Digital PCR Panel for Sensitive Hematopoietic Chimerism Quantification after Allogeneic Stem Cell Transplantation.

Accurate and sensitive determination of hematopoietic chimerism is a crucial diagnostic measure after allogeneic stem cell transplantation to monitor engraftment and potentially residual disease. Short tandem repeat (STR) amplification, the current "gold standard" for chimerism assessment facilitates reliable accuracy, but is hampered by its limited sensitivity (≥1%). Digital PCR (dPCR) has been shown to combine exact quantification and high reproducibility over a very wide measurement range with excellent sensitivity (routinely ≤0.1%) and thus represents a promising alternative to STR analysis. We here aimed at developing a whole panel of digital-PCR based assays for routine diagnostic. To this end, we tested suitability of 52 deletion/insertion polymorphisms (DIPs) for duplex analysis in combination with either a reference gene or a Y-chromosome specific PCR. Twenty-nine DIPs with high power of discrimination and good performance were identified, optimized and technically validated. We tested the newly established assays on retrospective patient samples that were in parallel also measured by STR amplification and found excellent correlation. Finally, a screening plate for initial genotyping with DIP-specific duplex dPCR assays was designed for convenient assay selection. In conclusion, we have established a comprehensive dPCR system for precise and high-sensitivity measurement of hematopoietic chimerism, which should be highly useful for clinical routine diagnostics.

Digital PCR to assess hematopoietic chimerism after allogeneic stem cell transplantation.

Analysis of hematopoietic chimerism after allogeneic stem cell transplantation represents a crucial method to evaluate donor-cell engraftment. Whereas sensitivity of classical approaches for chimerism monitoring is limited to ≥1%, quantitative polymerase chain reaction (qPCR)-based techniques readily detect one patient cell in >1,000 donor cells, thus facilitating application of chimerism assessment as a surrogate for minimal residual disease. However, due to methodologic specificities, qPCR combines its high sensitivity with limited resolution power in the state of mixed chimerism (e.g., >10% patient cells). Our aim was to overcome this limitation by employing a further development of qPCR, namely digital PCR (dPCR), for chimerism analysis. For proof-of-principle, we established more than 10 dPCR assays detecting Indel polymorphisms or Y-chromosome sequences and tested them on artificial cell mixtures and patient samples. Employing artificial cell mixtures, we found that dPCR allows exact quantification of chimerism over several orders of magnitude. Digital PCR results proved to be highly reproducible (deviation <5%), particularly in the "difficult" range of mixed chimerism. Excellent performance of the new assays was confirmed by analysis of multiple retrospective blood samples from patients after allogeneic stem cell transplantation, in comparison with established qPCR (14 patients) and short-tandem repeat PCR (4 patients) techniques. Finally, dPCR is easy to perform, needs only small amounts of DNA for chimerism assessment (65 ng corresponds to a sensitivity of approximately 0.03%), and does not require the use of standard curves and replicate analysis. In conclusion, dPCR represents a very promising method for routine chimerism monitoring.

Minimal residual disease diagnostics in patients with acute myeloid leukemia in the post-transplant period: comparison of peripheral blood and bone marrow analysis.

Considering the high relapse rates of AML after allogeneic hematopoietic stem cell transplant, research aims to improve post-transplant surveillance. To determine the value of peripheral blood (PB) for post-transplant minimal residual disease monitoring, we compared 38 PB and bone marrow (BM) sample pairs in 25 stem cell recipients with NPM1-mutated AML (12 males, 13 females, ages 21-73 years). NPM1A mutation levels and chimerism ratios were determined in non-separated BM/PB. We observed congruent results in 28/38 (74%). In 14/38 sample pairs (37%), BM and PB were negative for the NPM1A mutation. Fourteen sample pairs were positive in BM and PB, albeit at higher mutation levels in the BM in 11 cases (4- to 278-fold). Results were discordant in 10 cases (26%), with weakly positive mutation levels in the BM but negative levels in the PB. Cases with ≥0.2% NPM1A mutation level in BM were always positive in PB. Chimerism was concordant in BM and PB in 21/34 (62%) of sample pairs. In conclusion, MRD monitoring with qPCR for the NPM1 mutation and chimerism from non-separated PB contributes to surveillance in patients with AML in the post-transplant period, but even with highly sensitive qPCR there is a risk of failure to detect the mutation in PB.

Chimerism studies with quantitative real-time PCR in stem cell recipients with acute myeloid leukemia.

