Pileri, Stefano

ABSTRACTNodal follicular helper T‐cell (T FH) lymphoma of the angioimmunoblastic (AITL) subtype has a dismal prognosis. Using whole‐exome sequencing (n = 124), transcriptomic (n = 78), and methylation (n = 40) analysis, we identified recurrent mutations in known epigenetic drivers (TET2, DNMT3A, IDH2 R172 ) and novel ones (TET3, KMT2D). TET2, IDH2 R172 , DNMT3A co‐mutated AITLs had poor prognosis (p < 0.0001). Genes regulating T‐cell receptor (TCR) signaling (CD28, PLCG1, VAV1, FYN) or activation (RHOA G17V ) or regulators of the PI3K‐pathway (PIK(3)C members, PTEN, PHLPP1, PHLPP2) were mutated. CD28 mutation/fusion was associated with poor prognosis (p = 0.02). WES of purified, neoplastic T‐cell (CD3+PD1+) demonstrated high concordance with whole tumor biopsies and validated the presence of TET2 and DNMT3A in tumor and non‐lymphoid cells, but other mutations (CD28, RHOA G17V , IDH2 R172 , PLCG1) in neoplastic cells. Integrated DNA‐methylation and mRNA expression analysis revealed epigenetic alterations in genes regulating TCR, cytokines, PI3K‐signaling, and apoptosis. RNA‐seq analysis identified fusion transcripts regulating TCR‐activation (8%), revealed a restricted TCR‐repertoire (α = 87%, β = 72%), and showed the presence of Epstein–Barr virus transcriptome (73%). GEP demonstrated the association of B‐cells or dendritic cells in the tumor milieu with prognosis (p < 0.01). RNA‐seq and WES analysis of 12 AITL‐patient‐derived‐xenografts (PDX) showed that bi‐allelic TET2 and DNMT3A mutations or sub‐clonal mutations (PLCG1, PHLPP2) propagated in sequential passages, and gene signatures related to T FH and T CM (central‐memory) were well‐maintained through passages. Gene expression signatures associated with late PDX passages (3rd–5th) were enriched with proliferation and metabolic reprogramming‐related genes and predicted prognosis in an independent AITL series. Low PHLPP2 mRNA expression predicted poor prognosis (p = 0.05) and engineered PHLPP2 or TET2 loss in CD4+ T‐cells showed enhanced PI(3)K activation, thus uncovering a therapeutic target for clinical trials.

ABSTRACTBackgroundPrimary cutaneous anaplastic large cell lymphoma (pcALCL) and lymphomatoid papulosis (LyP) are indolent CD30‐positive lymphoproliferative disorders that rarely express TCR‐γδ. However, primary cutaneous gamma‐delta T‐cell lymphoma (pcGDTCL), characterized by TCR‐γδ expression on neoplastic cells, is a rare, aggressive cutaneous T‐cell lymphoma with a poor prognosis. Accurate differentiation is essential due to distinct clinical behavior and treatment.MethodsWe identified 11 TCR‐γδ‐positive pcALCL cases from internal and consultation files, verified by two cutaneous lymphoma experts, with clinicopathologic data recorded.ResultsThe median age was 68 years (range 38–95). Most cases presented as a single dermal lesion on the upper extremities. All were ALK‐negative, CD30‐positive (> 90% tumor cells), TCR‐βF1‐negative, and diffusely TCR‐γδ‐positive. CD4−/CD8− (54.5%) and CD4+/CD8− (45.5%) immunophenotypes were observed. CD2 (63.6%) and CD3 (54.5%) were the most common T‐cell antigens. Ulceration, inflammation (both 45.5%), and necrosis (36.4%) were frequent. Angiotropism, angiocentricity, and myxoid stroma appeared in one case; epidermotropism in two. Four of ten tested were DUSP22‐rearranged, and TP63 was negative in all eight tested.ConclusionsMost ALK‐negative TCR‐γδ‐positive pcALCL were CD4−/CD8−, followed by CD4+/CD8−. DUSP22 rearrangement occurred in 40% of cases, similar to reports in typical pcALCL. Its prognosis and the role of DUSP22 are yet to be clarified.

Lymphomatoid papulosis (LyP) has several histopathologic presentations. LyP featuring gamma-delta (γδ) T-cell receptor expression may masquerade as and may be misdiagnosed as aggressive cutaneous T-cell lymphoma, particularly primary cutaneous γδ T-cell lymphoma (PCGDTL) or γδ mycosis fungoides. We performed a clinicopathologic analysis of the largest series of LyP featuring γδ T-cell expression. We identified 26 patients with a diagnosis of LyP with γδ T cells from our institutions, as well as through a comprehensive review of the literature, and characterized these cases. Most cases were treated with topical steroids or not treated at all. The majority of cases showed a CD4-CD8+ phenotype and featured at least one cytotoxic marker. Histopathologic features included an intraepidermal or dermal infiltrate with large cells and frequent angiotropism. One case was initially misdiagnosed as PCGDTL, requiring further therapy. Our case series, the largest international cohort of γδ T cell predominant LyP cases, confirms marked clinicopathologic heterogeneity that may contribute to misdiagnosis, reasserting the need to identify classic clinical features, CD30+ T-cell components, and markers of cytotoxicity when dealing with this differential diagnosis. A limitation of this study includes somewhat limited follow-up, histologic, and immunophenotypic information for some cases.

