Sex-specific Genetic Determinants of Right Ventricular Structure and Function.

RATIONALE: While sex differences in right heart phenotypes have been observed, the molecular drivers remain unknown. OBJECTIVES: To provide biological insights into sex differences in the structure and function of the right ventricle (RV) using common genetic variation. METHODS: RV phenotypes were obtained from cardiac magnetic resonance imaging in 18,156 women and 16,171 men from the UK Biobank. Observational analyses and sex-stratified genome-wide association studies were performed. Candidate female-specific loci were evaluated against invasively measured cardiac performance in 479 female patients with idiopathic or heritable pulmonary arterial hypertension (PAH), recruited to the UK NIHR BioResource Rare Diseases study. MEASUREMENTS AND MAIN RESULTS: Sex was associated with differences in RV volumes and ejection fraction in models adjusting for left heart counterparts, blood pressure, lung function and sex hormone levels. Six genome-wide significant loci (13%) revealed heterogeneity of allelic effects between women and men, and significant sex-by-genotype interaction. These included two sex-specific candidate loci present in women only: a locus for RV ejection fraction in BMPR1A and a locus for RV end-systolic volume near DMRT2. Epigenetic data in RV tissue indicate that variation at the BMPR1A locus likely alters transcriptional regulation. In female patients with PAH, a variant located in the promoter of BMPR1A was significantly associated with cardiac index (effect size 0.16 l/min/m2), despite similar RV afterload. CONCLUSIONS: BMPR1A has emerged as a biologically plausible candidate gene for female-specific genetic determination of RV function, showing associations with cardiac performance under chronically increased afterload in female patients with PAH.

Using the Plasma Proteome for Risk Stratifying Patients with Pulmonary Arterial Hypertension.

Rationale: NT-proBNP (N-terminal pro-brain natriuretic peptide), a biomarker of cardiac origin, is used to risk stratify patients with pulmonary arterial hypertension (PAH). Its limitations include poor sensitivity to early vascular pathology. Other biomarkers of vascular or systemic origin may also be useful in the management of PAH. Objectives: Identify prognostic proteins in PAH that complement NT-proBNP and clinical risk scores. Methods: An aptamer-based assay (SomaScan version 4) targeting 4,152 proteins was used to measure plasma proteins in patients with idiopathic, heritable, or drug-induced PAH from the UK National Cohort of PAH (n = 357) and the French EFORT (Evaluation of Prognostic Factors and Therapeutic Targets in PAH) study (n = 79). Prognostic proteins were identified in discovery-replication analyses of UK samples. Proteins independent of 6-minute-walk distance and NT-proBNP entered least absolute shrinkage and selection operator modeling, and the best combination in a single score was evaluated against clinical targets in EFORT. Measurements and Main Results: Thirty-one proteins robustly informed prognosis independent of NT-proBNP and 6-minute-walk distance in the UK cohort. A weighted combination score of six proteins was validated at baseline (5-yr mortality; area under the curve [AUC], 0.73; 95% confidence interval [CI], 0.63-0.85) and follow-up in EFORT (AUC, 0.84; 95% CI, 0.75-0.94; P = 9.96 × 10-6). The protein score risk stratified patients independent of established clinical targets and risk equations. The addition of the six-protein model score to NT-proBNP improved prediction of 5-year outcomes from AUC 0.762 (0.702-0.821) to 0.818 (0.767-0.869) by receiver operating characteristic analysis (P = 0.00426 for difference in AUC) in the UK replication and French samples combined. Conclusions: The plasma proteome informs prognosis beyond established factors in PAH and may provide a more sensitive measure of therapeutic response.

Mining the Plasma Proteome for Insights into the Molecular Pathology of Pulmonary Arterial Hypertension.

