detection of EBV+ bystander B cells in TFH lymphomas, PTCL-NOS, and ATLL. | EBER in situ hybridization is the gold standard for EBV detection; LMP-1 immunohistochemistry is less sensitive. |
| CD56 (ICH, FC) | Cytotoxic and NK cell lymphomas. | Characteristically expressed in ENKTCL and MEITL; sometimes positive in primary nodal EBV+ PTCL; occasional aberrant expression in other T-cell lymphomas. |
| Other markers of T helper cell subsets: TBX21, GATA3, CXCR3, CCR4, FOXP3 (ICH, FC for cell surface markers) | Subsets of TH1 (TBX21+) and TH2 (GATA3+) PTCL-NOS. FOXP3 (Treg) expression in a subset of ATLLs. | Utility of these markers such as GATA3 or TBX21 is limited to specific diagnoses. Broad range of expression of GATA3 in nonhematologic malignancies and other lymphomas, including ALCLs. |
| CD25 (ICH, FC) | α-chain of the IL-2 receptor, expressed on normal T regulatory cells and usually expressed at high levels in ATLL cells, but also in ALCL and other PTCLs. | |
| Follicular dendritic cell (FDC) markers: CD21, CD23 (ICH) | Expansion of FDC characteristic of TFHL-AI; demonstration of follicular pattern in TFHL-follicular. | TFHL-AI pattern I has no FDC expansion. |
| B-cell markers: CD20, CD79a, PAX5 (IHC, FC) | Identification of a B-cell component or microenvironment in TFHL. Abundant B cells in association with a T-cell lymphoproliferation favor a reactive over a malignant process. | Some PTCLs may coexpress CD20 and/or other B-cell antigens. PAX5 positivity in a subset of ALCLs. |
| CD138, κ, λ (IHC) | Plasma cells. May be abundant in TFHL. | Monotypic or even monoclonal plasma cells in some TFHLs. |
| Antigen receptor gene rearrangements | ||
| TRB and TRG (PCR- or NGS-based assays) | Monoclonal gene rearrangements are detected in most T-cell neoplasms. In some circumstances, lack of monoclonal TR gene rearrangement favors NK over T-cell derivation. Monoclonal results support a diagnosis of TCL in cases with minimal involvement or when morphology and immunophenotyping are not definitively conclusive. | Useful to demonstrate T-cell lineage in T-cell lymphomas with extensive antigen loss. No correlation with TCRαβ+ vs TCRγδ+ phenotype. Monoclonal TRB or TRG rearrangements may be detected in reactive T-cell lymphoproliferations (eg, viral infections), but are usually small. |
| IGH, IGK, IGL (PCR- or NGS-based assays) | Monoclonal gene rearrangements in general indicative of a B-cell neoplasm. | Monoclonal IGH or IGK rearrangements may be detected in PTCLs with a B-cell component (especially TFHL). |
| Specific genetic alterations | ||
| ALK rearrangements (FISH) | 100% of ALCL ALK+. | Immunohistochemistry for ALK usually used as a surrogate for genetic testing. Rare cases of primary cutaneous ALCLs localized to the skin harbor ALK rearrangement and behave similarly to other ALK− primary cutaneous cases. |
| DUSP22 rearrangements (FISH) | Subset (25%-30%) of ALCL ALK−. | Also in a subset of primary cutaneous ALCLs (and rare cases of lymphomatoid papulosis). |
| TP63 rearrangements (FISH) | Small subset (<10%) of ALCL ALK−, and of PTCL-NOS. | Expression of TP63 does not reliably distinguish cases with TP63 rearrangement (TP63 IHC+) vs those without TP63 rearrangement (may also be TP63 IHC+). |
| JAK2 fusions (FISH or NGS) | Subset of ALCL ALK−, recurrent JAK2::STAT3 fusion in indolent clonal T-cell LPD of the gastrointestinal tract. | |
| TYK2 fusions (FISH or NGS) | Subset of ALCL ALK−, lymphomatoid papulosis and primary cutaneous ALCL. | Various fusions reported, including NFκB2::TYK2, PABPC4::TYK2 NPM1::TYK2. |