Introduction: Previously we demonstrated that HIV associated diffuse large B-cell lymphomas (DLBCL) of Germinal Center B-cell subtype (GCB) are more proliferative, with enhanced genomic stability and increased expression of DNA repair genes, particularly Fanconi anemia (FA) and homologous recombination (HR) genes compared to their HIV negative counterparts (Maguire et al, 2019). It is unknown if this signature is unique to the GCB subtype or if it is also observed in Activated B-cell (ABC) and Unclassified (UNC) DLBCL. Herein we validate our HIV associated GCB DLBCL findings in a larger independent cohort and report for the first time the aberrational profile of molecularly profiled HIV associated ABC DLBCL compared to their HIV negative counterparts respectively. Methods: An independent cohort of 353 DLBCL FFPE cases was assembled, including 125 HIV-DLBCL samples. All samples underwent hematopathological review to confirm diagnosis and determine tumor content. Samples with &lt;60% tumor content were macro-dissected before they were RNA/DNA extracted. Cell of origin (COO) profiles were assessed using the Lymph3Cx molecular assay which can distinguish primary mediastinal B-cell lymphoma (PMBL) like from GCB, ABC and UNC DLBCL (Mottok et al 2018). Copy number (CN) analyses were performed on samples with sufficient DNA (41 HIV neg, 32 HIV pos) using the OncoScan FFPE assay kit and the resulting data analyzed using Chromosome Analysis Suite (ChAS) v4.1 and the multisample viewer module of Reproductive Health Research Analysis Software (RHAS) v1.1. Additional digital gene expression profiling was performed using the Pan-Cancer panel (NanoString, Seattle, WA) customized to include 9 BCL2 family genes for a total of 739 genes and analyzed using the NanoString nSolver Advanced Analysis module. Results: GCB, ABC, UNC and PMBL-like proportions were 50%, 30%, 14% and 6% in HIV unassociated (HIV neg) and 50%, 23%, 25% and 2% in HIV pos tumors respectively, confirming both cohorts were GCB-enriched by molecular profiling. PMBL-like cases were excluded from further analyses. OncoScan CN analyses showed that HIV pos DLBCL were significantly less aberrant than HIV neg DLBCL (median CN per sample 0.56 vs 1.01, p&lt;0.001). HIV pos GCB tumors were significantly less aberrant than both HIV neg GCB tumors (median CN per sample 0.50 vs 1.11, p&lt;0.001), and HIV pos ABC tumors (median CN per sample 0.50 vs 0.81, p=0.009). HIV pos ABC tumors were also less aberrant than HIV neg ABC tumors, however, the difference was not significant overall (median CN per sample 0.81 vs 1.03, p=0.162) or for gain/amplifications (median CN per sample 0.38 vs 0.42, p=0.814), but was borderline significant for loss/deletions (median CN per sample 0.31 vs 0.47, p=0.06). GEP analyses identified Cell Cycle and DNA repair as the top two upregulated pathways in HIV pos GCB tumors compared to HIV neg GCB tumors. In contrast, Cell Cycle and DNA repair were downregulated in HIV pos ABC tumors compared to HIV neg ABC tumors. However, one gene ( RBX1) was identified as significantly upregulated in both pathways in HIV pos GCB (adjusted p-value 0.0215) and HIV pos ABC (adjusted p-value 0.0046) tumors compared to their HIV neg counterparts. A second gene ( SKP1) was also significantly upregulated in HIV pos GCB (adjusted p-value 3.94E-5) and HIV pos ABC (adjusted p-value 0.0031) tumors. Together RBX1 and SKP1 encode the two principal functional subunits of the SKP1, CUL1, F-box protein (SCF) complex which is part of the ubiquitin proteasome system (UPS) and has been reported as essential for maintaining genome and chromosome stability (Thompson et al 2021). Conclusions: These results demonstrate that HIV pos DLBCL tumors are less aberrant than their HIV neg counterparts irrespective of COO subtype. However, the degree of genomic stability differs by COO, with HIV pos GCB tumors being more genomically stable than HIV pos ABC tumors. Although both have enhanced expression of UPS related SCF genes ( RBX1, SKP1), only HIV pos GCB tumors have enhanced expression of key DNA repair genes as well including Fanconi Anemia ( BRCA2/FANCD1, FANCL) and mismatch repair ( MSH2, MSH6) genes (adjusted p-values of ≤ 0.0263). Given the reported role of SCF in genomic stability maintenance, the HIV pos COO CN differences could be due to the contributions of both DNA repair and SCF mechanisms in HIV pos GCB and mainly SCF mechanisms in HIV pos ABC tumors.

