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Mutations of the B-Cell Receptor Pathway Confer Chemoresistance in Primary Cutaneous Diffuse Large B-Cell Lymphoma Leg Type

Open ArchivePublished:May 28, 2019DOI:https://doi.org/10.1016/j.jid.2019.05.008
      Primary cutaneous diffuse large B-cell lymphoma, leg type (PCLBCL-LT) preferentially involves the lower limb in elderly subjects. A combination of polychemotherapy and rituximab has improved prognosis. However, about 50% of patients will experience progression or relapse without any predictive biologic marker of therapeutic response. The mutational profile of PCLBCL-LT has highlighted mutations contributing to constitutive NF-κB and B-cell receptor (BCR) signaling pathways but has not demonstrated clinical utility. Therefore, the mutational status of 32 patients with PCLBCL-LT (14 patients with complete durable response and 18 patients with relapsing or refractory disease) was determined with a dedicated lymphopanel. Tumor pairs at diagnosis and relapse or progression were analyzed in 14 relapsing or refractory patients. Patients with PCLBCL-LT harboring one mutation that targets one of the BCR signaling genes, CD79A/B or CARD11, displayed a reduced progression-free survival and specific survival (median 18 months, P = 0.002 and 51 months, P = 0.03, respectively, whereas median duration in the wild-type group was not reached) and were associated with therapeutic resistance (P = 0.0006). Longitudinal analyses revealed that MYD88 and CD79B were the earliest and among the most mutated genes. Our data suggest that evaluating BCR mutations in patients with PCLBCL-LT may help to predict first-line therapeutic response and to select targeted therapies.

      Abbreviations:

      ABC (activated B-cell), BCR (B-cell receptor), CR (complete durable response), DLBCL (diffuse large B-cell lymphoma), FFPE (formalin-fixed), paraffin-embedded (PCLBCL-LT), primary cutaneous diffuse large B-cell lymphoma (leg type), PFS (progression-free survival), RR (relapsing-refractory), R-PCT (rituximab and polychemotherapy), VAF (variant allele frequency)

      Introduction

      Primary cutaneous diffuse large B-cell lymphoma, leg type (PCLBCL-LT) has been individualized as an aggressive disease accounting for 5% of primary cutaneous lymphoma and for 20% of primary cutaneous B-cell lymphoma (
      • Willemze R.
      • Jaffe E.S.
      • Burg G.
      • Cerroni L.
      • Berti E.
      • Swerdlow S.H.
      • et al.
      WHO-EORTC classification for cutaneous lymphomas.
      ,
      • Willemze R.
      • Cerroni L.
      • Kempf W.
      • Berti E.
      • Facchetti F.
      • Swerdlow S.H.
      • et al.
      The 2018 update of the WHO-EORTC classification for primary cutaneous lymphomas.
      ). Occurring especially in elderly women, it presents mostly as tumor and nodules, preferentially involving the lower limb (
      • Grange F.
      • Beylot-Barry M.
      • Courville P.
      • Maubec E.
      • Bagot M.
      • Vergier B.
      • et al.
      Primary cutaneous diffuse large B-cell lymphoma, leg type: clinicopathologic features and prognostic analysis in 60 cases.
      ,
      • Willemze R.
      • Jaffe E.S.
      • Burg G.
      • Cerroni L.
      • Berti E.
      • Swerdlow S.H.
      • et al.
      WHO-EORTC classification for cutaneous lymphomas.
      ). PCLBCL-LT corresponds to a monotonous infiltrate of confluent sheets of centroblasts and immunoblasts typically expressing CD20, CD79a, Bcl-2, and MUM-1/IRF4, with usual negativity for CD5 and CD10 (
      • Menguy S.
      • Gros A.
      • Pham-Ledard A.
      • Battistella M.
      • Ortonne N.
      • Comoz F.
      • et al.
      MYD88 somatic mutation is a diagnostic criterion in primary cutaneous large B-cell lymphoma.
      ,
      • Swerdlow S.H.
      • Campo E.
      • Pileri S.A.
      • Harris N.L.
      • Stein H.
      • Siebert R.
      • et al.
      The 2016 revision of the World Health Organization classification of lymphoid neoplasms.
      ,
      • Willemze R.
      • Jaffe E.S.
      • Burg G.
      • Cerroni L.
      • Berti E.
      • Swerdlow S.H.
      • et al.
      WHO-EORTC classification for cutaneous lymphomas.
      ). Morphology and phenotype bring PCLBCL-LT close to the specific activated B-cell (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) which has been individualized by gene expression profiling from germinal center B-cell–like and primary mediastinal B-cell lymphoma (
      • Alizadeh A.A.
      • Eisen M.B.
      • Davis R.E.
      • Ma C.
      • Lossos I.S.
      • Rosenwald A.
      • et al.
      Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling.
      ). This ABC subtype corresponds to a post–germinal center cell of origin with constitutive activation of the NF-κB signaling pathway and a poorer outcome. Molecular profiling of PCLBCL-LT further supported its relationship with the ABC DLBCL subtype (
      • Hoefnagel J.J.
      • Dijkman R.
      • Basso K.
      • Jansen P.M.
      • Hallermann C.
      • Willemze R.
      • et al.
      Distinct types of primary cutaneous large B-cell lymphoma identified by gene expression profiling.
      ,
      • Pham-Ledard A.
      • Beylot-Barry M.
      • Barbe C.
      • Leduc M.
      • Petrella T.
      • Vergier B.
      • et al.
      High frequency and clinical prognostic value of MYD88 L265P mutation in primary cutaneous diffuse large B-cell lymphoma, leg-type.
      ). A combination of the CD20-directed monoclonal antibody rituximab and polychemotherapy (R-PCT) adapted to the age of patients has improved the prognosis (average 5-year survival, 73%) (
      • Grange F.
      • Joly P.
      • Barbe C.
      • Bagot M.
      • Dalle S.
      • Ingen-Housz-Oro S.
      • et al.
      Improvement of survival in patients with primary cutaneous diffuse large B-cell lymphoma, leg type, in France.
      ). After CD20 binding, rituximab exerts several antitumoral effects either by directly triggering antiproliferative effects through calcium release or influx, Src kinase activation and apoptosis through BCL2 downregulation, or by indirectly mediating complement-dependent cytotoxicity or antibody-dependent cell-mediated cytotoxicity/phagocytosis through Fc-γ binding by immune cells (
      • Bachy E.
      • Salles G.
      Are we nearing an era of chemotherapy-free management of indolent lymphoma?.
      ,
      • Chao M.P.
      Treatment challenges in the management of relapsed or refractory non-Hodgkin’s lymphoma - novel and emerging therapies.
      ). About half of the patients will experience cutaneous or extracutaneous relapse and progression with death because of the disease without any biologic marker that could predict R-PCT response (
      • Grange F.
      • Joly P.
      • Barbe C.
      • Bagot M.
      • Dalle S.
      • Ingen-Housz-Oro S.
      • et al.
      Improvement of survival in patients with primary cutaneous diffuse large B-cell lymphoma, leg type, in France.
      ,
      • Senff N.J.
      • Noordijk E.M.
      • Kim Y.H.
      • Bagot M.
      • Berti E.
      • Cerroni L.
      • et al.
      European Organization for Research and Treatment of Cancer and International Society for cutaneous Lymphoma consensus recommendations for the management of cutaneous B-cell lymphomas.
      ).
      Moreover, PCLBCL-LT has peculiar genetic features, with a dramatic prevalence of the MYD88L265P mutation in around 70% of cases (
      • Pham-Ledard A.
      • Cappellen D.
      • Martinez F.
      • Vergier B.
      • Beylot-Barry M.
      • Merlio J.P.
      MYD88 somatic mutation is a genetic feature of primary cutaneous diffuse large B-cell lymphoma, leg type.
      ,
      • Pham-Ledard A.
      • Prochazkova-Carlotti M.
      • Andrique L.
      • Cappellen D.
      • Vergier B.
      • Martinez F.
      • et al.
      Multiple genetic alterations in primary cutaneous large B-cell lymphoma, leg type support a common lymphomagenesis with activated B-cell-like diffuse large B-cell lymphoma.
      ). Mutations leading to the activation of the B-cell receptor (BCR) pathway and to NF-κB activation were first detected by targeted analyses (
      • Koens L.
      • Zoutman W.H.
      • Ngarmlertsirichai P.
      • Przybylski G.K.
      • Grabarczyk P.
      • Vermeer M.H.
      • et al.
      Nuclear factor-κB Pathway–Activating Gene Aberrancies in Primary cutaneous Large B-cell Lymphoma, Leg Type.
      ,
      • Pham-Ledard A.
      • Beylot-Barry M.
      • Barbe C.
      • Leduc M.
      • Petrella T.
      • Vergier B.
      • et al.
      High frequency and clinical prognostic value of MYD88 L265P mutation in primary cutaneous diffuse large B-cell lymphoma, leg-type.
      ). Using whole exome sequencing and a dedicated lymphopanel, a restricted set of highly recurrent mutations of MYD88, PIM1, CD79B, and MYC, as well as CDKN2A, PRDM1, and TNFAIP3 deletions, were found to be associated in 32 patients (
      • Mareschal S.
      • Pham-Ledard A.
      • Viailly P.J.
      • Dubois S.
      • Bertrand P.
      • Maingonnat C.
      • et al.
      Identification of somatic mutations in primary cutaneous diffuse large B-cell lymphoma, leg type by massive parallel sequencing.
      ). Indeed, the PCLBCL-LT mutational profile is closer to that of primary central nervous system lymphoma than to other DLBCL subtypes, a finding recently confirmed by another whole exome sequencing study of 19 cases of PCLBCL-LT (
      • Mareschal S.
      • Pham-Ledard A.
      • Viailly P.J.
      • Dubois S.
      • Bertrand P.
      • Maingonnat C.
      • et al.
      Identification of somatic mutations in primary cutaneous diffuse large B-cell lymphoma, leg type by massive parallel sequencing.
      ,
      • Zhou X.A.
      • Louissaint A.
      • Wenzel A.
      • Yang J.
      • Martinez-Escala M.E.
      • Moy A.P.
      • et al.
      Genomic analyses identify recurrent alterations in immune evasion genes in diffuse large B-cell lymphoma, leg type.
      ).
      This study aimed to evaluate if the mutational status of 36 genes involved in B-cell lymphomagenesis may help to differentiate among 32 patients with PCLBCL-LT, after R-PCT, those who experienced complete durable response (CR) and those who presented with relapsing-refractory (RR) disease. Our second objective was to compare tumor pairs at diagnosis and at relapse or progression in 14 patients.

