Identification of high‐risk <i> <scp>DUSP</scp> 22 </i> ‐rearranged <scp>ALK</scp> ‐negative anaplastic large cell lymphoma

Recent studies have identified two mutually exclusive recurrent rearrangements in anaplastic lymphoma kinase (ALK)-negative anaplastic large cell lymphoma (ALCL) that have important clinical significance (Feldman et al, 2011) (Vasmatzis et al, 2012). The DUSP22 rearrangement, which involves the DUSP22-IRF4 locus on 6p25.3, most commonly occurs as a t(6;7)(p25.3;q32.3)(2) and the TP63 rearrangement, which results from a TP63-TBL1XR1 inversion (Vasmatzis et al, 2012). In the first clinical report, DUSP22 rearrangements occurred in ~30% of all ALK-negative ALCL and were associated with a very favourable prognosis [5-year overall survival (OS) 90%] whereas TP63 rearrangements occurred in 8% and were associated with a dismal prognosis (5-year OS 17%) (Parrilla Castellar et al, 2014). The majority of ALK-negative ALCL were ‘triple negative', lacking any known rearrangements, and had an intermediate prognosis (5-year OS 42%) (Parrilla Castellar et al, 2014). More recently, five cases of DUSP22-rearranged ALK-negative ALCL from a Danish series (Pedersen et al, 2017a) and eight cases (including one PTCL-not otherwise specified) from an upfront transplant Phase 2 study (Pedersen et al, 2017b), were evaluated, with similar favourable outcomes (5 year OS >80%). These data have led to treatment guideline modifications, but represent a limited number of cases (NCCN 2018). Herein, we evaluated the frequency, clinical features and outcome of previously defined ALCL genetic subgroups in an independent series of systemic ALCL. In addition, the prognostic significance of immunohistochemical (IHC) markers was explored. All cases of newly diagnosed ALCL were identified in the British Columbia Cancer Lymphoid Cancer database and confirmed by expert haematopathologists based on the World Health Organization classification (GWS, PF). A tissue microarray was constructed and IHC and fluorescence in situ hybridisation (FISH) was performed as previously described using in-house bacterial artificial chromosome break-apart probes for DUSP22 and TP63 loci (Figs S1 and S2) (Scott et al, 2012). Of 62 ALK-negative ALCL cases evaluated, 12 (19%) harboured a DUSP22 rearrangement, one (2%) had a TP63 rearrangement and the remainder were triple negative (n = 49, 79%). All DUSP22 rearrangements were verified on whole sections and at an independent laboratory (AF). Most patients (78%) received CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone)/CHOP-like chemotherapy (92% in DUSP22) (Table 1, Table SI). Some high-risk clinical features were noted in the DUSP22-rearranged cases: Median age 61·5 years; extranodal involvement (67%); bone/bone marrow involvement (42%); high lactate dehydrogenase (42%) (Table 1). The median follow-up for all living patients was 8·6 years (range 1·8–34 years). Consistent with prior studies, outcomes in ALK-negative ALCL were inferior to those with ALK-positive ALCL [5-year progression-free survival (PFS) 23% vs. 62%, P < 0·002; 5-year OS 32% vs. 69%, P < 0·01] (Fig S3A,B). Of note, survival estimates for ALCL in our study are lower than some, but not all other series (Hapgood & Savage, 2015), possibly reflecting the population-based nature of this analysis. Surprisingly, the outcome of DUSP22-rearranged ALK-negative ALCL cases was lower than that observed in published series, with a 5-year PFS and OS of 40% (Fig 1A,B) and 5-year disease-specific survival of 45%, with similar findings when only those treated with curative intent chemotherapy are evaluated (DUSP22-rearranged, n = 11), 5-year PFS and OS 44%. One patient had a central nervous system (CNS) parenchymal relapse (Table SI). Interestingly, five of the relapses occurred over 1 year from diagnosis, including two ≥4 years. Further details on the clinical course are provided in the Supplementary Material. Of note, the 5-year PFS and OS estimates were poor for triple negative ALK-negative ALCL (19% and 28%, respectively) but comparable to the Danish series (n = 20, 5-year OS 33%) (Fig 1A,B). The sole case with a TP63 rearrangement died within 6 months of diagnosis. Excluding the case with a TP63 rearrangement, multivariable Cox proportional hazard models were used to estimate hazard ratios (HR) using ALK-positive ALCL as the reference group (Table SII). There was no statistical difference in OS and PFS in both crude and adjusted [for international prognostic index (IPI) and age] analyses. Similarly, no differences were observed using triple negative cases as the reference group (results not shown). This may reflect the challenge of analysing small datasets with limited power. Regardless, the outcome observed in DUSP22-rearranged