| CD28::ICOS and CD28::CTLA4 fusions (NGS) | Subset of TFHL, rare in PTCL-NOS, cutaneous lymphomas, and ATLL. | |
| RHOAG17V (NGS, AS-PCR, ddPCR) | Hot spot mutation in TFHL. | Other RHOA variants occasional in TFH lymphomas and frequent in ATLL. |
| IDH2 (NGS, AS-PCR, IHC) | Hotspot mutations at R172 residue in one-third of TFHL-AI. | Expression of mutant proteins can be demonstrated by IHC with antibodies specific of IDH2 variants. Correlation with clear cell morphology. |
| TET2, DNMT3 (NGS) | Inactivating mutations, often multiple, very frequent in TFHL and less common in other PTCLs. | Mutations also associated to clonal hematopoiesis and therefore not necessarily indicative of a T-cell neoplasm. |
| CD28, PLCG1, FYN, CARD11, VAV1,… (NGS) | Gain-of-function mutations recurrent in TFHL, ATLL, and cutaneous T-cell lymphomas, and rarely in other TCL. | |
| SETD2 (NGS and FISH) | Loss-of-function mutations and/or deletions characteristic of MEITL and HSTL. | Loss of SETD2 function translates to loss of H3K36me3 by IHC. |
| JAK1, JAK3, STAT3, STAT5B (NGS) | Gain-of-function mutations in various PTCL entities, especially frequent in primary intestinal TCLs, HSTL, ENKTCL, ALCL ALK−, BIA-ALCL, leukemic NK-cell, and T-cell disorders. | |
| Iso(7q) (FISH) | Iso(7q) detected in >80% of HSTL. | Characteristic but not specific of HSTL, also reported in acute myeloid and lymphoblastic leukemia, myelodysplastic syndrome, rare cases of ENKTCL, and ALCL. |
| Marker . | Diagnostic utility . | Interpretation and comments . |
|---|---|---|
| Antigens assessed by immunohistochemistry (IHC) or flow cytometry (FC) | ||
| Pan-T-cell antigens: CD2, CD3, CD5, CD7, CD43 (IHC, FC) | Identification T-cell (or NK) populations. Aberrant loss of T-cell antigen expression is a phenotypic marker favoring neoplastic over benign lymphoproliferation. | NK and T cells are distinguished by the expression of surface CD3 (assessed on fresh cells by FC), positive in T cells and negative in NK cells, whereas both T and NK cells express cytoplasmic CD3 (assessed by IHC on routinely processed tissues); in addition, CD5 is constitutively absent in NK cells (and in TCRγδ+ T cells) Extensive loss of T-cell antigens and possibly «null» immunophenotype is characteristic of ALK+ and to a lesser extent ALK− ALCL. CD7 loss is typical of ATLL. |
| CD4 and CD8 (IHC, FC) | Characterization of T-cell proliferations according to the CD4+CD8− or CD4−CD8+ lineages. | Double negative expression may indicate immature lymphoid cells, TCRγδ+ or NK cells, or aberrant phenotype in mature TCRαβ+ T cells, found in ALPS or lymphomas. Double positive expression may indicate immature lymphoid cells, or aberrant phenotype in mature TCRαβ+ T cells, usually indicative of malignancy. |
| TCRβ and TCRδ chains (IHC, FC) | Identification of TCRαβ+ vs TCRγδ+ T cells. | Most nodal PTCLs derive from TCRαβ+ T cells. PTCL may show TCR downregulation, or silencing of TCR expression. |
| TRBC1 (IHC, FC) | Identification of clonal T-cell population by monotypic expression pattern. | Only applicable if surface CD3 and TCRαβ expression is retained. |
| CD30 (ICH, FC) | Activation marker, not lineage-specific. Strong uniform expression in ALCLs, heterogeneous expression in a proportion of cases in many TCL entities, usually in a subset of the cells. | Only scattered cells may be positive in small-cell and histiocyte-rich patterns of ALK+ ALCL. CD30 expression in bystander Reed–Sternberg-like cells in TFHL and PTCL-NOS. May be targetable by antibody–drug conjugates. |
| CD15 (IHC, FC) | Glycan determinant, marker of myeloid cells. May be positive in the neoplastic cells in ALCL and a subset of PTCL-NOS. | Coexpression of CD30 and CD15 in bystander HRS B cells in various PTCLs. |