Rationale: Pulmonary arterial hypertension (PAH) is characterized by structural remodeling of pulmonary arteries and arterioles. Underlying biological processes are likely reflected in a perturbation of circulating proteins. Objectives: To quantify and analyze the plasma proteome of patients with PAH using inherited genetic variation to inform on underlying molecular drivers. Methods: An aptamer-based assay was used to measure plasma proteins in 357 patients with idiopathic or heritable PAH, 103 healthy volunteers, and 23 relatives of patients with PAH. In discovery and replication subgroups, the plasma proteomes of PAH and healthy individuals were compared, and the relationship to transplantation-free survival in PAH was determined. To examine causal relationships to PAH, protein quantitative trait loci (pQTL) that influenced protein levels in the patient population were used as instruments for Mendelian randomization (MR) analysis. Measurements and Main Results: From 4,152 annotated plasma proteins, levels of 208 differed between patients with PAH and healthy subjects, and 49 predicted long-term survival. MR based on cis-pQTL located in proximity to the encoding gene for proteins that were prognostic and distinguished PAH from health estimated an adverse effect for higher levels of netrin-4 (odds ratio [OR], 1.55; 95% confidence interval [CI], 1.16-2.08) and a protective effect for higher levels of thrombospondin-2 (OR, 0.83; 95% CI, 0.74-0.94) on PAH. Both proteins tracked the development of PAH in previously healthy relatives and changes in thrombospondin-2 associated with pulmonary arterial pressure at disease onset. Conclusions: Integrated analysis of the plasma proteome and genome implicates two secreted matrix-binding proteins, netrin-4 and thrombospondin-2, in the pathobiology of PAH.

[Targeted therapy for pulmonary arterial hypertension in patients without comorbidities]. / Gezielte medikamentöse Therapie der pulmonalarteriellen Hypertonie bei Patient*innen ohne Komorbiditäten.

The 2022 guidelines on pulmonary hypertension from the European Society of Cardiology (ESC) and the European Respiratory Society (ERS) provide therapeutic strategies that account for the variability in the clinical presentation of newly diagnosed patients. We summarize treatment recommendations for pulmonary arterial hypertension (PAH) in patients without significant comorbidities, particularly for idiopathic, hereditary, drug/toxin-induced, or connective tissue disease-associated PAH. In this group of patients, multidimensional assessments for short-term mortality risk guide initial treatment decisions and treatment decisions during follow-up. Upfront dual combination therapy (phosphodiesterase type-5 inhibitor and endothelin receptor antagonist) is recommended for low- and intermediate-risk patients, and triple therapy including a parenteral prostacyclin should be considered in high- or intermediate-high-risk patients. If a low or intermediate-low-risk profile cannot be achieved during therapy, sequential add-on therapy escalation with parenteral prostacyclin or a prostacyclin receptor agonist should be considered, and switching from a phosphodiesterase type-5 inhibitor to a guanylate cyclase stimulator may also be considered.

[Genetic diagnostics and molecular approaches in pulmonary arterial hypertension]. / Genetische Diagnostik und molekulare Ansätze bei pulmonalarterieller Hypertonie.

The recently published new European guidelines for diagnosis and treatment of pulmonary hypertension now offer the so far most extensive description of genetic testing and counselling for pulmonary arterial hypertension patients. In addition, the importance of a clinical screening of healthy mutation carriers is highlighted as well as the genetic testing of patients with a suspicion of pulmonary veno-occlusive disease. We frame the respective parts of the guidelines on genetic testing and counselling in the context of recent data and provide comments. Finally, we give an outlook on novel molecular approaches starting from Sotatercept, addressing ion channels and novel therapeutic developments.

Mendelian randomisation and experimental medicine approaches to interleukin-6 as a drug target in pulmonary arterial hypertension.

