Comparative bioinformatic analysis of KRAS, STK11 and KEAP1 (co-)mutations in non-small cell lung cancer with a special focus on KRAS G12C.

OBJECTIVES: Mutations in STK11 (STK11MUT) and KEAP1 (KEAP1MUT) occur frequently in non-small cell lung cancer (NSCLC) and are often co-mutated with KRAS. Several studies linked the co-occurrence of KRASMUT + STK11MUT, as well as KRASMUT + KEAP1MUT to reduced response to immune checkpoint inhibitors (ICI) and even a negative impact on survival. Data focusing STK11 + KEAP1 co-mutations or the triple mutation (KRAS + STK11 + KEAP1) are scarce. The recent availability of KRAS-G12C inhibitors increases the clinical relevance of those co-mutations in KRAS-mutated NSCLC. MATERIALS AND METHODS: We present a comprehensive bioinformatic analysis encompassing six datasets retrieved from cBioPortal. RESULTS: Independent of the treatment, triple mutations and STK11MUT + KEAP1MUT were significantly associated with a reduced overall survival (OS). Across treatments, OS of patients with a KRAS G12C triple mutation was significantly reduced compared to patients with KRAS G12C-only. Under ICI-therapy, there was no significant difference in OS between patients harboring the KRAS G12C-only and patients with the KRAS G12C triple mutation, but a significant difference between patients harboring KRAS non-G12C and KRAS non-G12C triple mutations. Triple mutated primary tumors showed a significantly increased frequency of distant metastases to bone and adrenal glands compared to KRAS-only mutated tumors. Additionally, our drug response analysis in cancer cell lines harboring the triple mutations revealed the WNT pathway inhibitor XAV-939 as a potential future drug candidate for this mutational situation. CONCLUSION: The triple mutation status may serve as a negative prognostic and predictive factor across treatments compared to KRASMUT-only. KRAS G12C generally seems to be a negative predictive marker for ICI-therapy.

Is iron deficiency caused by BMPR2 mutations or dysfunction in pulmonary arterial hypertension patients?

Iron deficiency is common in idiopathic and heritable pulmonary arterial hypertension patients (I/HPAH). A previous report suggested a dysregulation of the iron hormone hepcidin, which is controlled by BMP/SMAD signaling involving the bone morphogenetic protein receptor 2 (BMPR-II). Pathogenic variants in the BMPR2 gene are the most common cause of HPAH. Their effect on patients' hepcidin levels has not been investigated. The aim of this study was to assess whether iron metabolism and regulation of the iron regulatory hormone hepcidin was disturbed in I/HPAH patients with and without a pathogenic variant in the gene BMPR2 compared to healthy controls. In this explorative, cross-sectional study hepcidin serum levels were quantified by enzyme-linked immunosorbent assay. We measured iron status, inflammatory parameters and hepcidin modifying proteins such as IL6, erythropoietin, and BMP2, BMP6 in addition to BMPR-II protein and mRNA levels. Clinical routine parameters were correlated with hepcidin levels. In total 109 I/HPAH patients and controls, separated into three groups, 23 BMPR2 variant-carriers, 56 BMPR2 noncarriers and 30 healthy controls were enrolled. Of these, 84% had iron deficiency requiring iron supplementation. Hepcidin levels were not different between groups and corresponded to the degree of iron deficiency. The levels of IL6, erythropoietin, BMP2, or BMP6 showed no correlation with hepcidin expression. Hence, iron homeostasis and hepcidin regulation was largely independent from these parameters. I/HPAH patients had a physiologically normal iron regulation and no false elevation of hepcidin levels. Iron deficiency was prevalent albeit independent of pathogenic variants in the BMPR2 gene.

Phenotyping of idiopathic pulmonary arterial hypertension: a registry analysis.

