Long-term risk associated with clonal hematopoiesis in patients with severe aortic valve stenosis undergoing TAVR.

BACKGROUND: Mutations in the clonal hematopoiesis of indeterminate potential (CHIP)-driver genes DNMT3A and TET2 have been previously shown to be associated with short-term prognosis in patients undergoing TAVR for aortic valve stenosis. We aimed to extend and characterize these findings on long-term outcome in a large cohort. METHODS: A total of 453 consecutive patients undergoing TAVR were included in an up to 4-year follow-up study. Next-generation sequencing was used to identify DNMT3A- and/or TET2-CHIP-driver mutations. Primary endpoint was all-cause mortality. Since CHIP-driver mutations appear to be closely related to DNA methylation, results were also assessed in patients who never smoked, a factor known to interfere with DNA methylation. RESULTS: DNMT3A-/TET2-CHIP-driver mutations were present in 32.4% of patients (DNMT3A n = 92, TET2 n = 71), and were more frequent in women (52.4% vs. 38.9%, p = 0.007) and older participants (83.3 vs. 82.2 years, p = 0.011), while clinical characteristics or blood-derived parameters did not differ. CHIP-driver mutations were associated with a significantly higher mortality up to 4 years after TAVR in both univariate (p = 0.031) and multivariate analyses (HR 1.429, 95%CI 1.014-2.013, p = 0.041). The difference was even more pronounced (p = 0.011) in never smokers. Compared to TET2 mutation carriers, patients with DNMT3A mutations had significantly less frequently concomitant coronary and peripheral artery disease. CONCLUSION: DNMT3A- and TET2-CHIP-driver mutations are associated with long-term mortality in patients with aortic valve stenosis even after a successful TAVR. The association is also present in never smokers, in whom no biasing effect from smoking on DNA methylation is to be expected.

Low Circulating Musclin is Associated With Adverse Prognosis in Patients Undergoing Transcatheter Aortic Valve Implantation at Low-Intermediate Risk.

Background Musclin is an activity-stimulated and cardioprotective myokine that attenuates pathological cardiac remodeling. Musclin deficiency, in turn, results in reduced physical endurance. The aim of this study was to assess the prognostic value of circulating musclin as a novel, putative biomarker to identify patients undergoing transcatheter aortic valve implantation (TAVI) who are at a higher risk of death. Methods and Results In this study, we measured systemic musclin levels in 368 patients undergoing TAVI who were at low to intermediate clinical risk (median EuroSCORE [European System for Cardiac Operative Risk Evaluation] II: 3.5; quartile 1-quartile, 2.2%-5.3%), whereby 209 (56.8%) patients were at low and 159 (43.2%) were at intermediate risk. Median preprocedural musclin levels were 2.7 ng/mL (quartile 1-quartile 3, 1.5-4.6 ng/mL). Musclin levels were dichotomized in low (<2.862 ng/mL, n=199 [54.1%]) or high (≥ 2.862 ng/mL, n=169 [45.9%]) groups using cutoff values determined by classification and regression tree analysis. The primary end point was 1-year overall survival. Patients with low circulating musclin levels exhibited a significantly higher prevalence of frailty, low albumin values, hypertension, and history of stroke as well as higher N-terminal pro-B-type natriuretic peptide. Low musclin levels significantly predicted risk of death in univariable (hazard ratio, 1.81; 95% CI, 1.00-3.53 [P=0.049]) and multivariable (adjusted hazard ratio, 2.45; 95% CI, 1.06-5.69 [P=0.037]) Cox regression analyses. Additionally, low musclin levels in combination with conventional EuroSCORE II suggested improved risk stratification in patients undergoing TAVI who were at low to intermediate clinical risk into subgroups with reduced 1-year survival rates by log-rank test (P for trend=0.003). Conclusions Circulating musclin is an independent predictor of 1-year overall survival in patients undergoing TAVI. Combined with EuroSCORE II, circulating musclin might help to improve prediction of mortality in patients undergoing TAVI who are at low to intermediate clinical risk.