BACKGROUND: Inflammation and dysregulated immunity are important in the development of pulmonary arterial hypertension (PAH). Compelling preclinical data supports the therapeutic blockade of interleukin-6 (IL-6) signalling. METHODS: We conducted a phase 2 open-label study of intravenous tocilizumab (8 mg·kg-1) over 6 months in patients with group 1 PAH. Co-primary end-points were safety, defined by incidence and severity of adverse events, and change in pulmonary vascular resistance. Separately, a mendelian randomisation study was undertaken on 11 744 individuals with European ancestry including 2085 patients with idiopathic/heritable disease for the IL-6 receptor (IL6R) variant (rs7529229), known to associate with circulating IL-6R levels. RESULTS: We recruited 29 patients (male/female 10/19; mean±sd age 54.9±11.4 years). Of these, 19 had heritable/idiopathic PAH and 10 had connective tissue disease-associated PAH. Six were withdrawn prior to drug administration; 23 patients received at least one dose of tocilizumab. Tocilizumab was discontinued in four patients owing to serious adverse events. There were no deaths. Despite evidence of target engagement in plasma IL-6 and C-reactive protein levels, both intention-to-treat and modified intention-to-treat analyses demonstrated no change in pulmonary vascular resistance. Inflammatory markers did not predict treatment response. Mendelian randomisation did not support an effect of the lead IL6R variant on risk of PAH (OR 0.99, p=0.88). CONCLUSION: Adverse events were consistent with the known safety profile of tocilizumab. Tocilizumab did not show any consistent treatment effect.

Respiratory muscle dysfunction in long-COVID patients.

PURPOSE: Symptoms often persistent for more than 4 weeks after COVID-19-now commonly referred to as 'Long COVID'. Independent of initial disease severity or pathological pulmonary functions tests, fatigue, exertional intolerance and dyspnea are among the most common COVID-19 sequelae. We hypothesized that respiratory muscle dysfunction might be prevalent in persistently symptomatic patients after COVID-19 with self-reported exercise intolerance. METHODS: In a small cross-sectional pilot study (n = 67) of mild-to-moderate (nonhospitalized) and moderate-to-critical convalescent (formerly hospitalized) patients presenting to our outpatient clinic approx. 5 months after acute infection, we measured neuroventilatory activity P0.1, inspiratory muscle strength (PImax) and total respiratory muscle strain (P0.1/PImax) in addition to standard pulmonary functions tests, capillary blood gas analysis, 6 min walking tests and functional questionnaires. RESULTS: Pathological P0.1/PImax was found in 88% of symptomatic patients. Mean PImax was reduced in hospitalized patients, but reduced PImax was also found in 65% of nonhospitalized patients. Mean P0.1 was pathologically increased in both groups. Increased P0.1 was associated with exercise-induced deoxygenation, impaired exercise tolerance, decreased activity and productivity and worse Post-COVID-19 functional status scale. Pathological changes in P0.1, PImax or P0.1/PImax were not associated with pre-existing conditions. CONCLUSIONS: Our findings point towards respiratory muscle dysfunction as a novel aspect of COVID-19 sequelae. Thus, we strongly advocate for systematic respiratory muscle testing during the diagnostic workup of persistently symptomatic, convalescent COVID-19 patients.

[Organ-specific sequelae of COVID-19 in adults]. / Organbezogene Folgeerscheinungen von COVID­19 bei Erwachsenen.

Organ-specific sequelae after COVID-19 occur frequently and are highly diverse in their features. Sequelae and symptoms persisting for more than four weeks after COVID-19 define the condition "long COVID."Organ-specific sequelae of COVID-19 generally occur more often after severe disease. Yet, duration and intensity of organ-specific sequelae are highly variable. While pulmonary sequelae typically persist after more severe acute disease, COVID-19 sequelae may also develop weeks after infection and can affect any organ. The degree of SARS-CoV­2 specificity of COVID-19 sequelae, however, remains unclear. Thus, diagnosis and treatment of COVID-19 sequelae represent an interdisciplinary challenge. Diagnostic and therapeutic approaches are guided by type, extent, and cause of the specific sequelae as targeted therapy options for long COVID are lacking.In the present work, we review current knowledge regarding the prevalence/incidence, duration, specificity, type, and extent of organ-specific COVID-19 sequelae and summarize current diagnostic and therapeutic strategies (as of November 2021).

[Intimal pulmonary sarcoma: A case report of a rare differential diagnosis of chronic thromboembolic pulmonary hypertension]. / Das intimale Sarkom der Pulmonalarterie ­ Fallbericht einer seltenen Differenzialdiagnose der chronisch thromboembolischen pulmonalen Hypertonie.

