Establishment and characterization of a novel patient-derived TP63-rearranged anaplastic large-cell lymphoma model PTCL-S1

Abstract

Anaplastic large-cell lymphoma (ALCL) accounts for 15% of all peripheral T-cell lymphomas globally and can be further divided into subcategories, of which patients with ALK-negative ALCL have dismal prognosis and overall survival. We established a patient-derived xenograft (PDX) and in vitro model (designated PTCL-S1) of TP63-rearranged ALK-negative ALCL from the primary tumour site of a 55-year old Chinese woman. Whole genome sequencing of the patient's tumour identified various mutations including AKT1 and NOTCH1, as well as the TP63-TBL1XR1 gene fusion. RNA sequencing followed by Sanger sequencing confirmed the gene rearrangement in original tumour, PDX and PTCL-S1 cell line. Immunohistochemistry profiling of the PDX model and cell-line were consistent with the patient's primary tumour sample (CD3 + /CD30 + /CD79a-). Cytotoxic agents (doxorubicin, etoposide and gemcitabine) commonly used in ALCL treatment exhibited potent anti-proliferative activity in the cell-line. In conclusion, the established PTCL-S1 cell line can be a useful tool for further investigation of the understanding of TP63-rearranged ALK-negative ALCL.

Keywords: Anaplastic large cell lymphoma (ALCL); Patient-derived xenograft; TP63 rearrangement; TP63-TBL1XR1 fusion; Tumour models.

Fig. 1

Clinical and pathological features of patient with ALK-negative ALCL. a 18F-fluorodeoxyglucose Positron Emission Tomography/Computed Tomography (FDG-PET/CT) imaging taken at time of relapse showed hypermetabolic supradiaphragmatic and infradiaphragmatic lymphadenopathy (red arrows), including the right inguinal, left supraclavicular, subcarinal, peri-hilar and para-aortic regions, as well as FDG-avid bony lesions indicative of marrow infiltration (green arrow) (scale bar: 2 cm). Transverse diameter of dominant nodes: (Top Left to Right) 2–1.2 cm (Bottom Left to Right) 1.7–5 cm. b IHC of patient tumour tissue revealed to be strongly and uniformly positive for CD30 and CD3, while being negative for CD79a (scale bar: 100 µm).

Fig. 2

PDX establishment from patient tissue. a Solid lymphoma developed 8 days following subcutaneous implantation of tumour tissue into NSG mice. b IHC of established PDX revealed similar characteristics to the original patient tumour (scale bar: 100 µm)

Fig. 3

Characterization of PTCL-S1 cell-line. a Brightfield image of PTCL-S1 at 10 × magnification (scale bar: 100 µm) at passage 31. PTCL-S1 cells are mostly spherical with some irregularities in their shape, appearing to be elongated, and they tend to grow in clumps. b PTCL-S1 cells grow readily in RPMI-1640 supplemented with 10% FBS, 10% HS and has an estimated doubling time of 22.26 ± 0.82 h. c IHC of established cell line at passage 10. IHC revealed that PTCL-S1 highly expresses CD30 and CD3 and is negative for CD79a (scale bar: 100 µm). d STR profiling of PTCL-S1 demonstrated a unique DNA fingerprint of the cell line, with no evidence of cross-contamination with cell-lines from known databases

Fig. 4

Genomic characterization of PTCL-S1. Validation of genomic characteristics in PDX and cell line was performed by sanger sequencing at passage 4 and 58 respectively. a NOTCH1 and AKT1 somatic mutations were detected by Whole Genome Sequencing (WGS). b Both somatic mutations were verified and validated in the PTCL-S1 PDX and cell-line by Sanger sequencing. c TP63-TBL1XR1 fusion was identified following WGS of the primary tumour and RNA-Seq of PTCL-S1 cell-line passage 40. d Verification and validation of the TP63-TBL1XR1 fusion in the PTCL-S1 PDX and cell-line by sanger sequencing

Fig. 5

Drug response characterization of PTCL-S1 to standard PTCL treatment regimens. PTCL-S1 treated with the various drugs showed reduction of viability in a dose-dependent manner with reasonable sensitivity to each drug. All drug response experiments were performed using cells between passage 30–40