Gene Expression Signatures for the Accurate Diagnosis of Peripheral T-Cell Lymphoma Entities in the Routine Clinical Practice

Abstract

Purpose: Peripheral T-cell lymphoma (PTCL) includes heterogeneous clinicopathologic entities with numerous diagnostic and treatment challenges. We previously defined robust transcriptomic signatures that distinguish common PTCL entities and identified two novel biologic and prognostic PTCL-not otherwise specified subtypes (PTCL-TBX21 and PTCL-GATA3). We aimed to consolidate a gene expression-based subclassification using formalin-fixed, paraffin-embedded (FFPE) tissues to improve the accuracy and precision in PTCL diagnosis.

Materials and methods: We assembled a well-characterized PTCL training cohort (n = 105) with gene expression profiling data to derive a diagnostic signature using fresh-frozen tissue on the HG-U133plus2.0 platform (Affymetrix, Inc, Santa Clara, CA) subsequently validated using matched FFPE tissues in a digital gene expression profiling platform (nCounter, NanoString Technologies, Inc, Seattle, WA). Statistical filtering approaches were applied to refine the transcriptomic signatures and then validated in another PTCL cohort (n = 140) with rigorous pathology review and ancillary assays.

Results: In the training cohort, the refined transcriptomic classifier in FFPE tissues showed high sensitivity (> 80%), specificity (> 95%), and accuracy (> 94%) for PTCL subclassification compared with the fresh-frozen-derived diagnostic model and showed high reproducibility between three independent laboratories. In the validation cohort, the transcriptional classifier matched the pathology diagnosis rendered by three expert hematopathologists in 85% (n = 119) of the cases, showed borderline association with the molecular signatures in 6% (n = 8), and disagreed in 8% (n = 11). The classifier improved the pathology diagnosis in two cases, validated by clinical findings. Of the 11 cases with disagreements, four had a molecular classification that may provide an improvement over pathology diagnosis on the basis of overall transcriptomic and morphological features. The molecular subclassification provided a comprehensive molecular characterization of PTCL subtypes, including viral etiologic factors and translocation partners.

Conclusion: We developed a novel transcriptomic approach for PTCL subclassification that facilitates translation into clinical practice with higher precision and uniformity than conventional pathology diagnosis.

FIG 1.

PTCL molecular classifier. (A) Study design and schematics of the molecular diagnosis of PTCL. A molecular classifier for PTCL subclassification was derived using HG-U133plus2.0 array data from PTCL with FF tissue (n = 109), designated as the training cohort. This molecular classifier had 442 distinct genes, including housekeeping and other genes involved in T-cell biology. This transcriptomic signature was considered for NanoString analysis using corresponding matched FFPE samples. Transcripts that showed a high correlation between FF and FFPE data were selected. The algorithm was further refined to have the minimum number of transcripts for subclassification and mimic the FF predictor score withthe nCounter platform (see the Methods and Materials for details). The final diagnostic model resulted in 153 transcripts (99 diagnostic, five viral, 16 housekeeping, and 33 T-cell biology–related) and was validated in an independent cohort of PTCL cases rigorously characterized by pathology and other ancillary methods. The classification algorithm was based on a series of several binary predictors to distinguish one entity from another, as detailed in the Data Supplement. (B) Heatmap of the finalized NanoString classifier in the training and validation cohorts. EBV and HTLV-1 viral transcripts commonly expressed in specific PTCL subtypes are shown, and housekeeping genes used for normalization and that do not vary between PTCL subtypes are displayed for comparison. (C) Kaplan-Meier curve of OS for 89 of the 105 training cohort cases and 66 of the 140 validation cases with available outcome data. (D) Kaplan-Meier curve of OS of PTCL subtypes included in the training cohort (molecular classification by the HG-U133plus2.0 array). (E) Kaplan-Meier curve of OS of PTCL entities included in the validation cohort (pathology classification). ^aFour cases excluded because of poor RNA quality. ^bIncludes classification, housekeeping, and important T-cell biology genes. ^cTwelve cases excluded because of reclassification as n-PTCL-T_FH. AITL, angioimmunoblastic T-cell lymphoma; ALCL, anaplastic large cell lymphoma; ATLL, adult T-cell lymphoma/leukemia; EBV, Epstein-Barr virus; ENKTCL, extranodal natural killer/T-cell lymphoma; FF, fresh-frozen; FFPE, formalin-fixed, paraffin-embedded; GEP, gene expression profiling; HBZ, HTLV-1 bZIP factor; HTLV-1, human T-lymphotropic virus; NOS, not otherwise specified; OS, overall survival; PTCL, peripheral T-cell lymphoma; T_FH, T follicular helper.

FIG 2.