Pulmonary angiosarcoma is a rare and malignant disease of the blood vessels. Initially, it can be misdiagnosed as chronic thromboembolic hypertension (CTEPH). In CTEPH, there is increased pressure and resistance of the pulmonary arteries following persistent obstruction of pulmonary circulation from (recurrent) thromboembolism despite adequate anticoagulative treatment.A 76-year-old patient was referred to our centre for pulmonary hypertension after a central, left-sided, subacute pulmonary thromboembolism had been observed 7 months earlier. It was treated with apixaban, but the patient described persistent dyspnoea and cough. We observed severely reduced diffusion capacity, ineffective ventilation during cardiopulmonary exercise testing and right heart strain on echocardiograph, signs that are in agreement with suspected CTEPH. Computer tomography of the chest showed a persistent, size-constant obliteration of the left main pulmonary artery, and ventilation perfusion scan confirmed complete interruption of perfusion. We suspected malignancy; PET-CT scan confirmed metabolically active lesions. Histopathological examination of a sample obtained from the lesion by endobronchial ultrasound-guided needle aspiration showed a sarcomatous tumour with amplification of the MDM2-gene. We diagnosed an intimal angiosarcoma of the left pulmonary artery and referred the patient to pneumectomy.Angiosarcoma of the pulmonary arteries is a rare differential diagnosis of persistent thrombotic lesion and suspected CTEPH. In 2015 there were less than 300 cases described.Pulmonary angiosarcoma should be considered if: lesion occupies the entire lumen of pulmonary arteries with dilatation, contrast enhancement and infiltration of the wall in radiological examination, FDG-PET CT reveals metabolically active lesions, no pulmonary thromboembolism was documented in the anamnesis, increase in size is seen despite anticoagulation, patient presents with B symptoms.Diagnosis confirmed by biopsy, resection of tumour and removal of metastases is the therapeutic standard. Median survival remains poor. Further research is needed for improved diagnosis and treatment.

Genetic evidence for a causative effect of airflow obstruction on left ventricular filling: a Mendelian randomisation study.

BACKGROUND: Observational studies on the general population have suggested that airflow obstruction associates with left ventricular (LV) filling. To limit the influence of environmental risk factors/exposures, we used a Mendelian randomisation (MR) approach based on common genetic variations and tested whether a causative relation between airflow obstruction and LV filling can be detected. METHODS: We used summary statistics from large genome-wide association studies (GWAS) on the ratio of forced expiratory volume in 1 s to forced vital capacity (FEV1/FVC) measured by spirometry and the LV end-diastolic volume (LVEDV) as assessed by cardiac magnetic resonance imaging. The primary MR was based on an inverse variance weighted regression. Various complementary MR methods and subsets of the instrument variables were used to assess the plausibility of the findings. RESULTS: We obtained consistent evidence in our primary MR analysis and subsequent sensitivity analyses that reducing airflow obstruction leads to increased inflow to the LV (odds ratio [OR] from inverse variance weighted regression 1.05, 95% confidence interval [CI] 1.01-1.09, P = 0.0172). Sensitivity analyses indicated a certain extent of negative horizontal pleiotropy and the estimate from biased-corrected MR-Egger was adjusted upward (OR 1.2, 95% CI 1.09-1.31, P < 0.001). Prioritisation of single genetic variants revealed rs995758, rs2070600 and rs7733410 as major contributors to the MR result. CONCLUSION: Our findings indicate a causal relationship between airflow obstruction and LV filling in the general population providing genetic context to observational associations. The results suggest that targeting (even subclinical) airflow obstruction can lead to direct cardiac improvements, demonstrated by an increase in LVEDV. Functional annotation of single genetic variants contributing most to the causal effect estimate could help to prioritise biological underpinnings.

Whole-Blood RNA Profiles Associated with Pulmonary Arterial Hypertension and Clinical Outcome.