cases remains of clinical significance. Despite the aggressive clinical course in some patients, the IHC features of DUSP22-rearranged cases were in keeping with prior reports and highlight that it is a defined entity. CD2 and CD3 expression was frequent and all cases were EMA negative (Table SIII) (Parrilla Castellar et al, 2014). Most cases were cytotoxic marker-negative and all were negative for pSTAT3 and PDL1 (Luchtel et al, 2018) (Table SIII). Taken together, this data suggests that there may be further genetic and/or biological heterogeneity that impacts prognosis, which can only be captured in larger datasets. Despite usually good outcomes, higher risk groups have been noted in ALK-positive ALCL, including older patients, multiple IPI risk factors and CD3+ tumours (Sibon et al, 2017). CD3 positivity was also associated with an inferior outcome in triple negative cases in our cohort, a finding that has not been previously reported (5-year OS 18% vs. 40%, P = 0·01; 5-year PFS 7% vs. 28%, P = 0·05 in the CD3+ and CD3− groups, respectively) (Fig S4A,B). This was consistent after adjusting for the IPI (OS: HR 2·31 (95% confidence interval 1·16, 4·61, P = 0·017); PFS: HR 1·825 (95% confidence interval 0·95, 3·51, P = 0·07). Our data suggests that triple negative cases may also not be a homogeneous group and further studies are needed to confirm these findings and investigate the functional consequence. In summary, in this comprehensive clinico-pathological and genetic analysis, we confirm that DUSP22-rearranged ALK-negative ALCL have unique pathological features. However, similar to prior observations in ALK-positive ALCL, some can present with high risk clinical features and have an aggressive course, including CNS relapse. CD3+ triple negative ALK-negative ALCL is associated with a dismal outcome, supporting additional heterogeneity in the largest subgroup. Additional large-scale studies are needed to fully understand the full disease spectrum of ALK-negative ALCL. The authors thank Kathryn E. Pierce, laboratory development co-ordinator, Mayo Clinic. FISH studies at the Mayo Clinic were supported by R01 CA177734 from the National Cancer Institute. This work is supported by a Terry Fox Research Institute team grant (#1023 and #1061) to RDG and CS. We thank the British Columbia Cancer Foundation for their support. CS is supported by a career investigator award from the Michael Smith Foundation for Health Research. KJS and GH wrote the manuscript. KJS conceived and designed the study. SBN and GH interpreted all fluorescence in situ hybridisation testing (FISH) for all cases; AF interpreted the DUSP22 FISH testing on all 12 cases; PF, GWS and AM performed the pathology review; RDG, CS, GWS and PF provided the samples. DL performed statistical analysis; GH, KJS, DV, LHS, JMC and DS contributed to the clinical data collection. All authors contributed to data interpretation and have approved final manuscript. Table SI. Clinical features at diagnosis and outcome of twelve cases of DUSP22 rearranged ALK-negative ALCL. Table SII. Multivariate Cox regression model for overall survival and progression-free survival: Unadjusted and adjusted analysis. Table SIII. Immunohistochemical features in ALCL by genetic subtype. Fig S1. (A) Bacterial artificial chromosome (BACs) used for fluorescence in situ hybridization DUSP22 break-apart assays with hg 19 co-ordinates. (B) Partial karyotype showing the expected site of chromosomal localization (6p25.3) on normal metaphases. (C) Schematic diagram indicating the alignment of in house BAC probes to the DUSP22 locus of UCSC Hg19. (D) A representative example of interphases with DUSP22 break-apart in an ALCL case demonstrating a rearrangement, with 1 normal fusion and separation of the red and green signal (arrows). Fig S2. (A) Bacterial Artificial Chromosomes (BACs) used for fluorescence in situ hybridization break-apart assays detect TP63 rearrangements with hg 19 coordinates. (B) Partial karyotype showing the expected site of chromosomal localization (3q28) on normal metaphases. (C) Schematic diagram indicating the alignment of in house BAC probes to the TP63 locus of UCSC Hg19. (D) Sole case of TP63 break-apart rearrangement, with 1 normal fusion and separation of the red and green signal (arrows) indicating a rearrangement. Fig S3. (A) Progression-free survival ALK-positive (pos) versus ALK-negative (neg) ALCL. (B) Overall survival of ALK-positive versus ALK-negative. Fig S4. (A) Progression free survival in triple negative ALK-negative (neg) ALCL by CD3 expression. (B) Overall survival in triple negative ALK-negative (neg) ALCL by CD3 expression. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

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