Single-cell RNA-sequencing reveals profound changes in circulating immune cells in patients with heart failure.

AIMS: Identification of signatures of immune cells at single-cell level may provide novel insights into changes of immune-related disorders. Therefore, we used single-cell RNA-sequencing to determine the impact of heart failure on circulating immune cells. METHODS AND RESULTS: We demonstrate a significant change in monocyte to T-cell ratio in patients with heart failure, compared to healthy subjects, which were validated by flow cytometry analysis. Subclustering of monocytes and stratification of the clusters according to relative CD14 and FCGR3A (CD16) expression allowed annotation of classical, intermediate, and non-classical monocytes. Heart failure had a specific impact on the gene expression patterns in these subpopulations. Metabolically active genes such as FABP5 were highly enriched in classical monocytes of heart failure patients, whereas ß-catenin expression was significantly higher in intermediate monocytes. The selective regulation of signatures in the monocyte subpopulations was validated by classical and multifactor dimensionality reduction flow cytometry analyses. CONCLUSION: Together this study shows that circulating cells derived from patients with heart failure have altered phenotypes. These data provide a rich source for identification of signatures of immune cells in heart failure compared to healthy subjects. The observed increase in FABP5 and signatures of Wnt signalling may contribute to enhanced monocyte activation.

[Detection of disseminated tumor cells in the bone marrow of breast cancer patients with a new molecular method]. / Detekcja rozsianych komórek nowotworowych w szpiku kostnym u pacjentek z rakiem piersi z zastosowaniem nowej metody molekularnej.

OBJECTIVES: Numerous studies have shown that the presence of clinically occult disseminated tumor cells (DTC) in the bone marrow (BM) of breast cancer patients is associated with an unfavourable clinical outcome. Immunocytochemistry (ICC) remains the gold standard for their detection. Assays based on RT-PCR are available; however so far they have not been used for routine detection of DTC. Therefore, the purpose of this study was to evaluate a newly established molecular method for the detection of DTC. MATERIALS AND METHODS: BM aspirates from 405 patients were examined. Half of the samples were immediately inserted into ICC and the other half was examined with our newly established molecular method based on RT-PCR. Immunocytochemistry was performed according to the Consensus Recommendations of the German, Austrian, and Swiss Societies of Senology and ISHAGE Working Group (A45B-B3 antibody). RT-PCR was conducted as a one-step real-time assay Cytokeratin 19-mRNA was amplified. RESULTS: In 142 of 405 (35%) aspirates disseminated tumor cells were detected by RT-PCR. In 34% of patients DTC were detected by ICC. 48% of the BM samples were positive by at least one method. In 73% of the patients identical results were obtained (p<0,001). CONCLUSION: Our newly established molecular assay for the detection of disseminated tumor cells, and thus minimal residual disease, is sensitive, fast and reproducible, and has a potential to be used as a confirmatory or alternative test for DTC detection.

Evaluation of a RT-PCR based routine screening tool for the detection of disseminated epithelial cells in the bone marrow of breast cancer patients.

PURPOSE: Numerous studies have shown that the presence of clinically occult disseminated tumor cells (DTC's) in the bone marrow (BM) of breast cancer patients is associated with an unfavourable clinical outcome. Immunocytochemistry (ICC) remains the gold standard for their detection. While assays based on RT-PCR are available, they have not been used for routine detection of DTC's. METHODS: To assess the quality of the assay, we performed a direct comparison of DTC detection rates in a large cohort of 385 patients using both standardized ICC and real-time RT-PCR protocols. Correlation rates were assessed, and results were compared with clinical data. RESULTS: A significant correlation between ICC and RT-PCR was observed (P < 0.01). Positivity rates were similar (both 35%) and the results of both methods agreed in 73% of cases (280/385). CONCLUSIONS: We describe a real-time RT-PCR based protocol for DTC-detection that has been specifically designed for routine clinical laboratory use. As such, RT-PCR has the potential to become an alternative testing method for BM evaluation in breast cancer patients.