OBJECTIVE: Chimerism is well-established for surveillance of acute myeloid leukemia (AML) patients after allogeneic hematopoietic stem cell transplantation (HSCT), but interpretation of the results and techniques is not standardized. MATERIALS AND METHODS: We correlated chimerism in 75 AML patients (38 male, 37 female) who underwent myeloablative (n = 36)/reduced (n = 39) allo-HSCT with the risk of relapse and survival. Chimerism was evaluated by quantitative real-time polymerase chain reaction for donor/recipient specific polymorphisms/Y-specific sequences. RESULTS: After HSCT, 40 patients (53%) achieved stable complete donor chimerism (≥ 99.0% of donor alleles), while 35 (47%) failed to achieve stable donor chimerism. Thirty-one patients (41%) showed decreasing donor alleles after having first achieved complete donor chimerism. To investigate the kinetics of mixed chimerism, patients were separated whether they showed subsequent increasing or decreasing donor alleles. Subsequent decrease of donor alleles was associated with relapses in 17 of 18 cases (94%), while no patient with subsequent increasing donor alleles relapsed (p < 0.001). Patients with mixed chimerism and increasing donor alleles had better 2-year disease-free survival (85%) than those with decreasing donor alleles (0%; p < 0.001). CONCLUSIONS: The kinetics of mixed chimerism as assessed by quantitative real-time polymerase chain reaction is an important prognostic predictor in the post-transplantation period of AML patients.

NY-CO-58/KIF2C is overexpressed in a variety of solid tumors and induces frequent T cell responses in patients with colorectal cancer.

NY-CO-58/KIF2C has been identified as a tumor antigen by screening antibody responses in patients with colorectal cancer. However, expression had not consequently been examined, and nothing was known about its ability to induce spontaneous T cell responses, which have been suggested to play a role in the development of colorectal cancer. We analyzed 5 colorectal cancer cell lines, and tumor samples and adjacent healthy tissues from 176 patients with epithelial cancers for the expression of NY-CO-58/KIF2C by RT-PCR and Western Blot. T cell responses of 43 colorectal cancer patients and 35 healthy donors were evaluated by ELISpot following stimulation with 30mer peptides or full-length protein. All cell lines and tumor samples from colorectal cancer patients expressed NY-CO-58/KIF2C on the protein and RNA level, and expression levels correlated strongly with Ki-67 expression (r = 0.69; p = 0.0003). Investigating NY-CO-58/KIF2C-specific T cell responses, CD8(+) T cells directed against 1 or more peptides were found in less than 10% of patients, whereas specific CD4(+) T cells were detected in close to 50% of patients. These T cells were of high avidity, recognized the naturally processed antigen and secreted IFN-gamma and TNF-alpha. Depletion of CD4(+)CD25(+) T cells before stimulation significantly increased the intensity of the preexisting response. NY-CO-58/KIF2C is significantly overexpressed in colorectal and other epithelial cancers and expression levels correlate with the proliferative activity of the tumor. Importantly, NY-CO-58/KIF2C was able to induce spontaneous CD4(+) T cell responses of the Th1-type, which were tightly controlled by peripheral T regulatory cells.

Cancer-testis antigens MAGE-C1/CT7 and MAGE-A3 promote the survival of multiple myeloma cells.

BACKGROUND: Multiple myeloma is a life-threatening disease and despite the introduction of stem cell transplantation and novel agents such as thalidomide, lenalidomide, and bortezomib most patients will relapse and develop chemoresistant disease. Therefore, alternative therapeutic modes for myeloma are needed and cancer-testis antigens such as MAGE-C1/CT7 and MAGE-A3 have been suggested to represent a class of tumor-specific proteins particularly suited for targeted immunotherapies. Surprisingly, the biological role of cancer-testis genes in myeloma remains poorly understood. DESIGN AND METHODS: We performed the first investigation of the function of two cancer-testis antigens most commonly expressed in myeloma, MAGE-C1/CT7 and MAGE-A3, using an RNA interference-based gene silencing model in myeloma cell lines. Functional assays were used to determine changes in proliferation, cell adhesion, chemosensitivity, colony formation, and apoptosis resulting from gene-specific silencing. RESULTS: We show that the investigated genes are not involved in regulating cell proliferation or adhesion; however, they play an important role in promoting the survival of myeloma cells. Accordingly, knock-down of MAGE-C1/CT7 and MAGE-A3 led to the induction of apoptosis in the malignant plasma cells and, importantly, both genes were also essential for the survival of clonogenic myeloma precursors. Finally, silencing of cancer-testis genes further improved the response of myeloma cells to conventional therapies. CONCLUSIONS: Cancer-testis antigens such as MAGE-C1/CT7 and MAGE-A3 play an important role in promoting the survival of myeloma cells and clonogenic precursors by reducing the rate of spontaneous and chemotherapy-induced apoptosis and might, therefore, represent attractive targets for novel myeloma-specific therapies.

