A single-tube, EuroClonality-inspired, TRG clonality multiplex PCR aids management of patients with enteropathic diseases, including from formaldehyde-fixed, paraffin-embedded tissues

Celiac disease is a chronic inflammation of the small intestine with villous atrophy that can become refractory to a gluten-free diet. Two categories of refractory celiac disease can be distinguished by the phenotype of intraepithelial lymphocytesand the status of TRG genes. Their distinction is important because 30% to 50% of type II but only 0% to 14% of type I evolve to an aggressive enteropathy-associated T-cell lymphomaand therefore require intensive treatment. Currently, differentialdiagnosis integratesimmunohistochemistry, immunophenotyping, and TRG clonality analyses, but each has limitations. A single-tube multiplex TRG PCR (ECN) was prospectively compared to an in-house two-tube TRG PCR (N2T) in 73 samples, including 67 cryopreservedintestine tissues. Thirteen formalin-fixed, paraffin-embedded (FFPE) samples were also analyzed retrospectively. The ECN PCR had comparable efficiency to detect major clonal rearrangements in highly infiltrated tissues from T-cell lymphoproliferative disordersand type II refractory celiac disease and to detect the persistence of minor clones in type II refractory celiac disease follow-up samples. The ECN PCR abolished the risk of amplification of false-positive weak clonal rearrangements in cryopreservedspecimens and allowed improved detection of clonal rearrangements in DNA from FFPE samples. The ECN PCR allows robust assessment of cryopreservedand FFPE digestive tissues at diagnosis and follow-up of enteropathieswith villous atrophy, thus guiding therapeutic management.