AITL classification. (A) Scatterplot of the AITL diagnostic score versus the average expression of five T_FH-related genes in training and validation AITLs. (B) Boxplot of EBER and EBNA1 transcript expression in the AITLs (training/validation cohort). (C) Kaplan-Meier curve of OS of AITLs in the combined cohorts by CD20 mRNA expression. Solid lines are cases classified as AITL by molecular classification (P = .02); dotted lines are AITL by pathology (P = .06). CD20 expression in representative low- and high-expression AITL cases (400×). (D) Mutation status of cases with available sequencing data. Cases that were AITL-PTCL-NOS intermediate or did not classify as AITL on the NanoString classifier are noted with an asterisk. (E) Violin and dot plot of AITL classification diagnostic scores in AITL and PTCL-NOS cases profiled on the nCounter. Cases that were discordant between AITL and PTCL-NOS are given in red, and intermediate AITL cases in gray. (F) Heatmaps of AITL showing disagreement by NanoString classification in the validation cohort. The mean signature of the concordant cases is shown. For cases labeled intermediate, those diagnosed as AITL by consensus pathology review are on the left and intermediate cases diagnosed as PTCL-NOS are on the right. Two PTCL-NOS classified as AITL by nCounter. (G-I) Shown focal expression of BCL-6 and ICOS (400x; G, H, and I; G, H&E, H, BCL-6 and I, ICOS) seen in a PTCL-NOS case that was classified as AITL by the nCounter platform. AITL, angioimmunoblastic T-cell lymphoma; BCL-6, B-cell lymphoma 6; ENKTCL, extranodal natural killer/T-cell lymphoma; ICOS, inducible T cell co-stimulator; NOS, not otherwise specified; OS, overall survival; PTCL, peripheral T-cell lymphoma; T_FH, T follicular helper; WT, wild type.

FIG 3.

ALCL classification. (A) Violin and dot plot of ALCL classification scores versus PTCL-NOS. (B) ALK-positive ALCL versus ALK-negative ALCL on the nCounter platform. Cases that were discordant between ALCL and PTCL-NOS or ALK-negative and ALK-positive are given in red. (C) Heatmaps of ALK-negative and ALK-positive ALCL cases showing disagreement by NanoString classification in the validation cohort. H&E and CD30 and ALK immunostains of a representative ALK-negative ALCL case that was classified as ALK-positive ALCL by NanoString assay are shown (400×). (D) Kaplan-Meier curve of OS of ALCLs in the training and validation cohort by NanoString classification. (E) Heatmap of CD30 and cytotoxic transcript expression in ALCL and PTCL-NOS cases. Discrepant cases are noted with red lines. ALCL, anaplastic large cell lymphoma; H&E, hematoxylin and eosin; NOS, not otherwise specified; OS, overall survival; PTCL, peripheral T-cell lymphoma.

FIG 4.

ATLL and ENKTCL classification. (A) Violin and dot plot of ATLL classification scores in ATLL and PTCL-NOS cases profiled on the nCounter. (B) Heatmaps of ATLL cases discordant by nCounter classification in the validation cohort. H&E stain and CD4 staining for a case diagnosed as PTCL-NOS but classified at ATLL by nCounter (lower panel). (C) Scatterplot of expression of the HTLV-1–specific transcripts HBZ versus ATLL score in training and validation ATLL cases. The solid fitted line represents training data, and the dashed line validation data. (D) Heatmap of HBZ and Tax-1 expression in the ATLL and PTCL-NOS validation cohorts. The discrepant cases are noted by a red asterisk. (E) Scatterplot of HBZ expression measured by qRT-PCR versus nCounter. (F and G) Violin and dot plots of (F) ENKTCL classification scores or (G) EBER scores in ENKTCLs and PTCL-NOS cases profiled in the training and validation cohorts on the nCounter. Discordant cases are given in red. (H) Heatmap of expression of CD3 gamma and delta and EBV transcripts in ENKTCL and PTCL-NOS cases in the training and validation cohorts. (I) Heatmap of expression of relevant signatures in the ENKTCL-discordant case compared with average signatures in the validation cohort. ATLL, adult T-cell leukemia/lymphoma; EBV, Epstein-Barr virus; ENKTCL, extranodal natural killer/T-cell lymphoma; H&E, hematoxylin and eosin; HBZ, HTLV-1 bZIP factor; HTLV-1, human T-lymphotropic virus; NOS, not otherwise specified; PTCL, peripheral T-cell lymphoma; qRT-PCT, quantitative real-time polymerase chain reaction.

FIG 5.

PTCL-NOS subclassification. (A) Violin and dot plots of PTCL-GATA3 classification scores in PTCL-NOS cases profiled in the training and validation cohorts on the nCounter. (B) Mutation status of the PTCL-NOS cohort with available sequencing data. (C) Heatmaps of expression of CD4, CD8, CD20, and cytotoxic genes in the training (upper) and validation (lower) cases. In CD4-positive lymphomas, the CD8 expression is likely contributed from the tumor microenvironment. (D) Scatterplot of the average expression of the cytotoxic genes versus CD20 in cases that classified as PTCL-TBX21 by NanoString. (E) H&E and IHC stains for one representative PTCL-GATA3 case showing GATA3 (left) and one PTCL-TBX21 case showing TBX21 and CD8 expression (right). (F) Kaplan-Meier curve of OS for PTCL-NOS cases with available outcome data in the combined training and validation cohorts classified as PTCL-GATA3 or PTCL-TBX21 NanoString. AITL, angioimmunoblastic T-cell lymphoma; ALCL, anaplastic large cell lymphoma; ATLL, adult T-cell lymphoma/leukemia; ENKTCL, extranodal natural killer/T-cell lymphoma; IHC, immunohistochemistry; NOS, not otherwise specified; OS, overall survival; PTCL, peripheral T-cell lymphoma; WT, wild-type.