      results

       Clinicopathological features

      Our cohort comprised 32 patients, mostly women (59%), with a median age at diagnosis of 81 years (range, 61–97), preferentially involving the legs (27 of 32, 84%) (Table 1). All PCLBCL-LT cases expressed BCL2, MUM1, and CD20, and were negative for CD10. Most cases were positive for BCL6 (72%) (Supplementary Table S1 online). This profile indicated an ABC subtype according to the modified Hans’ algorithm (
      • Menguy S.
      • Beylot-Barry M.
      • Marie P.
      • Pham Ledard A.
      • Frison E.
      • Comoz F.
      • et al.
      Primary cutaneous large B-cell lymphomas: relevance of the 2017 World Health Organization classification: clinicopathological and molecular analyses of 64 cases.
      ). After initial therapy by R-PCT, 14 patients (44%) reached CR at a median of 39 months (range, 9–117). Alternatively, 13 patients (41%) experienced relapse after initial complete response with a median of 22 months (range, 8–45) and were considered as relapsing patients, together with 5 patients (15%) refractory to treatment (RR, n = 18). The median follow-up was 38 months (range, 8–117). The median disease free period was 23 months (range, 0–117). Patients had a poor prognosis, with the 5-year overall survival rate at 38% (95% confidence interval, 18–58%). Half of the patients (n = 16) died during the follow-up period, 9 (56%) related to lymphoma and 7 (44%) unrelated to the disease.
      Table 1Main Findings at Diagnosis and Follow-Up Data According to CD79A/B Mutation and CD79A/B or CARD11 Mutation (BCR Mutation)
      CharacteristicsCD79A/BBCR mutationP-value
      Indicates the difference between patients exhibiting CD79A/B and CARD11 WT and those with a mutation in CD79A or CD79B or in CARD11 gene (BCR mutation).
      TotalWTMutationP-value
      Indicates the difference between patients exhibiting CD79A/B WT and those with a mutation in the CD79A/B gene.
      WTMutation
      Patients, n (%)32 (100)12 (38)20 (62)10 (31)22 (69)
      Age at diagnosis (y), median (range)81 (61-97)82 (61-97)81 (62-94)79.5 (61-97)82.5 (62-94)
      Female sex, n (%)19 (59)9 (75)10 (50)0.278 (80)11 (50)0.14
      Localization, n (%)
       Lower limb27 (84)10 (83)17 (85)0.998 (80)19 (86)0.64
       Other site5 (16)2 (17)3 (15)2 (20)3 (14)
      LDH
       Normal23 (72)10 (83)13 (65)0.429 (90)14 (64)0.21
       High9 (28)2 (16)7 (35)1 (10)8 (36)
      T stage, n (%)
       T19 (28)5 (42)4 (20)0.215 (50)4 (18)0.12
       T220 (63)7 (58)13 (65)5 (50)15 (68)
       T33 (9)0 (0)3 (15)0 (0)3 (14)
      Extracutaneous spreading, n (%)9 (28)2 (16)7 (35)0.701 (10)8 (36)0.27
      Type of treatment regimens, n (%)
       R-PCT with anthracycline22 (69)9 (75)13 (65)0.708 (80)14 (64)0.44
       R-PCT without anthracycline10 (31)3 (25)7 (35)2 (20)8 (36)
      Outcome, n (%)
       CR14 (44)9 (75)5 (25)0.019 (90)5 (23)0.0006
       RR18 (56)3 (25)15 (75)1 (10)17 (77)
       Relapse after CR13 (41)3 (25)10 (50)0.521 (10)12 (54)0.99
       Primary refractory5 (15)0 (0)5 (25)0 (0)5 (23)
      Status at last follow-up, n (%)
       Alive disease free12 (38)6 (50)6 (30)0.726 (60)6 (27)0.12
       Alive with disease4 (12)1 (8)3 (15)1 (10)3 (14)
       Died of lymphoma9 (28)2 (17)4 (20)0 (0)9 (41)
       Died unrelated7 (22)3 (25)7 (35)3 (30)4 (18)
      Specific survival rates, %
       3-year survival7480700.33
      P-values correspond to the comparison of survival curves.
      100630.03
      P-values correspond to the comparison of survival curves.
       5-year survival59805010045
      Overall survival rates, %
       3-year survival6562660.65
      P-values correspond to the comparison of survival curves.
      76590.21
      P-values correspond to the comparison of survival curves.
       5-year survival3842375133
      Progression-free survival rates, %
       3-year survival4282240.02
      P-values correspond to the comparison of survival curves.
      100220.002
      P-values correspond to the comparison of survival curves.
       5-year survival3261187516
      Abbreviations: BCR, B-cell receptor; CR, complete durable response; LDH, lactate dehydrogenase; R-PCT, rituximab and polychemotherapy; RR, relapsing-refractory; WT, wild-type.
      Boldface indicates statistical significance at P < 0.05.
      a Indicates the difference between patients exhibiting CD79A/B WT and those with a mutation in the CD79A/B gene.
      b Indicates the difference between patients exhibiting CD79A/B and CARD11 WT and those with a mutation in CD79A or CD79B or in CARD11 gene (BCR mutation).
      c P-values correspond to the comparison of survival curves.