| ALK (IHC) | Demonstration of ALK expression is essential for the diagnosis of ALK+ ALCL. IHC is an excellent surrogate to ALK gene fusion detection by genetic assays. | Variable patterns of subcellular staining according to the partner gene of ALK in the fusion. Nuclear and cytoplasmic expression is characteristic of NPM1::ALK, which is the most common fusion. |
| Cytotoxic molecules: TIA-1, perforin, granzyme B (IHC) | Identification of a nonactivated (TIA1+ alone) or activated (perforin+ or granzyme B+) cytotoxic phenotype, characteristically seen in ALCL and a subset of PTCL-NOS. | Many reactive cytotoxic cells may be present in noncytotoxic TCL (eg, TFHL-AI). |
| TFH cell markers: PD1, ICOS, CXCL13, CD10, BCL6 (most commonly used), CD57, CXCR5, SAP, MAF, CD200 (IHC, FC for cell surface markers) | TFH immunophenotype is defined by the expression of at least 2 and ideally 3 TFH markers in CD4+ cells. | None of the TFH markers are in isolation sensitive or specific for the TFH phenotype. Intensity of expression should be similar to that of reactive TFH cells, in a significant proportion of the presumed neoplastic cells. |
| EBV (ISH, IHC) | Detection mandatory to diagnose EBV-associated lymphomas. Detection of EBV+ bystander B cells in TFH lymphomas, PTCL-NOS, and ATLL. | EBER in situ hybridization is the gold standard for EBV detection; LMP-1 immunohistochemistry is less sensitive. |
| CD56 (ICH, FC) | Cytotoxic and NK cell lymphomas. | Characteristically expressed in ENKTCL and MEITL; sometimes positive in primary nodal EBV+ PTCL; occasional aberrant expression in other T-cell lymphomas. |
| Other markers of T helper cell subsets: TBX21, GATA3, CXCR3, CCR4, FOXP3 (ICH, FC for cell surface markers) | Subsets of TH1 (TBX21+) and TH2 (GATA3+) PTCL-NOS. FOXP3 (Treg) expression in a subset of ATLLs. | Utility of these markers such as GATA3 or TBX21 is limited to specific diagnoses. Broad range of expression of GATA3 in nonhematologic malignancies and other lymphomas, including ALCLs. |
| CD25 (ICH, FC) | α-chain of the IL-2 receptor, expressed on normal T regulatory cells and usually expressed at high levels in ATLL cells, but also in ALCL and other PTCLs. | |
| Follicular dendritic cell (FDC) markers: CD21, CD23 (ICH) | Expansion of FDC characteristic of TFHL-AI; demonstration of follicular pattern in TFHL-follicular. | TFHL-AI pattern I has no FDC expansion. |
| B-cell markers: CD20, CD79a, PAX5 (IHC, FC) | Identification of a B-cell component or microenvironment in TFHL. Abundant B cells in association with a T-cell lymphoproliferation favor a reactive over a malignant process. | Some PTCLs may coexpress CD20 and/or other B-cell antigens. PAX5 positivity in a subset of ALCLs. |
| CD138, κ, λ (IHC) | Plasma cells. May be abundant in TFHL. | Monotypic or even monoclonal plasma cells in some TFHLs. |
| Antigen receptor gene rearrangements | ||
| TRB and TRG (PCR- or NGS-based assays) | Monoclonal gene rearrangements are detected in most T-cell neoplasms. In some circumstances, lack of monoclonal TR gene rearrangement favors NK over T-cell derivation. Monoclonal results support a diagnosis of TCL in cases with minimal involvement or when morphology and immunophenotyping are not definitively conclusive. | Useful to demonstrate T-cell lineage in T-cell lymphomas with extensive antigen loss. No correlation with TCRαβ+ vs TCRγδ+ phenotype. Monoclonal TRB or TRG rearrangements may be detected in reactive T-cell lymphoproliferations (eg, viral infections), but are usually small. |