Follicular helper T-cell lymphoma of the angioimmunoblastic type (AITL) is associated with dismal prognosis. We performed functional genomic approaches including whole-exome sequencing (WES; n=119), transcriptomic (n=78) and methylation (n=40) analysis. We identified recurrent mutations in known epigenetic drivers ( TET2, DNMT3A, IDH2 R172), and also identified novel ones (TET3, KMT2D). Somatic mutation of all three epigenetic drivers ( TET2, IDH2, and DNMT3A) was associated with poor prognosis (p&lt;.001). Mutations in genes regulating T-cell receptor (TCR) signaling ( CD28 VAV1, FYN, PLCG1) or activation ( RHOA G17V), and regulators of the PI3K pathway (PIK(3)C members, PTEN,PHLPP-1/-2) were also found. Genome-wide DNA-methylation analysis integrated with mRNA expression profiling also revealed epigenetic alterations in genes regulating TCR-RHOA/B/C or PI3K-signaling. TET2 loss was noted in 85% AITLs and was significantly associated with RHOA G17V, CD28 and IDH2 R172mutations. AITLs lacking RHOA G17V tended to have mutations regulating the JAK-STAT pathway ( JAK2, JAK3, STAT1, STAT3, SOCS1). RNA-seq analysis identified fusion transcripts in genes regulating TCR activation (8%), revealed a restricted TCR repertoire in the majority of cases (a=87%, b=72%), and showed the presence of Epstein-Barr virus transcriptome (73%). GEP demonstrated association of B-cells in the tumor-milieu with better prognosis (p=.006), while dendritic cells were associated with worse prognosis (p=.001), which was further validated by immunohistochemistry using CD20, CD68, and CD163 antibodies. RNA-seq and corresponding WES analysis of 12 AITL patient-derived-xenografts (PDX) showed that bi-allelic TET2 mutations, DNMT3A mutations or sub-clonal mutations ( PLCG1 PHLPP2) werepropagated in sequential passages. Gene signatures related to T FH (follicular helper) and T CM (central memory) were also well-maintained in secondary passages in PDX models. Gene signatures of late PDX passages (3 rd-5 th) were enriched with genes related to proliferation and metabolic reprogramming, and in an independent cohort of AITLs, high expression of T3/T5 related signatures was associated with worse outcome (p=0.02/p=0.009). Low mRNA expression of PHLPP2 predicted poor prognosis (p=.03) and engineered PHLPP2 loss showed enhanced PI(3)K activation and FOXO1 inactivation in CD4+ T-cells in-vitro. Thus, we defined the genomic landscape for AITL, which is largely characterized by epigenetic alterations, TCR signaling and PI3K/AKT dysregulation, which may be amenable for therapeutic targeting.

Abstract With the introduction of large-scale molecular profiling methods and high-throughput sequencing technologies, the genomic features of most lymphoid neoplasms have been characterized at an unprecedented scale. Although the principles for the classification and diagnosis of these disorders, founded on a multidimensional definition of disease entities, have been consolidated over the past 25 years, novel genomic data have markedly enhanced our understanding of lymphomagenesis and enriched the description of disease entities at the molecular level. Yet, the current diagnosis of lymphoid tumors is largely based on morphological assessment and immunophenotyping, with only few entities being defined by genomic criteria. This paper, which accompanies the International Consensus Classification of mature lymphoid neoplasms, will address how established assays and newly developed technologies for molecular testing already complement clinical diagnoses and provide a novel lens on disease classification. More specifically, their contributions to diagnosis refinement, risk stratification, and therapy prediction will be considered for the main categories of lymphoid neoplasms. The potential of whole-genome sequencing, circulating tumor DNA analyses, single-cell analyses, and epigenetic profiling will be discussed because these will likely become important future tools for implementing precision medicine approaches in clinical decision making for patients with lymphoid malignancies.

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.

Abstract Since the publication of the Revised European-American Classification of Lymphoid Neoplasms in 1994, subsequent updates of the classification of lymphoid neoplasms have been generated through iterative international efforts to achieve broad consensus among hematopathologists, geneticists, molecular scientists, and clinicians. Significant progress has recently been made in the characterization of malignancies of the immune system, with many new insights provided by genomic studies. They have led to this proposal. We have followed the same process that was successfully used for the third and fourth editions of the World Health Organization Classification of Hematologic Neoplasms. The definition, recommended studies, and criteria for the diagnosis of many entities have been extensively refined. Some categories considered provisional have now been upgraded to definite entities. Terminology for some diseases has been revised to adapt nomenclature to the current knowledge of their biology, but these modifications have been restricted to well-justified situations. Major findings from recent genomic studies have impacted the conceptual framework and diagnostic criteria for many disease entities. These changes will have an impact on optimal clinical management. The conclusions of this work are summarized in this report as the proposed International Consensus Classification of mature lymphoid, histiocytic, and dendritic cell tumors.