       Lymphopanel sequencing at diagnosis

      Lymphopanel analysis was performed by next-generation sequencing of the 32 PCLBCL-LT primary samples. For eight patients, sequencing data of both tumoral and matched normal DNA at diagnosis were already available (
      • Mareschal S.
      • Pham-Ledard A.
      • Viailly P.J.
      • Dubois S.
      • Bertrand P.
      • Maingonnat C.
      • et al.
      Identification of somatic mutations in primary cutaneous diffuse large B-cell lymphoma, leg type by massive parallel sequencing.
      ). The median overall sequencing depth was 1614× (range, 813–2716). When frozen biopsy was available (21 of 32 samples), comparison of next-generation sequencing data from formalin-fixed, paraffin-embedded (FFPE) DNA and frozen DNA at diagnosis showed an increase in C to T transitions in FFPE samples related to formalin-induced changes (
      • Spencer D.H.
      • Sehn J.K.
      • Abel H.J.
      • Watson M.A.
      • Pfeifer J.D.
      • Duncavage E.J.
      Comparison of clinical targeted next-generation sequence data from formalin-fixed and fresh-frozen tissue specimens.
      ). Such artifactual variants had a low variant allele frequency (VAF) fewer than 5%.
      The median of mutations per patient was 6 (range, 0–16). The median VAF was 34% with a range of 6% to 85%, and the discrepancy of VAF in the same tumor sample also suggested the combination of clonal and subclonal mutations (Supplementary Table S2 online). As expected, we observed a high prevalence of mutations in PIM1 (69%), MYD88 (69%), and IRF4 (16%) leading to NF-κB pathway activation, and in CD79B (56%) leading to BCR pathway activation (Figure 1). Most MYD88 mutations corresponded to the mutational hotspot p.L265P except for two patients with p.V217F and p.S219C mutations, previously reported in nodal DLBCL (
      • Dubois S.
      • Viailly P.J.
      • Mareschal S.
      • Bohers E.
      • Bertrand P.
      • Ruminy P.
      • et al.
      Next-generation sequencing in diffuse large B-cell lymphoma highlights molecular divergence and therapeutic opportunities: a LYSA study.
      ,
      • Ngo V.N.
      • Young R.M.
      • Schmitz R.
      • Jhavar S.
      • Xiao W.
      • Lim K.H.
      • et al.
      Oncogenically active MYD88 mutations in human lymphoma.
      ). PIM1, IRF4, GNA13, CD58, ARID1A, and MYC frequently displayed more than one somatic mutation per patient, and we investigated if aberrant somatic hypermutation involved the following genes by searching single nucleotide variations at the activation-induced cytidine deaminase motif (DGYW/WRCH) (
      • Rogozin I.B.
      • Pavlov Y.I.
      The cytidine deaminase AID exhibits similar functional properties in yeast and mammals.
      ): IRF4, XPO1, MYC, PIM1, BCL2, CARD11, CD58, MYD88, CD79B, ARID1A, and GNA13. Indeed, 33% to 100% of observed single nucleotide variations appeared as a fingerprint of activation-induced cytidine deaminase activity for IRF4, XPO1, MYC, PIM1, BCL2, CARD11, and CD58 genes, but not for MYD88, CD79B, GNA13, and ARID1A (Supplementary Figure S1).
      Figure thumbnail gr1
      Figure 1Overview of the somatic mutations and CNV alterations detected by the lymphopanel. (a) Each line represents one patient and each column represents one gene organized by symbols:
      ❶ missense; in the middle, the number of mutations observed is specified
      ⧫ nonframeshift indel
      ✖ stop-gain comprising frameshift and nonsense mutations
      ★ splicing
      Shading indicates CNV observed for each gene: red shading indicates deletions, homozygous (dark) or heterozygous (light), blue shading indicates gains. (b) Graph represents proportion of affected patients for each gene by mutations (black) or CNV (gray). CNV, copy number variation; CRx, durable complete responder patients; indel, insertion–deletion; RRx, relapsing/refractory patients.
      CDKN2A deletion was the most frequent copy number variation observed (21 of 32 patients), with 11 homozygous and 10 heterozygous deletions (Figure 1). CDKN2B was also deleted in nine cases (28%), with five heterozygous and four homozygous deletions. Other deletions involved PRDM1 (28%), TNFAIP3 (19%), and TP53 (13%). Interestingly, one heterozygous deletion of either PRDM1 or TNFAIP3 was associated with a stop-gain mutation on the second copy, leading to complete inactivation of the gene in each patient (n = 2).
      Copy gains detected for BCL2 (19%) and MYC (6%) were confirmed by interphase fluorescence in situ hybridization. Copy number variation analysis also indicated the presence of an amplification of MYC (> 25 copies) and BCL2 (> 6 copies), in an RR patient (RR16).

       RR patients to R-PCT displayed mutations targeting BCR pathway

      Mutations of CD79A/B encoding for BCR subunits were frequent (n = 20, 62%). Two RR patients had a splice mutation in the CD79A ITAM region, consisting of a deletion across the intron 4/exon 5 that removes a part of the ITAM domain, including the first tyrosine. CD79B mutations detected in 18 patients exclusively involved the ITAM domain and targeted the first tyrosine residue (Y196), except in one patient with an E197D mutation (Supplementary Figure S2). CARD11 mutations on the coiled-coil domain detected in two patients were mutually exclusive with those of CD79A/B (Supplementary Figure S2).
      At presentation, the three patients at T3 stage displayed a mutation involving the BCR pathway (Table 1).
      A reduced progression-free survival (PFS) was observed in patients with a CD79B mutation (median, 18 months vs. 45 months in wild-type group, log-rank, P = 0.05) (Figure 2a). A mutation among CD79A or CD79B genes was also associated with a reduced PFS (median, 22 months vs. not reached in the wild-type group, log-rank, P = 0.02) (Figure 2b) and a lower proportion of response to R-PCT (75% vs. 25%, P = 0.01) (Table 1). Altogether, patients with a mutation of either CD79A/B or CARD11 encoding for members of the BCR signaling pathway displayed a reduced PFS and specific survival (median 18 months, log-rank, P = 0.002, and median 51 months, log-rank, P = 0.03 respectively; median durations were not reached in the wild-type group) (Figure 3) but not a reduced overall survival (median 48 months vs. not reached in the wild-type group, log-rank, P = 0.21) (Supplementary Figure S3 online). Alternatively, a wild-type status of these genes was associated with R-PCT response (90% vs. 23%, P = 0.0006) (Table 1). There was no statistically significant difference of baseline treatment regimen (R-PCT with or without anthracycline) according to therapeutic response and BCR mutational status (P = 1.00 and P = 0.44, respectively) (Table 1). The MYD88 mutated status had no adverse impact on PFS or overall survival, but might be associated with a better specific survival (P = 0.05) (Supplementary Figure S4 online). In the different MYD88wt/MYD88mt/BCRwt/BCRmt status combinations, a negative impact on specific survival, overall survival, and PFS was only observed for patients with BCRmt status regardless of MYD88 status, although the small size of each subgroup did not permit it to reach statistical significance.
      Figure thumbnail gr2
      Figure 2Kaplan–Meier survival curves for PFS in 32 patients with PCLBCL-LT according to the mutational status of CD79B or CD79A or B. (a) A CD79B mutation was detected in 18 of 32 patients with statistically significant difference for PFS (P = 0.05). (b) A CD79A/B mutation was present in 20 of 32 patients, with statistically significant difference for PFS (P = 0.02). Group 0 corresponds to WT patients; Group 1 corresponds to MT patients. MT, mutated; PCLBCL-LT, primary cutaneous diffuse large B-cell lymphoma, leg type; PFS, progression-free survival; WT, wild-type.
      Figure thumbnail gr3
      Figure 3Kaplan–Meier survival curves of 32 patients with PCLBCL-LT according to the mutational status of the three genes implicated in the BCR signaling pathway (CARD11 and CD79A/B). (a) PFS and (b) specific survival. A BCR mutation was present in 22 of 32 patients, with statistically significant difference for PFS (P = 0.002) and specific survival (P = 0.03). Group 0 corresponds to WT patients; Group 1 corresponds to MT patients. BCR, B-cell receptor; MT, mutated; PCLBCL-LT, primary cutaneous diffuse large B-cell lymphoma, leg type; PFS, progression-free survival; WT, wild-type.