Rationale: Idiopathic and heritable pulmonary arterial hypertension (PAH) are rare but comprise a genetically heterogeneous patient group. RNA sequencing linked to the underlying genetic architecture can be used to better understand the underlying pathology by identifying key signaling pathways and stratify patients more robustly according to clinical risk.Objectives: To use a three-stage design of RNA discovery, RNA validation and model construction, and model validation to define a set of PAH-associated RNAs and a single summarizing RNA model score. To define genes most likely to be involved in disease development, we performed Mendelian randomization (MR) analysis.Methods: RNA sequencing was performed on whole-blood samples from 359 patients with idiopathic, heritable, and drug-induced PAH and 72 age- and sex-matched healthy volunteers. The score was evaluated against disease severity markers including survival analysis using all-cause mortality from diagnosis. MR used known expression quantitative trait loci and summary statistics from a PAH genome-wide association study.Measurements and Main Results: We identified 507 genes with differential RNA expression in patients with PAH compared with control subjects. A model of 25 RNAs distinguished PAH with 87% accuracy (area under the curve 95% confidence interval: 0.791-0.945) in model validation. The RNA model score was associated with disease severity and long-term survival (P = 4.66 × 10-6) in PAH. MR detected an association between SMAD5 levels and PAH disease susceptibility (odds ratio, 0.317; 95% confidence interval, 0.129-0.776; P = 0.012).Conclusions: A whole-blood RNA signature of PAH, which includes RNAs relevant to disease pathogenesis, associates with disease severity and identifies patients with poor clinical outcomes. Genetic variants associated with lower SMAD5 expression may increase susceptibility to PAH.

Mendelian randomisation analysis of red cell distribution width in pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a rare disease that leads to premature death from right heart failure. It is strongly associated with elevated red cell distribution width (RDW), a correlate of several iron status biomarkers. High RDW values can signal early-stage iron deficiency or iron deficiency anaemia. This study investigated whether elevated RDW is causally associated with PAH.A two-sample Mendelian randomisation (MR) approach was applied to investigate whether genetic predisposition to higher levels of RDW increases the odds of developing PAH. Primary and secondary MR analyses were performed using all available genome-wide significant RDW variants (n=179) and five genome-wide significant RDW variants that act via systemic iron status, respectively.We confirmed the observed association between RDW and PAH (OR 1.90, 95% CI 1.80-2.01) in a multicentre case-control study (cases n=642, disease controls n=15 889). The primary MR analysis was adequately powered to detect a causal effect (odds ratio) between 1.25 and 1.52 or greater based on estimates reported in the RDW genome-wide association study or from our own data. There was no evidence for a causal association between RDW and PAH in either the primary (ORcausal 1.07, 95% CI 0.92-1.24) or the secondary (ORcausal 1.09, 95% CI 0.77-1.54) MR analysis.The results suggest that at least some of the observed association of RDW with PAH is secondary to disease progression. Results of iron therapeutic trials in PAH should be interpreted with caution, as any improvements observed may not be mechanistically linked to the development of PAH.

Reduced plasma levels of small HDL particles transporting fibrinolytic proteins in pulmonary arterial hypertension.

BACKGROUND: Aberrant lipoprotein metabolism has been implicated in experimental pulmonary hypertension, but the relevance to patients with pulmonary arterial hypertension (PAH) is inconclusive. OBJECTIVE: To investigate the relationship between circulating lipoprotein subclasses and survival in patients with PAH. METHODS: Using nuclear magnetic resonance spectroscopy, 105 discrete lipoproteins were measured in plasma samples from two cohorts of patients with idiopathic or heritable PAH. Data from 1124 plasma proteins were used to identify proteins linked to lipoprotein subclasses. The physical presence of proteins was confirmed in plasma lipoprotein subfractions separated by ultracentrifugation. RESULTS: Plasma levels of three lipoproteins from the small high-density lipoprotein (HDL) subclass, termed HDL-4, were inversely related to survival in both the discovery (n=127) and validation (n=77) cohorts, independent of exercise capacity, comorbidities, treatment, N-terminal probrain natriuretic peptide, C reactive protein and the principal lipoprotein classes. The small HDL subclass rich in apolipoprotein A-2 content (HDL-4-Apo A-2) exhibited the most significant association with survival. None of the other lipoprotein classes, including principal lipoprotein classes HDL and low-density lipoprotein cholesterol, were prognostic. Three out of nine proteins identified to associate with HDL-4-Apo A-2 are involved in the regulation of fibrinolysis, namely, the plasmin regulator, alpha-2-antiplasmin, and two major components of the kallikrein-kinin pathway (coagulation factor XI and prekallikrein), and their physical presence in the HDL-4 subfraction was confirmed. CONCLUSION: Reduced plasma levels of small HDL particles transporting fibrinolytic proteins are associated with poor outcomes in patients with idiopathic and heritable PAH.