Longitudinal analysis and prognostic effect of cancer-testis antigen expression in multiple myeloma.

PURPOSE: Reliable data on the persistence of tumor expression of cancer-testis (CT) antigens over time and consequent analyses of the effect of CT antigen expression on the clinical course of malignancies are crucial for their evaluation as diagnostic markers and immunotherapeutic targets. EXPERIMENTAL DESIGN: Applying conventional reverse transcription-PCR, real-time PCR, and Western blot, we did the first longitudinal study of CT antigen expression in multiple myeloma analyzing 330 bone marrow samples from 129 patients for the expression of four CT antigens (MAGE-C1/CT7, MAGE-C2/CT10, MAGE-A3, and SSX-2). RESULTS: CT antigens were frequently and surprisingly persistently expressed, indicating that down-regulation of these immunogenic targets does not represent a common tumor escape mechanism in myeloma. We observed strong correlations of CT antigen expression levels with the clinical course of myeloma patients as indicated by the number of bone marrow-residing plasma cells and peripheral paraprotein levels, suggesting a role for CT antigens as independent tumor markers. Investigating the prognostic value of CT antigen expression in myeloma patients after allogeneic stem cell transplantation, we found that expression of genes, such as MAGE-C1, represents an important indicator of early relapse and dramatically reduced survival. CONCLUSIONS: Our findings suggest that CT antigens might promote the progression of multiple myeloma and especially MAGE-C1/CT7, which seems to play the role of a "gatekeeper" gene for other CT antigens, might characterize a more malignant phenotype. Importantly, our study also strongly supports the usefulness of CT antigens as diagnostic and prognostic markers as well as therapeutic targets in myeloma.

Molecular properties of Oep21, an ATP-regulated anion-selective solute channel from the outer chloroplast membrane.

The flux of phosphorylated carbohydrates, the major export products of chloroplasts, is regulated at the level of the inner and presumably also at the level of the outer membrane. This is achieved through modulation of the outer membrane Oep21 channel currents and tuning of its ion selectivity. Refined analysis of the Oep21 channel properties by biochemical and electrophysiological methods revealed a channel formed by eight beta-strands with a wider pore vestibule of dvest approximately 2.4 nm at the intermembrane site and a narrower filter pore of drestr approximately 1 nm. The Oep21 pore contains two high affinity sites for ATP, one located at a relative transmembrane electrical distance delta = 0.56 and the second close to the vestibule at the intermembrane site. The ATP-dependent current block and reduction in anion selectivity of the Oep21 channel is relieved by the competitive binding of phosphorylated metabolic intermediates like 3-phosphoglycerate and glycerinaldehyde 3-phosphate. Deletion of a C-terminal putative FX4K binding motif in Oep21 decreased the capability of the channel to tune its ion selectivity by about 50%, whereas current block remained unchanged.

Prediction of the plant beta-barrel proteome: a case study of the chloroplast outer envelope.

In the postgenomic era, the transformation of genetic information into biochemical meaning is required. We have analyzed the proteome of the chloroplast outer envelope membrane by an in silico and a proteomic approach. Based on its evolutionary relation to the outer membrane of Gram-negative bacteria, the outer envelope membrane should contain a large number of beta-barrel proteins. We therefore calculated the probability for the existence of beta-sheet, beta-barrel, and hairpin structures among all proteins of the Arabidopsis thaliana genome. According to the existence of these structures, a number of candidates were selected. This protein pool was analyzed by TargetP to discard sequences with signals that would direct the protein to other organelles different from chloroplasts. In addition, the pool was manually controlled for the presence of proteins known to function outside of the chloroplast envelope. The approach developed here can be used to predict the topology of beta-barrel proteins. For the proteomic approach, proteins of highly purified outer envelope membranes of chloroplasts from Pisum sativum were analyzed by ESI-MS/MS mass spectrometry. In addition to the known components, four new proteins of the outer envelope membranes were identified in this study.