       Clonal selection and heterogeneity between primary and secondary samples

      In 14 patients with RR PCLBCL-LT, the analysis was conducted at diagnosis and at relapse or progression. Corrected VAF of each mutation was adapted to the estimated fraction of tumoral cells (Figure 4).
      Figure thumbnail gr4
      Figure 4Variations of corrected variant allelic frequencies of mutations in 14 tumors pairs from the same patient at diagnosis (P) and at relapse or progression (R). The arrow indicates the time between the two biopsies in the same patient.
      In three patients (RR5, RR6, and RR15), no major variation in corrected VAF was observed between the biopsy pairs. VAF enrichment of primary mutations affecting CD79B (patients RR11, RR17, and RR18), CARD11 (patient RR2), and MYD88 (patients RR11 and RR17) was observed at relapse or progression, supporting a pivotal role in disease oncogenesis. Similarly, several mutants of SOCS1, MYC, and MYD88 had a very low VAF or even wild-type status at diagnosis and were enriched at relapse or progression. For example, an increase (from 1% to 58%) of the VAF of a SOCS1 mutation was observed at relapse in patient RR18. A similar increase from 1% to 48% of MYD88P258L mutant VAF was observed in patient RR9, who also gained at progression two MYC mutations at a VAF around 50%.
      Alternatively, a loss of mutation at relapse was observed for the following genes: B2M (RR3), IRF4 (RR7 and RR9), CREBBP (RR10), CD79B (RR10), CARD11 (RR13), and MEF2B (RR12) (Figure 4). To decipher the differences between diagnosis and relapse in patient RR13, DNA from three tumor-rich macrodissected areas of the primary biopsy and from two areas of the relapse biopsy was subjected to resequencing. The CARD11 mutation was detected at a VAF under 1% and equal to 9% in two areas of the primary biopsy and was absent in the third area of the primary biopsy, as opposed to 10% in global tissue. Other mutations of this case were homogeneously detected (Table 2). No CARD11 mutation was detected at relapse. Our data support both spatial intraindividual heterogeneity and temporal selection of subclones under treatment.
      Table 2Intratumor Heterogeneity in Primary and Relapse Biopsy
      Zone of extractionARID1ANOTCH2CCND3TNFAIP3CARD11NOTCH1
      PrimaryGlobal Tissuec.6100G>A ; p.(Glu2034Lys) ; 40c.7198C>T ; p.(Arg2400*) ; 71c.778C>T; p.(Gln260*) ; 40c.1880_1881del ; p.(Cys627Phefs*44) ; 65c.1071C>G ; p.(Asp357Glu) ; 10c.7538C>T ; p.(Ser2513Phe) ; 42
      Area 1c.6100G>A ; p.(Glu2034Lys) ; 43c.7198C>T ; p.(Arg2400*) ; 76c.778C>T ; p.(Gln260*) ; 42c.1880_1881del ; p.(Cys627Phefs*44) ; 63c.1071C>G ; p.(Asp357Glu) ; <1c.7538C>T ; p.(Ser2513Phe) ; 40
      Area 2c.6100G>A ; p.(Glu2034Lys) ; 41c.7198C>T ; p.(Arg2400*) ; 71c.778C>T ; p.(Gln260*) ; 35c.1880_1881del ; p.(Cys627Phefs*44) ; 71NA; 0 (0/2404 reads)c.7538C>T ; p.(Ser2513Phe) ; 38
      Area 3c.6100G>A ; p.(Glu2034Lys) ; 32c.7198C>T ; p.(Arg2400*) ; 66c.778C>T ; p.(Gln260*) ; 30c.1880_1881del ; p.(Cys627Phefs*44) ; 46c.1071C>G ; p.(Asp357Glu) ; 9c.7538C>T ; p.(Ser2513Phe) ; 34
      RelapseGlobal Tissuec.6100G>A ; p.(Glu2034Lys) ; 35c.7198C>T ; p.(Arg2400*) ; 55c.778C>T; p. .(Gln260*) ; 30c.1880_1881del ; p.(Cys627Phefs*44) ; 57NA; 0 (0/1786 reads)c.7538C>T ; p.(Ser2513Phe) ; 29
      Area 1c.6100G>A ; p.(Glu2034Lys) ; 33c.7198C>T ; p.(Arg2400*) ; 58c.778C>T ; p.(Gln260*) ; 34c.1880_1881del ; p.(Cys627Phefs*44) ; 43NA; 0 (0/2145 reads)c.7538C>T ; p.(Ser2513Phe) ; 35
      Area 2c.6100G>A ; p.(Glu2034Lys) ; 44c.7198C>T ; p.(Arg2400*) ; 72c.778C>T ; p.(Gln260*) ; 46c.1880_1881del ; p.(Cys627Phefs*44) ; 70NA; 0 (0/2567 reads)c.7538C>T ; p.(Ser2513Phe) ; 44
      Abbreviations: del, deletion; fs, frameshift; NA, Not applicable; ∗, stop codon.
      Resequencing of several areas from the biopsy of the patient RR13 at the diagnosis and at the relapse. The first line corresponds to the mutations observed from the global tissue. The other lines correspond to the mutation from a distinct zone of the sample. In each column gene, the mutation and the variant allele frequency (%) are specified. At diagnosis, the CARD11 mutation was detected in area 1 and 3 (low level), but was absent in area 2. At relapse, no CARD11 mutation was detected in any area.
      Finally, many patients displayed losses and gained mutations in the PIM1 gene, suggesting the presence of subclones carrying different PIM1 mutations (patients RR3, RR9, RR10, and RR18). The bioinformatics analysis also revealed that somatic hypermutation load was higher at relapse than at diagnosis, supporting an ongoing aberrant hypermutation process (Supplementary Figure S5 online).