The P2-receptor-mediated Ca2+ signalosome of the human pulmonary endothelium - implications for pulmonary arterial hypertension.

Dysfunction of the pulmonary endothelium is associated with most lung diseases. Extracellular nucleotides modulate a plethora of endothelial functions in the lung such as vessel integrity, vasodilatation, inflammatory, and thrombotic responses as well as survival and DNA repair, mostly via Ca2+ signaling pathways. However, a comprehensive analysis of the molecular components of the underlying P2 receptor-mediated Ca2+ signaling pathways in the lung has not been conducted so far. Therefore, our aim was to identify the principal P2 receptor Ca2+ signalosome in the human pulmonary endothelium and investigate potential dysregulation in pulmonary vascular disease. Comparative transcriptomics and quantitative immunohistochemistry were performed on publicly available RNA sequencing and protein datasets to identify the specific expression profile of the P2-receptor Ca2+ signalosome in the healthy human pulmonary endothelium and endothelial cells (EC) dysfunctional due to loss of or defective bone morphogenetic protein receptor (BMPR2). Functional expression of signalosome components was tested by single cell Ca2+ imaging. Comparative transcriptome analysis of 11 endothelial cell subtypes revealed a specific P2 receptor Ca2+ signalosome signature for the pulmonary endothelium. Pulmonary endothelial expression of the most abundantly expressed Ca2+ toolkit genes CALM1, CALM2, VDAC1, and GNAS was confirmed by immunohistochemistry (IHC). P2RX1, P2RX4, P2RY6, and P2YR11 showed strong lung endothelial staining by IHC, P2X5, and P2Y1 were found to a much lesser extent. Very weak or no signals were detected for all other P2 receptors. Stimulation of human pulmonary artery (HPA) EC by purine nucleotides ATP, ADP, and AMP led to robust intracellular Ca2+ signals mediated through both P2X and P2Y receptors. Pyrimidine UTP and UDP-mediated Ca2+ signals were generated almost exclusively by activation of P2Y receptors. HPAEC made dysfunctional by siRNA-mediated BMPR2 depletion showed downregulation of 18 and upregulation of 19 P2 receptor Ca2+ signalosome genes including PLCD4, which was found to be upregulated in iPSC-EC from BMPR2-mutant patients with pulmonary arterial hypertension. In conclusion, the human pulmonary endothelium expresses a distinct functional subset of the P2 receptor Ca2+ signalosome. Composition of the P2 receptor Ca2+ toolkit in the pulmonary endothelium is susceptible to genetic disturbances likely contributing to an unfavorable pulmonary disease phenotype found in pulmonary arterial hypertension.

Right Ventricular Index for Risk Stratification of Patients with Pulmonary Arterial Hypertension.

BACKGROUND: Right ventricular (RV) dysfunction is a major prognostic predictor in pulmonary arterial hypertension (PAH). OBJECTIVES: The objective of this study was to assess the prognostic impact of a newly developed index merging haemodynamic parameters into 1 variable. METHODS: We retrospectively assessed 2 cohorts of 248 patients (164 from Hamburg and 84 from Heidelberg) with invasively diagnosed PAH. During a median follow-up time of 3.6 years (3.1 and 4.0 years for Hamburg and Heidelberg, respectively), the composite endpoint of all-cause mortality and lung transplantation occurred in 57 patients (53 and 4 patients for Hamburg and Heidelberg, respectively). The RV index was developed in the Hamburg cohort and validated in the Heidelberg cohort: (right atrial pressure × pulmonary vascular resistance)/mixed venous oxygen saturation. RESULTS: Patients with a high RV index had a higher incidence of the combined endpoint in Kaplan-Meier analyses in the Hamburg and Heidelberg cohort (p = 0.017 and p = 0.034, respectively). The calculated RV index cut-off value was 91 and identified patients with a worse outcome in the Hamburg cohort and showed a trend in the Heidelberg cohort (p < 0.001 and p = 0.089, respectively). The RV index in Cox regression hazard models was an independent predictor of outcomes after adjustment for sex and age in both cohorts (Hamburg: hazard ratio [HR] 1.26 [95% CI 1.08, 1.47], p = 0.0027; Heidelberg: HR 2.27 [95% CI 1.46, 3.51], p < 0.001). A nomogram based on these results allowed risk stratification. CONCLUSION: Merging 3 haemodynamic variables into 1 variable, the RV index increased the prognostic power up to an independent risk factor. The RV index is easy to calculate and allows the construction of a nomogram for an individualized risk assessment.