ERalpha-status of disseminated tumour cells in bone marrow of primary breast cancer patients.

INTRODUCTION: Isolated disseminated tumour cells (DTC) are regarded as surrogate markers for minimal residual disease in breast cancer. Characterisation of these cells could help understand the known limitations of adjuvant therapy. Of particular interest is their oestrogen-receptor (ER) status because endocrine adjuvant therapy remains a cornerstone of breast cancer treatment. METHODS: Bone marrow (BM) aspirates from 254 patients with primary breast cancer were included in this study. A double immunofluorescence staining procedure was established for the identification of cytokeratin (CK) positive/Eralpha-positive cells. ERalpha status of the primary tumour was assessed immunohistochemically using the same antibody against ERalpha. RESULTS: In 107 of 254 (42%) breast cancer patients, CK-positive cells could be detected in the BM. More than one DTC in the BM was observed in 38 of the 107 patients. The number of detected cells ranged between 1 and 55 cells per 2 x 10(6) mononuclear cells. DTCs demonstrated ERalpha positivity in 12% of the patients. The ERalpha expression was heterogeneous in 10 of the 38 (26%) patients with more than one DTC. The concordance rate of ERalpha status between primary tumour and DTC was 28%. Only 12 of 88 patients with ERalpha-positive tumours also had ERalpha-positive DTCs. CONCLUSIONS: Primary tumours and DTCs displayed a concordant ERalpha status in only 28% of cases. Most of the DTCs were ERalpha negative despite the presence of an ERalpha-positive primary tumour. These findings further underline the distinct nature of DTCs and may explain the failure rates seen in conventional endocrine adjuvant therapy.

Primary systemic therapy does not eradicate disseminated tumor cells in breast cancer patients.

INTRODUCTION: The presence of disseminated tumor cells in the bone marrow of breast cancer patients has proven to be an independent prognostic factor. The aim of this study was to investigate the status of tumor cell dissemination after primary systemic therapy in relation to therapy response. METHODS: Bone marrow aspirates were obtained from 120 patients after completion of primary systemic therapy. Disseminated tumor cells were detected by immunocytochemistry using the APAAP method. Bone marrow status was correlated with clinicopathological factors as well as tumor response to primary systemic therapy. RESULTS: Sixty out of 120 patients had disseminated tumor cells in their bone marrow aspirates (50%). Response rates were 18% for pathologic complete remission, 52% for partial remission, 28% for no change and 3% for progression. Despite complete remission, 36% of these patients were bone marrow positive. In the partial remission group, the positivity rate was 48%. About 61% of patients with stable disease had disseminated tumor cells in their bone marrow. A trend to higher positivity rates was observed in the poor responder group compared to responders (61% vs. 38%, P = 0.1). CONCLUSION: Primary systemic therapy does not completely eradicate disseminated tumor cells in the bone marrow of breast cancer patients. The biological role of persistent disseminated tumor cells needs to be further investigated to optimize current and future therapeutic strategies.

Presence of apoptotic and nonapoptotic disseminated tumor cells reflects the response to neoadjuvant systemic therapy in breast cancer.

INTRODUCTION: Neoadjuvant systemic therapy (NST) is an established strategy to reduce tumor size in breast cancer patients prior to breast-conserving therapy. The effect of NST on tumor cell dissemination in these patients is not known. The aim of this study was to investigate the incidence of disseminated tumor cells (DTC), including apoptotic DTC, in breast cancer patients after NST, and to investigate the correlation of DTC status with therapy response. METHODS: Bone marrow aspiration was performed in 157 patients after NST. DTC were detected by immunocytochemistry using the A45-B/B3 anticytokeratin antibody. To detect apoptotic DTC the antibody M30 (Roche Diagnostics, Germany) was used, which detects a neo-epitope expressed only after caspase cleavage of cytokeratin 18 during early apoptosis. RESULTS: The incidence of DTC in breast cancer patients was 53% after completion of NST. Tumor dissemination was observed more frequently in patients with no change/progressive disease (69%) than in patients with partial remission or complete remission of the primary tumor (46%) (P < 0.05). Ten out of 24 patients with complete remission, however, were still bone marrow positive. Apoptotic DTC were present in 36 of 157 (23%) breast cancer patients. Apoptotic cells only were detected in 14% of the patients with partial remission or complete remission, but were detected in just 5% of the patients with stable disease. Apoptotic DTC were detectable in none of the patients with tumor progression. CONCLUSION: The pathological therapy response in breast cancer patients is reflected by the presence of apoptotic DTC. Patients with complete remission, however, may still have nonapoptotic DTC. These patients may also benefit from secondary adjuvant therapy.