      Discussion

      So far, the mutational profile of PCLBCL-LT had not been evaluated according to outcome or therapeutic response (
      • Mareschal S.
      • Pham-Ledard A.
      • Viailly P.J.
      • Dubois S.
      • Bertrand P.
      • Maingonnat C.
      • et al.
      Identification of somatic mutations in primary cutaneous diffuse large B-cell lymphoma, leg type by massive parallel sequencing.
      ,
      • Zhou X.A.
      • Louissaint A.
      • Wenzel A.
      • Yang J.
      • Martinez-Escala M.E.
      • Moy A.P.
      • et al.
      Genomic analyses identify recurrent alterations in immune evasion genes in diffuse large B-cell lymphoma, leg type.
      ). The comparison between 14 patients with CR to R-PCT and 18 patients with RR disease highlights the central role of mutations involving the BCR pathway that are enriched in patients with poor PFS and therapeutic resistance.
      In the RR group, a single case (RR16) harbored an amplification of MYC and BCL2 polysomy, which may account for therapeutic resistance (
      • Mareschal S.
      • Pham-Ledard A.
      • Viailly P.J.
      • Dubois S.
      • Bertrand P.
      • Maingonnat C.
      • et al.
      Identification of somatic mutations in primary cutaneous diffuse large B-cell lymphoma, leg type by massive parallel sequencing.
      ). Indeed, MYC and BCL2 coexpression, called dual expressor phenotype, identifies a subgroup of patients with PCLBCL-LT and adverse prognosis (
      • Menguy S.
      • Frison E.
      • Prochazkova-Carlotti M.
      • Dalle S.
      • Dereure O.
      • Boulinguez S.
      • et al.
      Double-hit or dual expression of MYC and BCL2 in primary cutaneous large B-cell lymphomas.
      ).
      Unlike our previous report on 58 PCLBCL-LT cases (
      • Pham-Ledard A.
      • Prochazkova-Carlotti M.
      • Andrique L.
      • Cappellen D.
      • Vergier B.
      • Martinez F.
      • et al.
      Multiple genetic alterations in primary cutaneous large B-cell lymphoma, leg type support a common lymphomagenesis with activated B-cell-like diffuse large B-cell lymphoma.
      ), no adverse prognostic value of the MYD88L265P mutation was found. Here, only patients treated by R-PCT were included, and diagnostic criteria for PCLBCL-LT have been improved (
      • Menguy S.
      • Gros A.
      • Pham-Ledard A.
      • Battistella M.
      • Ortonne N.
      • Comoz F.
      • et al.
      MYD88 somatic mutation is a diagnostic criterion in primary cutaneous large B-cell lymphoma.
      ,
      • Menguy S.
      • Frison E.
      • Prochazkova-Carlotti M.
      • Dalle S.
      • Dereure O.
      • Boulinguez S.
      • et al.
      Double-hit or dual expression of MYC and BCL2 in primary cutaneous large B-cell lymphomas.
      ,
      • Menguy S.
      • Beylot-Barry M.
      • Marie P.
      • Pham Ledard A.
      • Frison E.
      • Comoz F.
      • et al.
      Primary cutaneous large B-cell lymphomas: relevance of the 2017 World Health Organization classification: clinicopathological and molecular analyses of 64 cases.
      ).
      Alternatively, mutations involving the BCR pathway were found to be associated with disease aggressiveness. The BCR is composed of a membrane-anchored surface immunoglobulin with an antigen-binding site on the variable regions and heterodimeric subunits, CD79A and B, which transduces signals to the cell (
      • Davis R.E.
      • Ngo V.N.
      • Lenz G.
      • Tolar P.
      • Young R.M.
      • Romesser P.B.
      • et al.
      Chronic active B-cell-receptor signalling in diffuse large B-cell lymphoma.
      ). Antigenic stimulation of BCR signaling engages CARD11, a scaffolding protein that is part of the CBM multiprotein complex (CARD11/BCL10/MALT1), initiating NF-κB signaling (
      • Davis R.E.
      • Ngo V.N.
      • Lenz G.
      • Tolar P.
      • Young R.M.
      • Romesser P.B.
      • et al.
      Chronic active B-cell-receptor signalling in diffuse large B-cell lymphoma.
      ,
      • Ngo V.N.
      • Davis R.E.
      • Lamy L.
      • Yu X.
      • Zhao H.
      • Lenz G.
      • et al.
      A loss-of-function RNA interference screen for molecular targets in cancer.
      ). Cell survival of ABC DLBCL depends on “chronic active” BCR signaling (
      • Davis R.E.
      • Ngo V.N.
      • Lenz G.
      • Tolar P.
      • Young R.M.
      • Romesser P.B.
      • et al.
      Chronic active B-cell-receptor signalling in diffuse large B-cell lymphoma.
      ). CD79A/B mutations (20% of ABC DLBCL cases) increase “chronic BCR signaling” by enhancing surface BCR expression and attenuating LYN kinase, a negative regulator of BCR activation (
      • Davis R.E.
      • Ngo V.N.
      • Lenz G.
      • Tolar P.
      • Young R.M.
      • Romesser P.B.
      • et al.
      Chronic active B-cell-receptor signalling in diffuse large B-cell lymphoma.
      ,
      • Heizmann B.
      • Reth M.
      • Infantino S.
      Syk is a dual-specificity kinase that self-regulates the signal output from the B-cell antigen receptor.
      ). Chronic BCR signaling is also dependent on CARD11 mutations in 10% of ABC DLBCL cases, leading to activation of the antiapoptotic NF-κB pathway (
      • Davis R.E.
      • Ngo V.N.
      • Lenz G.
      • Tolar P.
      • Young R.M.
      • Romesser P.B.
      • et al.
      Chronic active B-cell-receptor signalling in diffuse large B-cell lymphoma.
      ,
      • Lenz G.
      • Davis R.E.
      • Ngo V.N.
      • Lam L.
      • George T.C.
      • Wright G.W.
      • et al.
      Oncogenic CARD11 mutations in human diffuse large B cell lymphoma.
      ). Addiction of ABC tumor cells to Bruton tyrosine kinase signaling was shown by genetic interference for CD79A/B, CARD11, or MYD88L265P mutations (
      • Davis R.E.
      • Ngo V.N.
      • Lenz G.
      • Tolar P.
      • Young R.M.
      • Romesser P.B.
      • et al.
      Chronic active B-cell-receptor signalling in diffuse large B-cell lymphoma.
      ,
      • Ngo V.N.
      • Davis R.E.
      • Lamy L.
      • Yu X.
      • Zhao H.
      • Lenz G.
      • et al.
      A loss-of-function RNA interference screen for molecular targets in cancer.
      ). Distinct subgroups of DLBCL have been defined by the cell of origin or differentiation stage from which they develop that also matches with initiating or acquired genetic lesions (
      • Schmitz R.
      • Wright G.W.
      • Huang D.W.
      • Johnson C.A.
      • Phelan J.D.
      • Wang J.Q.
      • et al.
      Genetics and pathogenesis of diffuse large B-cell lymphoma.
      ). Among them, the genetic subtype that corresponds to cases with both MYD88L265P and CD79B mutations was observed predominantly in the ABC subgroup of DLBCL with frequent extranodal secondary involvement or primary onset such as primary central nervous system lymphoma (
      • Schmitz R.
      • Wright G.W.
      • Huang D.W.
      • Johnson C.A.
      • Phelan J.D.
      • Wang J.Q.
      • et al.
      Genetics and pathogenesis of diffuse large B-cell lymphoma.
      ). This genetic subtype was associated with shorter PFS and overall survival than the other subgroups of DLBCL, but PCLBCL-LT was not evaluated (
      • Schmitz R.
      • Wright G.W.
      • Huang D.W.
      • Johnson C.A.
      • Phelan J.D.
      • Wang J.Q.
      • et al.
      Genetics and pathogenesis of diffuse large B-cell lymphoma.
      ). The genotype was present in 15 out of our 32 PCLBCL-LT cases (47%) and was observed both in the CR (n = 5, 36%) and the RR (n = 10, 56%) subgroups without prognostic impact. In fact, therapeutic resistance was associated with mutations targeting one of the BCR signaling genes (CD79A/B or CARD11).
      Ibrutinib is a selective inhibitor of Bruton tyrosine kinase that kills ABC DLBCL lines by reducing NF-κB pathway activity (
      • Davis R.E.
      • Ngo V.N.
      • Lenz G.
      • Tolar P.
      • Young R.M.
      • Romesser P.B.
      • et al.
      Chronic active B-cell-receptor signalling in diffuse large B-cell lymphoma.
      ,
      • Yang Y.
      • Shaffer A.L.
      • Emre N.C.T.
      • Ceribelli M.
      • Zhang M.
      • Wright G.
      • et al.
      Exploiting synthetic lethality for the therapy of ABC diffuse large B cell lymphoma.
      ). Interestingly, frequent responses were observed in ABC DLBCL cases with combined CD79A/B and MYD88L265P mutations (
      • Wilson W.H.
      • Young R.M.
      • Schmitz R.
      • Yang Y.
      • Pittaluga S.
      • Wright G.
      • et al.
      Targeting B cell receptor signaling with ibrutinib in diffuse large B cell lymphoma.
      ). Partial response to ibrutinib in PCLBCL-LT has also been reported in some RR PCLBCL-LT (
      • Fox L.C.
      • Yannakou C.K.
      • Ryland G.
      • Lade S.
      • Dickinson M.
      • Campbell B.A.
      • et al.
      Molecular mechanisms of disease progression in primary cutaneous diffuse large B-cell lymphoma, leg type during ibrutinib therapy.
      ,
      • Gupta E.
      • Accurso J.
      • Sluzevich J.
      • Menke D.M.
      • Tun H.W.
      Excellent outcome of immunomodulation or Bruton’s tyrosine kinase inhibition in highly refractory primary cutaneous diffuse large B-cell lymphoma, leg type.
      ). In 18 patients with primary central nervous system lymphoma, a phase Ib study of ibrutinib monotherapy followed by ibrutinib plus chemotherapy provided a good response rate in patients with CD79B and/or MYD88 mutations, but not those with CARD11 mutations, as also observed in nodal DLBCL (
      • Lionakis M.S.
      • Dunleavy K.
      • Roschewski M.
      • Widemann B.C.
      • Butman J.A.
      • Schmitz R.
      • et al.
      Inhibition of B cell receptor signaling by ibrutinib in primary CNS lymphoma.
      ,
      • Wilson W.H.
      • Young R.M.
      • Schmitz R.
      • Yang Y.
      • Pittaluga S.
      • Wright G.
      • et al.
      Targeting B cell receptor signaling with ibrutinib in diffuse large B cell lymphoma.
      ).
      Moreover, the assessment of tumor pairs at diagnosis and at relapse or progression in 14 patients showed subclonal variations. One half of the patients retained the same mutational profile at relapse, whereas the other half harbored genetic changes in the relapse beside common genetic driver mutations at high VAF of MYD88 and CD79B (
      • Mareschal S.
      • Pham-Ledard A.
      • Viailly P.J.
      • Dubois S.
      • Bertrand P.
      • Maingonnat C.
      • et al.
      Identification of somatic mutations in primary cutaneous diffuse large B-cell lymphoma, leg type by massive parallel sequencing.
      ). The study of primer pairs also confirms sustained activation-induced cytidine deaminase activity preferentially targeting the PIM1 gene (
      • Mareschal S.
      • Pham-Ledard A.
      • Viailly P.J.
      • Dubois S.
      • Bertrand P.
      • Maingonnat C.
      • et al.
      Identification of somatic mutations in primary cutaneous diffuse large B-cell lymphoma, leg type by massive parallel sequencing.
      ). The ongoing emergence of PIM1 mutations in PCLBCL-LT could also contribute to ibrutinib resistance observed in the PIM1-mutated ABC subtype of DLBCL (
      • Kuo H.P.
      • Ezell S.A.
      • Hsieh S.
      • Schweighofer K.J.
      • Cheung L.W.
      • Wu S.
      • et al.
      The role of PIM1 in the ibrutinib-resistant ABC subtype of diffuse large B-cell lymphoma.
      ).
      This study indicates that the mutational profile of PCLBCL-LT could help to predict therapeutic response to first-line R-PCT with resistance in patients bearing mutations of the BCR pathway. In such patients, ibrutinib could be proposed either in association with R-PCT or as a second-line therapy in patients without CARD11 or PIM1 mutations. Alternative therapies using lenalidomide, a modulator of NF-κB and IFN-β signaling, demonstrated effectiveness in some patients with RR PCLBCL-LT (
      • Beylot-Barry M.
      • Mermin D.
      • Maillard A.
      • Bouabdallah R.
      • Bonnet N.
      • Duval-Modeste A.B.
      • et al.
      A single-arm Phase II trial of lenalidomide in relapsing or refractory primary cutaneous large B-cell lymphoma, leg type.
      ). The toxicity and adverse effects of either ibrutinib or lenalidomide in this elderly population underscore the need for further deciphering mechanisms of therapeutic response.