| IGH, IGK, IGL (PCR- or NGS-based assays) | Monoclonal gene rearrangements in general indicative of a B-cell neoplasm. | Monoclonal IGH or IGK rearrangements may be detected in PTCLs with a B-cell component (especially TFHL). |
| Specific genetic alterations | ||
| ALK rearrangements (FISH) | 100% of ALCL ALK+. | Immunohistochemistry for ALK usually used as a surrogate for genetic testing. Rare cases of primary cutaneous ALCLs localized to the skin harbor ALK rearrangement and behave similarly to other ALK− primary cutaneous cases. |
| DUSP22 rearrangements (FISH) | Subset (25%-30%) of ALCL ALK−. | Also in a subset of primary cutaneous ALCLs (and rare cases of lymphomatoid papulosis). |
| TP63 rearrangements (FISH) | Small subset (<10%) of ALCL ALK−, and of PTCL-NOS. | Expression of TP63 does not reliably distinguish cases with TP63 rearrangement (TP63 IHC+) vs those without TP63 rearrangement (may also be TP63 IHC+). |
| JAK2 fusions (FISH or NGS) | Subset of ALCL ALK−, recurrent JAK2::STAT3 fusion in indolent clonal T-cell LPD of the gastrointestinal tract. | |
| TYK2 fusions (FISH or NGS) | Subset of ALCL ALK−, lymphomatoid papulosis and primary cutaneous ALCL. | Various fusions reported, including NFκB2::TYK2, PABPC4::TYK2 NPM1::TYK2. |
| CD28::ICOS and CD28::CTLA4 fusions (NGS) | Subset of TFHL, rare in PTCL-NOS, cutaneous lymphomas, and ATLL. | |
| RHOAG17V (NGS, AS-PCR, ddPCR) | Hot spot mutation in TFHL. | Other RHOA variants occasional in TFH lymphomas and frequent in ATLL. |
| IDH2 (NGS, AS-PCR, IHC) | Hotspot mutations at R172 residue in one-third of TFHL-AI. | Expression of mutant proteins can be demonstrated by IHC with antibodies specific of IDH2 variants. Correlation with clear cell morphology. |
| TET2, DNMT3 (NGS) | Inactivating mutations, often multiple, very frequent in TFHL and less common in other PTCLs. | Mutations also associated to clonal hematopoiesis and therefore not necessarily indicative of a T-cell neoplasm. |
| CD28, PLCG1, FYN, CARD11, VAV1,… (NGS) | Gain-of-function mutations recurrent in TFHL, ATLL, and cutaneous T-cell lymphomas, and rarely in other TCL. | |
| SETD2 (NGS and FISH) | Loss-of-function mutations and/or deletions characteristic of MEITL and HSTL. | Loss of SETD2 function translates to loss of H3K36me3 by IHC. |
| JAK1, JAK3, STAT3, STAT5B (NGS) | Gain-of-function mutations in various PTCL entities, especially frequent in primary intestinal TCLs, HSTL, ENKTCL, ALCL ALK−, BIA-ALCL, leukemic NK-cell, and T-cell disorders. | |
| Iso(7q) (FISH) | Iso(7q) detected in >80% of HSTL. | Characteristic but not specific of HSTL, also reported in acute myeloid and lymphoblastic leukemia, myelodysplastic syndrome, rare cases of ENKTCL, and ALCL. |
AI, angioimmunoblastic type; ALCL, anaplastic large cell lymphoma; ALK, anaplastic lymphoma kinase; ALPS, autoimmune lymphoproliferative syndrome; AS-PCR, allele-specific PCR; ATLL, adult T-cell leukemia/lymphoma; BIA, breast implant–associated; ddPCR, digital droplet PCR; EBERs, EBV-encoded small RNAs; EBV, Epstein-Barr virus; ENKTCL, extranodal NK/T-cell lymphoma, nasal type; F, follicular; FC, flow cytometry; FDC, follicular dendritic cell; FISH, fluorescent in situ hybridization; HSTL, hepatosplenic T-cell lymphoma; IHC, immunohistochemistry; IL-2, interleukin-2; LPD, lymphoproliferative disorder; LMP-1, latent membrane protein 1; MEITL, monomorphic epitheliotropic intestinal T-cell lymphoma; NGS, high-throughput sequencing; PCR, polymerase chain reaction; PTCL-NOS, peripheral TCL, not otherwise specified; TCL, T-cell lymphoma; TFHL, follicular helper TCL.