Chimerism Assay Using Single Nucleotide Polymorphisms Adjacent and in Linkage-Disequilibrium Enables Sensitive Disease Relapse Monitoring after Hematopoietic Stem-Cell Transplantation.

BACKGROUND: Short tandem repeat (STR)-based chimerism analysis has been widely used for chimerism monitoring after hematopoietic stem-cell transplantation (HSCT), but technical artifacts can be problematic. We designed a chimerism assay using single nucleotide polymorphisms (SNPs) adjacent and in linkage-disequilibrium (CASAL), which doubly checked for SNP pairs, and thus could reduce background errors and increase analytical sensitivity. METHODS: CASAL targeted 84 SNP pairs within 10 bp distance and in perfect linkage-disequilibrium. Using undiluted and serially diluted samples, baseline error rates, and linearity was calculated. Clinical performance of CASAL was evaluated in comparison with a conventional STR assay, using 191 posttransplant samples from 42 patients with HSCT. RESULTS: CASAL had ∼10 times lower baseline error rates compared to that of ordinary next-generation sequencing. Limit of detection and quantification of CASAL were estimated to be 0.09 and 0.39%, respectively, with a linear range of 0.1-100%. CASAL correlated well with STR assay (r2 = 0.99) and the higher sensitivity enabled detection of low-level recipient chimerism and earlier prediction of relapse. CONCLUSIONS: CASAL is a simple, analytically sensitive and accurate assay that can be used in clinical samples after HSCT with a higher performance compared to that of traditional assays. It should also be useful in other forensic and archeological testing.

Plasma DNA tissue mapping by genome-wide methylation sequencing for noninvasive prenatal, cancer, and transplantation assessments.

Plasma consists of DNA released from multiple tissues within the body. Using genome-wide bisulfite sequencing of plasma DNA and deconvolution of the sequencing data with reference to methylation profiles of different tissues, we developed a general approach for studying the major tissue contributors to the circulating DNA pool. We tested this method in pregnant women, patients with hepatocellular carcinoma, and subjects following bone marrow and liver transplantation. In most subjects, white blood cells were the predominant contributors to the circulating DNA pool. The placental contributions in the plasma of pregnant women correlated with the proportional contributions as revealed by fetal-specific genetic markers. The graft-derived contributions to the plasma in the transplant recipients correlated with those determined using donor-specific genetic markers. Patients with hepatocellular carcinoma showed elevated plasma DNA contributions from the liver, which correlated with measurements made using tumor-associated copy number aberrations. In hepatocellular carcinoma patients and in pregnant women exhibiting copy number aberrations in plasma, comparison of methylation deconvolution results using genomic regions with different copy number status pinpointed the tissue type responsible for the aberrations. In a pregnant woman diagnosed as having follicular lymphoma during pregnancy, methylation deconvolution indicated a grossly elevated contribution from B cells into the plasma DNA pool and localized B cells as the origin of the copy number aberrations observed in plasma. This method may serve as a powerful tool for assessing a wide range of physiological and pathological conditions based on the identification of perturbed proportional contributions of different tissues into plasma.

Monitoreo inmunológico: el comienzo de una nueva era en trasplantes / Inmune monitoring: the start of a new age in transplantation

La necesidad de ajustar la inmunosupresión en forma individualizada ha estimulado la emergencia de técnicas que permiten predecir eventos clínicos como rechazo agudo o tolerancia. Esta revisión analiza, considerando principalmente el trasplante renal, las limitantes actuales de la inmunosupresión para concluir que una terapia individualizada permitiría mejorar la sobrevida de pacientes y órganos trasplantados en el largo plazo. En segundo lugar describe los métodos diagnósticos que en forma más consistente han demostrado tener valor predictivo con importancia clínica. Entre ellos se cuentan ensayos funcionales, determinación de anticuerpos específicos y linfocitos reactivos contra el donante, así como el análisis de marcadores a nivel de proteínas o genómicos. Los avances logrados auguran el comienzo de una nueva eraen trasplantes.

Organ transplantation is often related to higher survival and lower morbidity than conservative treatments. Nevertheless, survival and morbidity could be optimized tailoring the immunosupression to the particular needs of each individual patient. The requirement to optimize immunosupression makes necessary to improve the immunologic assessment and therefore has promoted the development of new immunological diagnostic tools. This review addresses first the need to tailor immunosupression, and then focuses in the value of anti HLA antibodies, alloreactive T cells, phenotypic analysis of lymphocytes and cytokines, repertoire analysis and genetic approaches, as well as in vivo studies. Further validation and standardization of these tests are needed in order to enter the routine clinical practice. Accomplishment of these goals would signal the beginning of a new era in transplantation.