      Materials and Methods

       Inclusion criteria and number of subjects

      All patients with PCLBCL-LT were treated by R-PCT as first-line therapy and included between 01 January, 2004 and 07 January, 2017 in the database of the French Study group on cutaneous lymphomas. All patients have been included in previous observational retrospective studies with informed consent and/or included in a phase II clinical trial (NCT01556035, REVLEG) that evaluated lenalidomide in second line after R-PCT (
      • Beylot-Barry M.
      • Mermin D.
      • Maillard A.
      • Bouabdallah R.
      • Bonnet N.
      • Duval-Modeste A.B.
      • et al.
      A single-arm Phase II trial of lenalidomide in relapsing or refractory primary cutaneous large B-cell lymphoma, leg type.
      ). Clinical data were available or updated.
      At diagnosis, FFPE skin biopsy was available in 32 patients, with 21 matched tumor frozen material. For 14 out of the 18 RR patients after R-PCT therapy, we also had an FFPE skin biopsy at relapse before the second-line treatment. According to the French public Health and Bioethical Law, no ethics committee approval was needed for this observational study on already collected data and biological material (Article L1121-1 and Article R1121-3). Moreover, informed written consent was given by patients for use of their data and biological material for research purposes.

       Response to treatment

      Patients were followed during six R-PCT perfusions with an intermediate evaluation after three cures. Then, there was at least a half-yearly follow-up assessing potential relapse (
      • Cheson B.D.
      • Fisher R.I.
      • Barrington S.F.
      • Cavalli F.
      • Schwartz L.H.
      • Zucca E.
      • et al.
      Recommendations for initial evaluation, staging, and response assessment of Hodgkin and non-Hodgkin lymphoma: the Lugano classification.
      ). Patients were classified as follows:
      • 1.
        CR was defined by a complete durable response lasting at least 6 months—disappearance of all lesions by clinical and radiological evaluation and without relapse during the study.
      • 2.
        RR patients were either refractory patients with no CR under R-PCT (partial response, stability, or progression) or relapsing patients with apparition of new lesions after an initial complete response.

       Immunohistochemistry

      FFPE slides were subjected to automated immunolabeling (BOND-MAX, Leica, Wetzlar, Germany) with the following antibodies; BCL2 (clone 124, Dako, Carpinteria, CA), MUM1 (clone MUM1p, Dako), CD10 (clone 56C6, Leica), BCL6 (clone PGB6P, Dako), CD21 (clone 2G9, Leica), Ki67 (clone MM1, Leica), and CD20 (clone L26, Dako). The positivity cut-offs for BCL2, MUM1, or CD10 were ≥ 50%, ≥ 30%, and ≥ 30% of cells, respectively (
      • Menguy S.
      • Beylot-Barry M.
      • Marie P.
      • Pham Ledard A.
      • Frison E.
      • Comoz F.
      • et al.
      Primary cutaneous large B-cell lymphomas: relevance of the 2017 World Health Organization classification: clinicopathological and molecular analyses of 64 cases.
      ).

       Targeting next-generation sequencing using a lymphopanel comprising 36 genes

      DNA was extracted from FFPE and frozen tissues. A lymphopanel was designed to identify alterations within 36 genes important for lymphomagenesis, based on literature data and whole exome sequencing of RR DLBCL sequencing. A previous version has been used to characterize a PCLBCL-LT cohort (
      • Dubois S.
      • Viailly P.J.
      • Mareschal S.
      • Bohers E.
      • Bertrand P.
      • Ruminy P.
      • et al.
      Next-generation sequencing in diffuse large B-cell lymphoma highlights molecular divergence and therapeutic opportunities: a LYSA study.
      ,
      • Mareschal S.
      • Pham-Ledard A.
      • Viailly P.J.
      • Dubois S.
      • Bertrand P.
      • Maingonnat C.
      • et al.
      Identification of somatic mutations in primary cutaneous diffuse large B-cell lymphoma, leg type by massive parallel sequencing.
      ). The AmpliSeq panel (Thermo Fisher Scientific, Waltham, MA) covers 75.08 kilobases and was sequenced on Ion S5 (Thermo Fisher Scientific). Data analysis was performed using Torrent Suite version 5.6 software (Thermo Fisher Scientific). Reads were mapped to the human hg19 reference genome. The Variant Caller detected single nucleotide variation with VAF ≥ 2% and insertion–deletion with VAF ≥ 5%. VCF files were annotated by ANNOVAR (
      • Wang K.
      • Li M.
      • Hakonarson H.H.
      ANNOVAR: Functional annotation of genetic variants from high-throughput sequencing data.
      ). BAM sequences were also checked using Alamut Software (Interactive Biosoftware, Rouen, France). Copy number variation was screened using the ONCOCNV algorithm (
      • Boeva V.
      • Popova T.
      • Lienard M.
      • Toffoli S.
      • Kamal M.
      • Le Tourneau C.
      • et al.
      Multi-factor data normalization enables the detection of copy number aberrations in amplicon sequencing data.
      ) compared with seven control DNA patients.

       Interphase fluorescence in situ hybridization

      To confirm copy number variation detected by sequencing analysis, hybridization was performed to evaluate BCL2, MYC, and CDKN2A copy number (
      • Menguy S.
      • Prochazkova-Carlotti M.
      • Beylot-Barry M.
      • Saltel F.
      • Vergier B.
      • Merlio J.-P.
      • et al.
      PD-L1 and PD-L2 are differentially expressed by macrophages or tumor cells in primary cutaneous diffuse large B-cell lymphoma, leg type.
      ,
      • Pham-Ledard A.
      • Beylot-Barry M.
      • Barbe C.
      • Leduc M.
      • Petrella T.
      • Vergier B.
      • et al.
      High frequency and clinical prognostic value of MYD88 L265P mutation in primary cutaneous diffuse large B-cell lymphoma, leg-type.
      ). Slides were analyzed by two independent observers. Gain was defined by the presence of more than four copies, amplification by more than six copies, and a ratio to a control locus above two, and losses by less than two copies.

       Statistical analysis

      Comparison of clinical characteristics and response according to mutational status was performed using Fisher’s exact test. Patients without an event of interest were censored at their last study visit, with administrative end of follow-up in July 2018. Disease-specific survival was calculated from diagnosis to disease-related death, considering patients whose death was unrelated to lymphoma as censored. Overall survival duration was calculated from diagnosis to death from any cause and PFS from the diagnosis to recurrence or disease-related death, whichever came first. Kaplan–Meier survival curves were displayed. Statistical analyses were performed using Medcalc software (Ostend, Belgium) with a bilateral significance threshold of 0.05.

       Data availability statement

      Sequencing data were deposited in SRA database under NCBI accession PRJNA532525.

      Conflict of Interest

      Marie Beylot-Barry received research funding from Celgene; is an advisory board member for Roche and Takeda; and is a principal investigator for Celgene, Galderma, Kyowa Hakko, Millenium, Roche, Biocryst, and ArgenX. Fabrice Jardin received personal fees and travel accommodations from Janssen, Celgene, Roche, Amgen, and Gilead, and research funding from Roche and Celgene. All other authors state no conflict of interest.

      Acknowledgments

      The project was supported by the Société Française de Dermatologie with funding to Océane Ducharme and Ligue Contre le Cancer , Comité de Dordogne . We thank Elodie Laharanne for fluorescence in situ hybridization experiments, Martina Prochazkova-Carlotti for statistical analysis, and the cancer biobank of University Hospital of Bordeaux. The following members of the French Study Group of Cutaneous Lymphoma provided clinical data or pathological material: Laurent Mortier and Romain Dubois (CHU Lille, Lille, France), Stephane Dalle and Brigitte Balme (CHU Lyon, Lyon, France), Anne Benedicte Duval Deste and Philippe Courville (CHU Rouen, Rouen, France), Eve Maubec and Lydia Deschamps (CHU Bichat, Paris, France), and Saskia Oro and Nicolas Ortonne (CHU Henri-Mondor, Créteil, France).

