Identification of a New Subclass of ALK Negative Anaplastic Large Cell Lymphoma Expressing Aberrant Levels of ERBB4 Transcripts

Anaplastic Large Cell Lymphoma (ALCL) is a clinical and biological heterogeneous disease including the ALK+ and ALK- systemic forms. While ALK+ ALCL are molecularly characterized and can be readily diagnosed, no specific markers and molecular events leading to ALK- ALCL transformation have been identified so far.

To discover biomarkers and/or genes potentially involved in ALK- ALCL pathogenesis, we applied the Cancer Outlier Profile Analysis (COPA) algorithm to a large gene expression profiling (GEP) data set including 249 cases of T-NHLs and normal T-cells. Among the top outliers, ERBB4 and COL29A1 genes were exclusively expressed in a subset of ALK- ALCL cases, and resulted highly correlated. Differential analysis comparing the expression profiles of ERBB4/COL29A1+ to ALK+ ALCL patients identified 48 genes up- and 37 down-regulated (qval < 0.0076). The most significantly over-represented functional category included transcriptional regulators. Gene-set-enrichment-analysis (GSEA) indicated that ERBB4/COL29A1+ samples shared activation of specific pathways such as inflammatory response, TNF-NF-κB, JAK-STAT, cytokine, and angiogenesis. RNA sequencing and 5’RNA Ligase Mediated Rapid Amplification of cDNA Ends (RLM-RACE) identified two ERBB4 truncated transcripts (referred as I20ΔERBB4 and I12ΔERBB4), displaying Transcription Starting Sites (TSS) in intron 12 and 20, respectively. On the contrary, full length COL29A1 mRNA was expressed in ERBB4/COL29A1+ samples. RT-qPCR analysis performed on 170 T-NHL samples (51 PCTL-NOS, 44 ALK+ ALCL, and 75 ALK- ALCL) highlighted specific expression of ERBB4 aberrant transcripts in 25% of ALK- ALCL (18 out of 75). ERBB4+ patients showed higher expression of I20ΔERBB4 as compared to I12ΔERBB4 transcript. ERBB4 expression at protein level was confirmed by immunohistochemistry and Western Blotting on selected cases. Inspection of intronic regions spanning two hypothetical TSS revealed the presence of Human Endogenous Retrovirus (HERV) Long Terminal Repeats (LTR) displaying promoter activity, as demonstrated by luciferase assays.

In conclusion, we defined a new subclass of ALK- ALCL characterized by ectopic expression of ERBB4 and COL29A1 genes. To the best of our knowledge, ERBB4 truncated transcripts carrying intronic 5’UTR have never been described before. Further studies are required to address whether the expression of ERBB4 aberrant transcripts may contribute to the ALCL transformation and lymphoma maintenance. This information might lead to more rational therapeutic approaches for ERBB4+ ALCL patients.