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C.R. Soderquist et al.
enteropathy-associated T-cell lymphoma (EATL) and monomorphic epitheliotropic intestinal T-cell lymphoma (MEITL), are among the more common types of primary GI T-cell lymphomas, which are associated with high morbidity and mortality.1,4,5 In recent years, there has been a growing awareness of indolent T- and natural killer (NK)-cell lymphoproliferative disorders, which can also arise within the GI tract and involve a variety of GI organs.6,7 The pathogenesis of indolent NK-cell disorders is unclear and it is not yet known if they constitute neo- plastic proliferations of NK cells.7 Indolent T-cell lympho- proliferative disorders (ITLPD) of the GI tract, which con- stitute an immunophenotypically diverse group of clonal T-cell diseases, have been better characterized and hence included as provisional entities in the revised 4th edition of the World Health Organization (WHO) classification of lymphoid neoplasms.1 The clinical, morphological, and immunophenotypic features of ITLPD of the GI tract dif- fer from those of other types of primary GI T-cell lym- phomas6,8–16 and their cellular derivation, although not well established, is also considered to be distinct.9,11 Overlapping genomic and genetic alterations have been reported in EATL and MEITL.17–21 Limited data suggest a different spectrum of genomic aberrations in ITLPD of the GI tract,11,13 and until recently, no recurrent genetic abnormality had been identified in these disorders.15 However, the mutational landscape and molecular path- ways underlying the initiation and progression of ITLPD of the GI tract are unknown and the cell of origin of the different immunophenotypic subsets has not been defined. To gain further insights into the biology of these rare diseases, we performed comprehensive immunohis- tochemical, molecular and targeted next-generation sequencing analyses of a series of ten cases.
Methods
Case selection
The pathology department databases of multiple institutions were searched for primary GI T-cell lymphomas, over a 23-year period (1996-2018), to identify cases fulfilling histopathological and clinical criteria of ITLPD as defined in the revised WHO clas- sification.1 Clinical data, including therapy and outcomes, were obtained from the treating physicians or electronic medical records. The study was performed in accordance with the princi- ples of the Declaration of Helsinki and protocols approved by the Institutional Review Boards of the participating institutions.
Morphology and immunophenotypic analysis
Hematoxylin and eosin-stained formalin-fixed, paraffin-embed- ded (FFPE) biopsy sections were reviewed to assess cyto-architec- tural features. Immunohistochemical staining was performed using a comprehensive panel of antibodies, including those direct- ed against T-cell antigens, lineage-associated transcription factors, immune checkpoint molecules, histone modifications and cytokine signaling molecules (Online Supplementary Methods). The percentage of cells expressing nuclear T-bet and GATA3 was assessed in areas of dense lymphocytic infiltration determined by CD4 and CD8 staining. Cases with >50% cellular staining by both markers were deemed to co-express T-bet and GATA3. For pSTAT3 and pSTAT5, >10% nuclear staining was considered pos- itive. Flow cytometry was performed on cell suspensions prepared from tissue samples (Online Supplementary Methods).
T-cell receptor gene rearrangement analysis
Polymerase chain reaction (PCR) analysis to determine clonal T- cell receptor beta (TRB) and/or gamma (TRG) gene rearrangement was performed using the ‘Biomed-2’ primers on DNA extracted from fresh or FFPE GI biopsies, lymph nodes, peripheral blood, and bone marrow mononuclear cells, as previously described.22
Next-generation sequencing
Targeted next-generation sequencing of lesional and matched normal (control) tissue samples was performed using a custom panel of 465 cancer-associated genes, as previously described.23 Variant calling required a variant allelic fraction of at least 5% and at least ten variant reads. Variants with an allele prevalence >0.01% in gnomAD, those reported as benign or likely benign in ClinVar, and germline variants present in the normal samples or inferred from variant allelic fractions were excluded from the analysis. Non-synonymous variants that were not known driver mutations were analyzed by PolyPhen-2, SIFT, REVEL, and MetaSVM algorithms. Copy number changes were determined based on read depths using fragments per kilobase per million mapped reads24 normalized to a pool of sex-matched control sam- ples. The Fusion and Chromosomal Translocation Enumeration and Recovery Algorithm (FACTERA)25 was used to detect structur- al chromosomal alterations, which were confirmed by PCR using breakpoint-specific primers and Sanger sequencing of the PCR products (Online Supplementary Methods).
Fluorescent in-situ hybridization analysis
Fluorescent in-situ hybridization (FISH) analysis was performed
to assess for SETD2 and JAK2 alterations on FFPE tissue sections using custom designed hybridization probes and dual-color break- apart probes (Oxford Gene Technologies Inc, Tarrytown, NY, USA), respectively, as previously described.17,26 Hybridization pat- terns of at least 100 tumor nuclei were reviewed for each probe. Cases were considered to have SETD2 deletion if the percentage of nuclei with SETD2 locus deletion exceeded the cut-off value of 11.2%, and JAK2 rearrangement if the frequency of split-signals exceeded the cut-off value of 5.0%.
Results
Clinical characteristics and patients’ outcomes
Ten patients (male:female = 8:2) with ITLPD of the GI tract were identified at the contributing centers (cases 1, 2, and 4 were reported previously).11 The clinical features are summarized in Table 1. The median age at diagnosis was 45 years (range, 37-64 years). The ethnicity of eight patients for whom this information was available was: White (n=5), Hispanic (n=2), and Asian (n=1). The most common signs and symptoms were diarrhea (70%), weight loss (60%), and abdominal pain (50%), with dura- tions ranging from 2 to 16 years prior to diagnosis. Two patients lacked GI symptoms, with disease detected inci- dentally during routine colonoscopy and workup for inguinal lymphadenopathy. One patient had peptic ulcer disease, H. pylori infection and was serologically positive for hepatitis B and C viruses (case 9) and one patient (case 10) had a history of Crohn disease. Eight patients had been previously misdiagnosed as having celiac disease, seronegative and refractory to a gluten-free diet, and/or other types of lymphomas. The endoscopic findings included mucosal nodularity (70%), scalloping (40%), ery- thema (40%), decreased duodenal folds (30%), and polyps (20%). Common radiographic findings included abdomi-
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