      Author Contributions

      Conceptualization: OD, MBB, APL, FJ, JPM, AG; Data Curation: PJV, TB; Formal Analysis: OD, AG, EF; Funding Acquisition: OD, AG; Investigation: MBB, APL, OD, JPM, BV, NF, EB, AG; Writing - Original Draft Preparation: OD, MBB, JPM, AG; Writing - Review and Editing: OD, MBB, JPM, AG.

      Supplementary Material

      Figure thumbnail fx1
      Supplementary Figure S1Aberrant somatic hypermutation analysis. Percentage of total SNV (missense, nonsense, and splicing mutations) per genes targeted by aSHM, located at DGYW/WRCH AID target sites (
      • Rogozin I.B.
      • Pavlov Y.I.
      The cytidine deaminase AID exhibits similar functional properties in yeast and mammals.
      ), at diagnosis. AID, activation-induced cytidine deaminase; aSHM, aberrant somatic hypermutation; SNV, single nucleotide variation.
      Figure thumbnail fx2
      Supplementary Figure S2Schematics of genes annotated with oncogenic mutations found in PCLBCL-LT at diagnosis. PCLBCL-LT, primary cutaneous diffuse large B-cell lymphoma, leg type.
      Figure thumbnail fx3
      Supplementary Figure S3Overall survival for 32 patients with PCLBCL-LT according to the mutational status of the three genes implicated in the BCR signaling pathway (CARD11 and CD79A/B). Mutations involving the BCR were present in 22 patients and not detected in 10 patients. The difference was not statistically reached for OS (log-rank, P = 0.2125). BCR, B-cell receptor; MT, mutated; OS, overall survival; PCLBCL-LT, primary cutaneous diffuse large B-cell lymphoma, leg type; WT, wild-type.
      Figure thumbnail fx4
      Supplementary Figure S4Specific survival for 32 patients with PCLBCL-LT according to the mutational status of MYD88. Mutations involving MYD88 were present in 22 patients. The difference was not statistically reached for SS (log-rank, P = 0.0542). MT, mutated; PCLBCL-LT, primary cutaneous diffuse large B-cell lymphoma, leg type; SS, specific survival; WT, wild-type.
      Figure thumbnail fx5
      Supplementary Figure S5Distribution of mutations for each gene between primary and relapse or progression tumors for the 14 RR patients analyzed. Shading indicates different type of alterations observed for each gene: red shading indicates INDEL, blue shading indicates SNV not related to aSHM and green shading indicates SNV related to aSHM. aSHM, aberrant somatic hypermutation; INDEL, insertion–deletion; P, primary; R, relapse or progression; RR, relapsed-refractory; SNV, single nucleotide variation.
      Supplementary Table S1Summary of Immunophenotypical Features of 32 Patients with PCLBCL-LT Sequenced
      PatientBCL2MUM1BCL6CD10CD20CD21Ki67
      CR1111010100
      CR211101090
      CR311101080
      CR411101070
      CR511001060
      CR6111010100
      CR711Heterogeneous01090
      CR811101080
      CR911101080
      CR1011101080
      CR1111101080
      CR1211101090
      CR1311001090
      CR1411001090
      RR111101070
      RR211001ND90
      RR3111010100
      RR4111010ND
      RR5111010ND
      RR611101080
      RR711101090
      RR811101095
      RR911101080
      RR1011101080
      RR1111101NDND
      RR1211001ND100
      RR1311101090
      RR1411001080
      RR1511001080
      RR16110010ND
      RR1711101ND100
      RR1811101NDND
      Abbreviations: CR, complete durable response; ND, not down; PCLBCL-LT, primary cutaneous diffuse large B-cell lymphoma, leg type; RR, relapsed-refractory.
      1 = positive; 0 = negative. For Ki67, the percentage of positive cells is noted.
      Supplementary Table S2Mutations Observed at Diagnosis in the Lymphopanel for Each Patient
      Patient nameGeneNM_ ReferenceExonMutationVAF
      Only SNV and INDEL mutations with a VAF > 2% and 5% respectively are reported.
      Type
      CR1MYD88NM_0024685c.794T>C ; p.L265P25missense
      FOXO1NM_0020151c.203C>T ; p.A68V20missense
      IRF4NM_0024602c.54C>G ; p.S18R26missense
      GNA13NM_0065721c.271A>G ; p.N91D13missense
      1c.238G>C ; p.A80P14missense
      1c.224A>T ; p.D75V14missense
      2c.383G>T ; p.R128L17missense
      PIM1NM_0012431862c.383G>T ; p.R128L17missense
      2c.356G>A; p.G119D16missense
      4c.514C>G; p.P172A8missense
      4c.564_565delinsTA; p.S189T7nonframeshift indel
      4c.759_762delinsAATA; p.V254I15nonframeshift indel
      CR2MYD88NM_0024685c.794T>C ; p.L265P31missense
      BCL2NM_0006332c.167C>T; p.T56M35missense
      PIM1NM_0012431862c.462+1G>A ; p. ?23splicing
      4c.676G>A; p.E226K16missense
      CR3MYD88NM_0024685c.794T>C ; p.L265P24missense
      PIM1NM_0012431874c.564C>G; p.S188R23missense
      4c.676G>A; p.E226K24missense
      CR4PIM1NM_0012431861c.25C>T; p.L9F33missense
      1c.231C>G; p.S77R16missense
      4c.569G>A; p.G190D31missense
      SOCS1NM_0037452c.39_41delinsTTT ; p.S14F35nonframeshift indel
      MEF2BNM_001145785c.511C>T; p.R171*42nonsense
      CR5MYD88NM_0024685c.794T>C ; p.L265P14missense
      CD79BNM_0006265c.587A>G; p.Y196C11missense
      PIM1NM_0012431874c.566C>G; p.S189W7missense
      4c.646C>G; p.P216A22missense
      4c.652C>A; p.Q218K7missense
      4c.853A>G; p.K285E6missense
      4c.880+1G>A; p.?19splicing
      CR6MYD88NM_0024685c.794T>C ; p.L265P35missense
      CD79BNM_0006265c.587A>C; p.Y196S40missense
      EP300NM_00142931c.6567_6578del ; p.Q2189_Q2193del22nonframeshift indel
      GNA13NM_0065722c.493C>T ; p.R165C47missense
      PIM1NM_0012431861c.302_304delinsGCT; p.A101_H102delinsGY67nonframeshift indel
      4c.514_515delinsTT; p.P172F30nonframeshift indel
      4c.569G>A; p.G190D43missense
      4c.850C>T; p.L284F35missense
      CR7TNFRSF14NM_0038201c.2T>G; p?31splicing
      CR8MYD88NM_0024685c.794T>C ; p.L265P36missense
      CREBBPNM_00438029c.4865_4866dup; p.A1623Mfs*1337frameshift
      PIM1NM_0012431872c.388C>T; p.Q130*63nonsense
      4c.646C>T; p.P216S12missense
      CR9MYD88NM_0024683c.656C>G; p.S219C18missense
      CR10CD58NM_0017791c.2T>C ; p.?44splicing
      CR11MYD88NM_0024685c.794T>C ; p.L265P39missense
      IRF4NM_0024602c.59G>A; p.G20D42missense
      CD79BNM_0006265c.587A>T; p.Y196F83missense
      BCL2NM_0006332c.268C>T; p.P90S76missense
      CR12MYD88NM_0024685c.794T>C ; p.L265P39missense
      CD79BNM_0006265c.587A>G; p.Y196C85missense
      PIM1NM_0012431861c.230G>A; p.S77N16missense
      1c.276G>A; p.M92I36missense
      1c.323G>A; p.C108Y13missense
      2c.437G>A; p.G146D21missense
      3c.513+1G>A ; p. ?20splicing
      4c.655G>A; p.D219N9missense
      CR13MYD88NM_0024685c.794T>C ; p.L265P39missense
      CD79BNM_0006265c.586T>A; p.Y196N26missense