Tumor size, tumor location, and antitumor inflammatory response are associated with lymph node size in colorectal cancer patients.

Lymph node size affects lymph node retrieval in surgical specimen and is used as criterion for pre-operative radiological estimation of metastatic disease. However, factors determining lymph node size remain to be established. Therefore, the association between lymph node size and presence of metastatic cancer deposits as well as different primary tumor characteristics was analyzed in a prospective cross-sectional study. Visible and palpable nodes were harvested, and conventional histology, immunohistochemistry, and molecular analysis were performed. The study cohort comprised 148 patients (median age 69 years, range 36-92). Lymph node dissection rendered 4167 nodes. Mean lymph node count was 28 (median 26, range 9-67). Metastatic disease was detected in 320 (8%) nodes and was associated with lymph node size (P<0.001). Positive nodes measuring ≤2 mm caused upstaging within the N category in one third of cases, but did not identify patients as node-positive as all patients also had positive larger nodes. Large tumor size (P=0.001), right tumor location (P<0.001), and deep tumor penetration (P=0.024) were all independently associated with lymph node size, whereas high lymphocytic antitumor reaction just missed statistical significance (P=0.053) in multivariable analysis. Microsatellite instability had no influence on lymph node size when analysis was restricted to right-sided tumors. In conclusion, analysis of small lymph nodes may lead to upstaging within the N category, but they do not identify a patient as node-positive and do therefore not influence clinical decision-making in the adjuvant setting. The majority of enlarged lymph nodes, including those measuring >1 cm, are not involved by cancer. Different tumor characteristics, such as large primary tumor size, right tumor location, and deep tumor penetration are independently associated with lymph node size and need to be considered when interpreting enlarged nodes detected by radiological imaging.

Lymph node retrieval in colorectal cancer: determining factors and prognostic significance.

PURPOSE: The study aimed to analyze clinicopathological factors that determine the extent of lymph node retrieval and to evaluate its prognostic impact in patients with colorectal cancer (CRC). METHODS: The number of retrieved lymph nodes was analyzed in 381 CRC specimens. Lymph node count was related to different clinicopathological variables by binary logistic regression. Progression-free survival (PFS) and cancer-specific survival (CSS) were determined using the Kaplan-Meier method and Cox regression models. RESULTS: The median number of retrieved lymph nodes was 20 (mean 21 ± 10, range 1-65) in right-sided, 13 (16 ± 10, 1-66) in left-sided, and 15 (18 ± 11, 3-64) in rectal tumors. The number of retrieved lymph nodes was independently associated with T-classification (p < 0.001), N-classification (p = 0.014), and tumor size (p = 0.005) as well as right-sided tumor location (p = 0.012). There was no association with age, sex, tumor grade, mismatch-repair status, and lymph or blood vessel invasion. The longer the surgical specimen, the higher were the numbers of retrieved and positive lymph nodes (p < 0.001, respectively). In patients with locally advanced (T3/T4) tumors (n = 283), analysis of more than 12 lymph nodes was independently associated with PFS (HR = 0.63, p = 0.025) and CSS (HR = 0.54, p = 0.004). In the subset of T3/T4 N0 patients (n = 130), analysis of more than 12 lymph nodes similarly proved to be an independent predictor of outcome (PFS, HR = 0.48, p = 0.046; OS, HR = 0.41, p = 0.026). CONCLUSION: The number of retrieved lymph nodes is associated with higher tumor stage, tumor size, and right-sided location. Low lymph node count indicates adverse outcome in patients with locally advanced (T3/T4) disease.