Detection of cytokeratin-positive cells in the bone marrow of breast cancer patients undergoing adjuvant therapy.

The presence of cytokeratin-positive cells in the bone marrow of breast cancer patients has been proven to be an independent prognostic factor. Their fate in primary breast cancer patients undergoing adjuvant therapy is of particular interest. We investigated the bone marrow status of 112 patients undergoing postoperative adjuvant treatment before and after therapy. A total of 373 patients with histologically confirmed primary breast cancer underwent bone marrow aspiration at the time of primary surgery. All patients were informed of their bone marrow status and offered repeat aspiration after 12 months. All patients were then treated with adjuvant chemotherapy, endocrine therapy or both based on current treatment recommendations. About 112 patients returned for a second bone marrow aspiration after a mean interval of 12 months following the initiation of adjuvant treatment. In 93 of 112 patients (83%) disseminated tumor cells had been found in the bone marrow before initiation of systemic chemo/endocrine therapy. At the time of follow-up sampling, after surgery and completion of adjuvant chemotherapy, the positivity rate dropped to 24%. Positive bone marrow status during follow-up was only associated with grading (p=0.020). Adjuvant treatment regimens are not able to completely eliminate cytokeratin-positive cells from the bone marrow. Prospective studies need to evaluate, whether these cells could become targets for additional adjuvant therapy.

Her2 expression on disseminated tumor cells from bone marrow of breast cancer patients.

BACKGROUND: The presence of cytokeratin (CK)-positive cells in the bone marrow (BM) of breast cancer patients is an independent prognostic marker for long-term survival. While the exact nature of these cells remains under investigation, their persistence after chemotherapy is linked to decreased survival, making them a potential therapeutic target. Targeted therapy using the her2 antibody has been shown to improve survival in breast cancer patients. We studied the her2 expression in the BM of 105 patients with CK-positive cells present and compared it with results from the primary tumor. MATERIALS AND METHODS: BM cytospins were stained to detect CK-positive cells. In 105 patients with detectable CK-positive cells, additional staining with her2 antibody was performed. Using an automated imaging system, the her2 slides were evaluated based on the criteria of the International Society for Cellular Therapy. RESULTS were correlated with the her2 status of the primary tumor. Furthermore, the presence of her2 mRNA was examined in a subset of 27 patients using RT-PCR. RESULTS: On 22/105 (21%) cytospins with CK-positive cells, her2-positive cells could be detected. The positivity rate for RT-PCR was 15%. Her2 overexpression on the primary tumor was 26/105 (25%). Correlation with the BM status was as follows: Hercep score 0 to 1+: 79 patients, 10 with positive BM for her2 (12%). Hercep scores 2+ to 3+: 26 patients, 12 with her2-positive bone marrows (46%) (p=0.001). CONCLUSION: Her2-positive cells were detected in the BM of 15-21% of patients who were also CK-positive using immunocytochemistry and RT-PCR. Correlation exists between the presence of her2 on the primary tumor, the hercep score and the presence of her2- positive cells in the BM both with RT-PCR and immunocytochemistry. Despite this correlation, in 12.6% of patients with a her2-negative primary tumor, her2-positive cells could be detected in the BM.

Image analysis systems for the detection of disseminated breast cancer cells on bone-marrow cytospins.

