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Genetic alterations in indolent GI T-cell LPD
(Figure 3B, C, E, F). No pathogenic mutations or structural abnormalities were observed in two CD8+ ITLPD (cases 9 and 10), although a variant of uncertain significance was observed in one case (Online Supplementary Table S2).
Longitudinal analysis of five ITLPD (cases 1, 2, 4, 7, 8) revealed stable mutational profiles in four ITLPD. Accrual of mutations over time was noted in one CD4+ ITLPD (case 4). Only a KMT2D frameshift mutation was detect- ed in the first biopsy, obtained shortly after diagnosis. Additional mutations were identified at later time points, including a missense TP53 mutation prior to disease trans- formation. Of interest, biopsies at the first, second, and fourth time-points had shown different chromosome copy number changes (reported previously),11 but none of the altered regions corresponded to the loci of mutated genes.
Evaluation of the SETD2-H3K36me3 axis
No SETD2 mutations were observed by next-generation sequencing analysis and FISH did not detect any SETD2 deletions in the cases analyzed. Additionally, no loss of SETD2 protein or H3K36me3 was detected by immuno- histochemistry and H3K36me2 expression was observed in all analyzed cases (Figure 4A-C, Online Supplementary Table S3).
Evaluation of JAK-STAT pathway activation
Due to the presence of frequent and recurrent genetic alterations targeting the JAK-STAT pathway and IL2 genes, we evaluated pSTAT3-Y705 and pSTAT5-Y694 expression by immunohistochemistry to assess activation of the JAK-STAT signaling pathway. All nine tested cases only showed single scattered or small clusters of nuclear pSTAT3-Y705 and pSTAT5-Y694 positive cells (<10%) in all biopsies (Figure 4E, F, Online Supplementary Table S3).
Discussion
Despite an increasing awareness of ITLPD of the GI tract, deciphering their molecular pathogenesis and cellu- lar origins has been challenging, in part due to the rarity of these disorders. In this study, comprising one of the largest series of cases evaluated, we delineate novel genetic alter- ations, including recurrent mutations and rearrangements, suggest cellular origins, and expand the immunopheno- typic spectrum of these diseases.
The clinical presentations and disease course of our patients were largely congruent with previous descrip- tions.6,8-16 Of interest, the ITLPD were detected incidentally in two asymptomatic patients, which has rarely been doc- umented.10 A history of Crohn disease has been reported in some patients with CD8+ ITLPD,12,13 which was also the case for one patient in our series. Prior erroneous diag- noses of seronegative, refractory celiac disease in a high proportion (50%) of patients were deemed to be the con- sequence of misinterpretation of the histopathological changes and incomplete laboratory testing. Due to the rel- atively recent recognition of these disorders, it is not sur- prising that 40% of the ITLPD in the current study had been previously misdiagnosed as aggressive intestinal T- cell lymphomas (EATL and MEITL). Extra-GI disease was observed more frequently (40%) in our series than in pre- viously reported series, and transformation to aggressive lymphoma, which is considered rare,8,11,15,28 occurred in two patients, including one with a CD8+ ITLPD. These findings emphasize the need for comprehensive clinical and laboratory evaluation and long-term follow-up of individuals with these disorders.
Next-generation sequencing of the ITLPD revealed genetic alterations in 80% of the cases, including muta- tions in JAK-STAT signaling pathway genes, observed in
ABC
DEF
Figure 4. Analysis of the SETD2-H3K36me3 axis and JAK-STAT pathway activation. Immunohistochemical analysis of a CD4+ indolent T-cell lymphoproliferative dis- order with STAT3-JAK2 rearrangement (case 2) shows preserved (A) SETD2, (B) H3K36me2, and (C) H3K36me3 protein expression. The lymphocytes express (D) CD4. Only a few scattered (E) pSTAT3-Y705+ and (F) pSTAT5-Y694+ cells are noted (comprising <10% of the neoplastic lymphocytes).
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