Exploratory analysis of the neutrophil to lymphocyte ratio in patients with pulmonary arterial hypertension.

BACKGROUND: Chronic inflammation emerges as a feature of the pathogenesis of pulmonary arterial hypertension (PAH) in experimental models. Alterations of circulating cell subsets have been observed in patients with PAH. We aimed to assess associations of the white blood cell count with disease severity and outcome in patients with PAH. METHODS: The total and differential white blood cell count was related to functional parameters, pulmonary hemodynamics and transplantation-free survival in 77 patients with PAH in an observational single center study. RESULTS: An increased neutrophil/lymphocyte ratio was associated with poor World Health Organization functional class and shorter 6-minute walking distance, as well as with elevated right atrial pressure and high level of N-terminal prohormone of brain natriuretic peptide. During a median follow-up period of 31 months (range 16-56) 23 patients died and 2 patients were referred to lung transplantation. Using uni- and subsequent bivariate Cox proportional hazards analyses an increased neutrophil/lymphocyte ratio was associated with unfavorable transplantation-free survival independent of hemodynamic parameters and C-reactive protein. The prognostic implication sustained in subsets of patients with incident PAH and in the absence of cardiovascular risk factors. CONCLUSIONS: The results of this analysis indicate that a neutrophilic inflammation may be associated with clinical deterioration and poor outcome in patients with PAH. Assessing the composition of the differential white blood cell count may render prognostic information and could represent a step towards incorporating an inflammatory marker into the clinical management of patients with PAH.

Tumour budding with and without admixed inflammation: two different sides of the same coin?

BACKGROUND: Tumour budding is an adverse prognostic indicator in colorectal cancer (CRC). Marked overall peritumoural inflammation has been associated with favourable outcome and may lead to the presence of isolated cancer cells due to destruction of invading cancer cell islets. METHODS: We assessed the prognostic significance of tumour budding and peritumoural inflammation in a cohort of 381 patients with CRC applying univariate and multivariate analyses. RESULTS: Patients with high-grade budding and marked inflammation had a significantly better outcome compared with patients with high-grade budding and only mild inflammation. Outcome in these cases, however, was still worse compared with cases with low-grade budding, in which the extent of peritumoural inflammation had no further prognostic effect. CONCLUSIONS: Tumour budding proved to be a powerful prognostic variable in patients with CRC. Scattering of invading cancer cell islets by marked overall peritumoural inflammation seems to have a minor role.

Inclusion of cytological features in tumor grading improves prognostic stratification of patients with colorectal cancer.

PURPOSE: Tumor grade is a traditional prognostic parameter in colorectal cancer. Remarkably, however, there is still no generally accepted consensus how to perform tumor grading. In this study, we systematically compared the prognostic value of traditional grading based upon histological features, that is, gland formation alone with grading based upon both histological and cytological features, such as nuclear pleomorphism and anaplasia ("alternative grade"). METHODS: Three hundred eighty-one tumors of randomly selected patients were retrospectively reviewed. Traditional and alternative tumor grades were related to various clinicopathological features and to progression-free and cancer-specific survival applying both univariate and multivariate testing. RESULTS: Traditional and alternative tumor grades were significantly associated with T and N classification, tumor size, lymphovascular invasion, as well as both progression-free and cancer-specific survival. In Cox's proportional hazards regression models, the alternative grade was superior to the traditional tumor grade and was significantly associated with progression-free survival (hazard ratio 1.57, 95% confidence interval 1.04-2.35; p = 0.031), independent of patients' age and gender, T and N classification, and lymphovascular invasion. Likewise, patients with tumors with high alternative grade were more likely to die of disease (hazard ratio 1.30, 95% confidence interval 0.85-2.00), but this difference was not statistically significant (p = 0.22). CONCLUSIONS: Tumor grade based upon both histological and cytological features was superior to grade based upon histological features alone and proved to be an independent prognostic parameter. Thus, tumor grade based upon both histological and cytological features may help to improve prognostic stratification and may thereby affect clinical decision-making and patient management.