Abstract Peripheral T-cell lymphomas (PTCLs) are heterogenous T-cell neoplasms often associated with epigenetic dysregulation. We investigated de novo DNA methyltransferase 3A (DNMT3A) mutations in common PTCL entities, including angioimmunoblastic T-cell lymphoma and novel molecular subtypes identified within PTCL–not otherwise specified (PTCL-NOS) designated as PTCL-GATA3 and PTCL-TBX21. DNMT3A-mutated PTCL-TBX21 cases showed inferior overall survival (OS), with DNMT3A-mutated residues skewed toward the methyltransferase domain and dimerization motif (S881–R887). Transcriptional profiling demonstrated significant enrichment of activated CD8+ T-cell cytotoxic gene signatures in the DNMT3A-mutant PTCL-TBX21 cases, which was further validated using immunohistochemistry. Genomewide methylation analysis of DNMT3A-mutant vs wild-type (WT) PTCL-TBX21 cases demonstrated hypomethylation in target genes regulating interferon-γ (IFN-γ), T-cell receptor signaling, and EOMES (eomesodermin), a master transcriptional regulator of cytotoxic effector cells. Similar findings were observed in a murine model of PTCL with Dnmt3a loss (in vivo) and further validated in vitro by ectopic expression of DNMT3A mutants (DNMT3A-R882, -Q886, and -V716, vs WT) in CD8+ T-cell line, resulting in T-cell activation and EOMES upregulation. Furthermore, stable, ectopic expression of the DNMT3A mutants in primary CD3+ T-cell cultures resulted in the preferential outgrowth of CD8+ T cells with DNMT3AR882H mutation. Single-cell RNA sequencing(RNA-seq) analysis of CD3+ T cells revealed differential CD8+ T-cell subset polarization, mirroring findings in DNMT3A-mutated PTCL-TBX21 and validating the cytotoxic and T-cell memory transcriptional programs associated with the DNMT3AR882H mutation. Our findings indicate that DNMT3A mutations define a cytotoxic subset in PTCL-TBX21 with prognostic significance and thus may further refine pathological heterogeneity in PTCL-NOS and suggest alternative treatment strategies for this subset.

Abstract Purpose: Peripheral T-cell lymphoma (PTCL) is a heterogeneous group of non–Hodgkin lymphomas with aggressive clinical behavior. We performed comprehensive miRNA profiling in PTCLs and corresponding normal CD4+ Th1/2 and TFH-like polarized subsets to elucidate the role of miRNAs in T-cell lymphomagenesis. Experimental Design: We used nCounter (NanoString Inc) for miRNA profiling and validated using Taqman qRT-PCR (Applied Biosystems, Inc). Normal CD4+ T cells were polarized into effector Th subsets using signature cytokines, and miRNA significance was revealed using functional experiments. Results: Effector Th subsets showed distinct miRNA expression with corresponding transcription factor expression (e.g., BCL6/miR-19b, -106, -30d, -26b, in IL21-polarized; GATA3/miR-155, miR-337 in Th2-polarized; and TBX21/miR-181a, -331-3p in Th1-polarized cells). Integration of miRNA signatures suggested activation of TCR and PI3K signaling in IL21-polarized cells, ERK signaling in Th1-polarized cells, and AKT–mTOR signaling in Th2-polarized cells, validated at protein level. In neoplastic counterparts, distinctive miRNAs were identified and confirmed in an independent cohort. Integrative miRNA–mRNA analysis identified a decrease in target transcript abundance leading to deregulation of sphingolipid and Wnt signaling and epigenetic dysregulation in angioimmunoblastic T-cell lymphoma (AITL), while ERK, MAPK, and cell cycle were identified in PTCL subsets, and decreased target transcript abundance was validated in an independent cohort. Elevated expression of miRNAs (miR-126-3p, miR-145-5p) in AITL was associated with poor clinical outcome. In silico and experimental validation suggest two targets (miR-126→ SIPR2 and miR-145 → ROCK1) resulting in reduced RhoA-GTPase activity and T–B-cell interaction. Conclusions: Unique miRNAs and deregulated oncogenic pathways are associated with PTCL subtypes. Upregulated miRNA-126-3p and miR-145-5p expression regulate RhoA-GTPase and inhibit T-cell migration, crucial for AITL pathobiology.