We assessed the accuracy of automated cell imaging systems when compared to manual evaluation of cytospin slides in determining the presence of cytokeratin-positive, disseminated breast cancer cells in bone marrow aspirates. A total of 298 cytospin slides of bone marrow aspirates were first evaluated by individual screening by one expert immunocytologist. Subsequently, all 298 slides were evaluated by the Automated Cell Imaging System (ACIS) by ChromaVisiontrade mark. Two separate analysis cycles were performed using ACIS. The results of the two ACIS analysis cycles were almost identical: in 293 out of 298 samples (98.3%), identical numbers of disseminated breast cancer cells were detected. In the remaining five samples (1.7%), the result of the two ACIS analysis cycles differed by only one tumor cell. By using the manual technique, 120 cytospin samples were found to be positive. ACIS was able to detect additional tumor cells in 64 cases. Not once did ACIS miss tumor cells when compared to the manual technique. Using ACIS, we were able to determine the bone marrow status of patients with nonmetastatic breast cancer faster, with greater accuracy, and with greater reproducibility than with the manual technique.

Survival of Ugandan infants with subtype A and D HIV-1 infection (HIVNET 012).

Virologic factors may influence survival of HIV-1-infected infants. We compared survival of Ugandan infants with subtype A and subtype D HIV-1 infection. This study was performed in the context of the Ugandan clinical trial HIVNET 012, which compared the efficacy of single-dose nevirapine (NVP) and short-course zidovudine (AZT) for prevention of HIV-1 mother-to-child transmission. HIV-1 subtypes were determined by phylogenetic analysis of HIV-1 protease and reverse transcriptase sequences from 32 women in the NVP arm and 54 women in the AZT arm of HIVNET 012 whose infants were HIV-1 infected by 6 to 8 weeks of age. We found no association between HIV-1 subtype (A vs. D) and infant survival in this cohort. Further studies are needed to evaluate whether HIV-1 subtype influences clinical outcome in pediatric HIV-1 infection.

Analysis of length variation in the V1-V2 region of env in nonsubtype B HIV type 1 from Uganda.

We optimized an assay for analysis of length variation in the V1-V2 region of HIV-1 env in plasma samples from Uganda. V1-V2 env length variation was analyzed in 31 plasma samples containing subtype A, C, D, or A/D recombinant HIV-1. DNA corresponding to the V1-V2 region was amplified by nested PCR. One of the primers in the second step of the PCR was fluorescently labeled. Successful amplification was confirmed by agarose gel electrophoresis. V1-V2 length variation of PCR products was analyzed with an ABI PRISM 3100 genetic analyzer and GeneScan software. A diversity score was generated for each sample on the basis of the degree of fragment length variation. The V1-V2 region was successfully amplified from 30 of 31 samples. Fragment length analysis was successful for all of those 30 samples. The diversity score and lengths of V1-V2 fragments were unique for each sample. This assay can be used for analysis of V1-V2 length variation in subtypes commonly found in Uganda. This assay may be helpful for studies examining the impact of env length diversity on HIV-1 transmission and pathogenesis in regions where these subtypes are prevalent.

Identification of Ugandan HIV type 1 variants with unique patterns of recombination in pol involving subtypes A and D.

Most HIV-1 infections in Uganda are caused by subtypes A and D. The prevalence of recombination and the sites of specific breakpoints between these subtypes have not been reported. HIV-1 pol sequences encoding protease (amino acids 1-99) and reverse transcriptase (amino acids 1-324) from 102 pregnant Ugandan women were analyzed by the Recombinant Identification Program, SimPlot, and examination of phylogenetically informative sites to identify sites of recombination between sequence segments belonging to different subtypes. Thirteen percent (13 of 102) of the pol sequences contained strong evidence of recombination between subtypes A and D. At least nine different patterns of recombination were observed. Five women infected with a recombinant virus transmitted the recombinant virus perinatally. In this population-based study, intersubtype recombinants were common. The large number of different types of pol recombinants identified suggests that recombination occurs readily in the pol region. Perinatal transmission of the recombinant viruses demonstrates their evolutionary stability.