      PIM1NM_0012431864c.559G>T; p.V187L23missense
      4c.563_565delinsA; p.S188Nfs*815frameshift
      4c.676G>A; p.E226K25missense
      PRDM1NM_0011982c.291G>C; p.E97D38missense
      CREBBPNM_00438027c.4508A>G; p.Y1503C29missense
      CR14MYD88NM_0024683c.649G>T; p.V217F26missense
      PIM1NM_0012431864c.574T>G; p.S192A27missense
      CCND3NM_0017605c.869T>A ; p.I290K34missense
      RR1MYD88NM_0024685c.794T>C ; p.L265P31missense
      PIM1NM_0012431862c.356G>A; p.G119D16missense
      4c.646C>G; p.P216A19missense
      4c.676G>A; p.E226K26missense
      4c.822G>C; p.K274N34missense
      CD79ANM_0017835c.568-2_610del ; p.?21splicing
      RR2CARD11NM_0324155c.645G>C; p.K215N23missense
      MYCNM_0024672c.62G>C; p.S21T61missense
      2c.379A>C; p.N127H59missense
      2c.386G>A; p.S129N58missense
      2c.490C>G; p.L164V62missense
      2c.569G>A; p.S190N63missense
      2c.654C>G; p.S218R51missense
      RR3B2MNM_0040481c.2T>A ; p.?51splicing
      CD79BNM_0006265c.587A>G; p.Y196C32missense
      MYD88NM_0024685c.794T>C ; p.L265P41missense
      PIM1NM_0012431863c.510G>C ; p.E170D18missense
      2c.460C>G ; p.P154A27missense
      4c.796A>G ; p.I266V36missense
      1c.319C>A ; p.P107T44missense
      4c.514C>T ; p.P172S43missense
      4c.514-2_514delinsGGCGCCCTCGCCCCTGCAGT ; p.P172Gfs*813frameshift
      1c.165C>G ; p.S55R60missense
      1c.171C>A ; p.S57R32missense
      4c.753_754delinsTC ; p.C252R25nonframeshift indel
      SOCS1NM_0037452c.165C>A ; p.F55L34missense
      RR4CD79BNM_0006265c.586T>C; p.Y196H30missense
      CREBBPNM_00438027c.4481C>G ; p.P1494R25missense
      MYD88NM_0024685c.794T>C ; p.L265P14missense
      PIM1NM_0012431864c.676G>A; p.E226K16missense
      RR5CD79BNM_0006265c.586T>A; p.Y196N61missense
      RR6NOTCH2NM_02440827c.4942G>C; p.A1648P35missense
      MYD88NM_0024685c.794T>C ; p.L265P36missense
      PIM1NM_0012431861c.230G>A; p.S77N31missense
      1c.277C>G; p.L93V41missense
      2c.360_361delinsCC; p.K120_E121delinsNQ58nonframeshift indel
      2c.462+1G>A; p.?28splicing
      3c.475C>T; p.H159Y38missense
      3c.497C>A; p.S166Y10missense
      4c.676_686delinsAAAAGGGGAGA; p.E226_A229delinsKRGD57nonframeshift indel
      4c.724G>C; p.V242L42missense
      4c.823C>G; p.L275V36missense
      4c.845_862del; p.A282_T287del26nonframeshift indel
      5c.918C>A; p.Y306*36nonsense
      CREBBPNM_00438026c.4337G>A; p.R1446H37missense
      CIITANM_00024611c.1240C>T; p.R414*37nonsense
      CD79BNM_0006265c.586T>A; p.Y196N35missense
      RR7CD79BNM_0006265c.587A>C; p.Y196S78missense
      MYD88NM_0024685c.794T>C ; p.L265P36missense
      IRF4NM_0024602c.61_70del; p.N21Sfs*511frameshift
      PIM1NM_0012431861c.194G>A; p.S65N45missense
      1c.250T>A; p.C84S45missense
      2c.356G>A; p.G119D49missense
      4c.649_651delinsATA; p.V217I34nonframeshift indel
      PRDM1NM_0011987c.1951C>T; p.R651*58nonsense
      TP53NM_0005468c.821T>G; p.V274G63missense
      RR8CD79BNM_0006265c.586T>G; p.Y196D27missense
      MYD88NM_0024685c.794T>C; p.L265P28missense
      NOTCH2NM_02440827c.4999G>A; p.V1667I52missense
      XPO1NM_00340015c.1711G>A; p.E571K18missense
      IRF4NM_0024602c.53G>A; p.S18N31missense
      PIM1NM_0012431863c.512T>A; p.L171Q49missense
      5c.891_906delinsATATAGTCCTCCAGAGTGGATCCTC; p.I304_R305insLWI10nonframeshift indel
      6c.1082_1097delinsTCTTGGCCCTTCAGATAGGCCA; p.Cys361_Pro366delinsFLALQIGQ23nonframeshift indel
      MYCNM_0024671c.4_30del; p.D2_Q10del24nonframeshift indel
      ARID1ANM_0060151c.202C>G; p.P68A26missense
      1c.490_504del; p.A164_V168del28nonframeshift indel
      1c.507C>A; p.F169L40missense
      RR9CD79BNM_0006265c.586T>C; p.Y196H38missense
      IRF4NM_0024602c.59G>A; p.G20D38missense
      2c.171_173delinsAAA; p.G58N36nonframeshift indel
      PIM1NM_0012431861c.134_135delinsAT; p.S45N48nonframeshift indel
      1c.212G>A; p.G71D32missense
      1c.227_230delinsTCAA; p.G76_S77delinsVN46nonframeshift indel
      1c.272G>A; p.G91E67missense
      3c.497C>T; p.S166F38missense
      4c.549_555del; p.L184Rfs*240frameshift
      4c.569G>A; p.G190D43missense
      4c.646C>G; p.P216A48missense
      4c.652C>T; p.Q218*48nonsense
      4c.676G>A; p.E226K61missense
      RR10MYD88NM_0024685c.794T>C; p.L265P57missense
      CD58NM_0017793c.556C>T; p.Q186*28nonsense
      3c.406G>T; p.G136*29nonsense
      PIM1NM_0012431862c.361G>T; p.E121*28nonsense
      1c.140G>A; p.S47N37missense
      4c.711_712insTTCTTCTGGCAGGTGTTGGA; p.E244Dfs*12825frameshift
      CREBBPNM_00438026c.4337G>A; p.R1446H30missense
      CD79BNM_0006265c.587A>G; p.Y196C32missense
      RR11MYD88NM_0024685c.794T>C; p.L265P18missense
      PIM1NM_0012431862c.387C>A; p.Y129*19nonsense
      CD79BNM_0006265c.586T>C; p.Y196H18missense
      RR12CD79ANM_0017835c.568-2_610del ; p.?47splicing
      MEF2BNM_0011457853c.232A>G; p.S78G42missense
      RR13 (1/2)ARID1ANM_00601520c.6100G>A; p.E2034K40missense
      NOTCH2NM_02440834c.7198C>T; p.R2400*71nonsense
      CCND3NM_0017605c.778C>T; p.Q260*40nonsense
      TNFAIP3NM_0012705077c.1880_1881del; p.C627Ffs*4465frameshift
      RR13 (2/2)CARD11NM_0324158c.1071C>G; p.D357E10missense
      NOTCH1NM_01761734c.7538C>T; p.S2513F42missense
      RR14MYD88NM_0024685c.794T>C; p.L265P38missense
      PIM1NM_0012431864c.823C>T; p.L275F40missense
      MYCNM_0024672c.560C>T; p.S187F42missense
      2c.791G>A; p.S264N39missense
      3c.977C>T; p.A326V44missense
      CD79BNM_0006265c.586T>G; p.Y196D38missense
      RR15XPO1NM_00340015c.1711G>A; p.E571K18missense
      CD79BNM_0006265c.587A>C; p.Y196S26missense
      RR16
      RR17CXCR4NM_0010085401c.1033_1053del; p.T346_S352del12nonframeshift indel
      MYD88NM_0024685c.794T>C; p.L265P39missense
      PIM1NM_0012431861c.226_247del; p.G76Pfs*10230frameshift
      4c.823C>T; p.L275F34missense
      PLCG2NM_00266118c.1873G>A; p.A625T54missense
      CD79BNM_0006265c.591G>C; p.E197D39missense
      RR18MYD88NM_002468c.794T>C; p.L265P70missense
      PIM1NM_0012431862c.368_393dup; p.G132Sfs*6211frameshift
      4c.691C>T; p.Q231*37nonsense
      4c.711C>A; p.S237R35missense
      4c.823C>T; p.L275F39missense
      FOXO1NM_0020151c.153C>A; p.N51K46missense
      CD79BNM_0006265c.586T>C; p.Y196H44missense
      Abbreviations: CR, complete durable response; indel, insertion–deletion; RR, relapsed-refractory; SNV, single nucleotide variation; VAF, variant allele frequency.
      a Only SNV and INDEL mutations with a VAF > 2% and 5% respectively are reported.

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