BACKGROUND: Among patients meeting diagnostic criteria for idiopathic pulmonary arterial hypertension (IPAH), there is an emerging lung phenotype characterised by a low diffusion capacity for carbon monoxide (DLCO) and a smoking history. The present study aimed at a detailed characterisation of these patients. METHODS: We analysed data from two European pulmonary hypertension registries, COMPERA (launched in 2007) and ASPIRE (from 2001 onwards), to identify patients diagnosed with IPAH and a lung phenotype defined by a DLCO of less than 45% predicted and a smoking history. We compared patient characteristics, response to therapy, and survival of these patients to patients with classical IPAH (defined by the absence of cardiopulmonary comorbidities and a DLCO of 45% or more predicted) and patients with pulmonary hypertension due to lung disease (group 3 pulmonary hypertension). FINDINGS: The analysis included 128 (COMPERA) and 185 (ASPIRE) patients with classical IPAH, 268 (COMPERA) and 139 (ASPIRE) patients with IPAH and a lung phenotype, and 910 (COMPERA) and 375 (ASPIRE) patients with pulmonary hypertension due to lung disease. Most patients with IPAH and a lung phenotype had normal or near normal spirometry, a severe reduction in DLCO, with the majority having no or a mild degree of parenchymal lung involvement on chest computed tomography. Patients with IPAH and a lung phenotype (median age, 72 years [IQR 65-78] in COMPERA and 71 years [65-76] in ASPIRE) and patients with group 3 pulmonary hypertension (median age 71 years [65-77] in COMPERA and 69 years [63-74] in ASPIRE) were older than those with classical IPAH (median age, 45 years [32-60] in COMPERA and 52 years [38-64] in ASPIRE; p<0·0001 for IPAH with a lung phenotype vs classical IPAH in both registries). While 99 (77%) patients in COMPERA and 133 (72%) patients in ASPIRE with classical IPAH were female, there was a lower proportion of female patients in the IPAH and a lung phenotype cohort (95 [35%] COMPERA; 75 [54%] ASPIRE), which was similar to group 3 pulmonary hypertension (336 [37%] COMPERA; 148 [39%] ASPIRE]). Response to pulmonary arterial hypertension therapies at first follow-up was available from COMPERA. Improvements in WHO functional class were observed in 54% of patients with classical IPAH, 26% of patients with IPAH with a lung phenotype, and 22% of patients with group 3 pulmonary hypertension (p<0·0001 for classical IPAH vs IPAH and a lung phenotype, and p=0·194 for IPAH and a lung phenotype vs group 3 pulmonary hypertension); median improvements in 6 min walking distance were 63 m, 25 m, and 23 m for these cohorts respectively (p=0·0015 for classical IPAH vs IPAH and a lung phenotype, and p=0·64 for IPAH and a lung phenotype vs group 3 pulmonary hypertension), and median reductions in N-terminal-pro-brain-natriuretic-peptide were 58%, 27%, and 16% respectively (p=0·0043 for classical IPAH vs IPAH and a lung phenotype, and p=0·14 for IPAH and a lung phenotype vs group 3 pulmonary hypertension). In both registries, survival of patients with IPAH and a lung phenotype (1 year, 89% in COMPERA and 79% in ASPIRE; 5 years, 31% in COMPERA and 21% in ASPIRE) and group 3 pulmonary hypertension (1 year, 78% in COMPERA and 64% in ASPIRE; 5 years, 26% in COMPERA and 18% in ASPIRE) was worse than survival of patients with classical IPAH (1 year, 95% in COMPERA and 98% in ASPIRE; 5 years, 84% in COMPERA and 80% in ASPIRE; p<0·0001 for IPAH with a lung phenotype vs classical IPAH in both registries). INTERPRETATION: A cohort of patients meeting diagnostic criteria for IPAH with a distinct, presumably smoking-related form of pulmonary hypertension accompanied by a low DLCO, resemble patients with pulmonary hypertension due to lung disease rather than classical IPAH. These observations have pathogenetic, diagnostic, and therapeutic implications, which require further exploration. FUNDING: COMPERA is funded by unrestricted grants from Acceleron, Bayer, GlaxoSmithKline, Janssen, and OMT. The ASPIRE Registry is supported by Sheffield Teaching Hospitals NHS Foundation Trust, Sheffield, UK.

Gene panel diagnostics reveals new pathogenic variants in pulmonary arterial hypertension.

BACKGROUND: A genetic predisposition can lead to the rare disease pulmonary arterial hypertension (PAH). Most mutations have been identified in the gene BMPR2 in heritable PAH. However, as of today 15 further PAH genes have been described. The exact prevalence across these genes particularly in other PAH forms remains uncertain. We present the distribution of mutations across PAH genes identified at the largest German referral centre for genetic diagnostics in PAH over a course of > 3 years. METHODS: Our PAH-specific gene diagnostics panel was used to sequence 325 consecutive PAH patients from March 2017 to October 2020. For the first year the panel contained thirteen PAH genes: ACVRL1, BMPR1B, BMPR2, CAV1, EIF2AK4, ENG, GDF2, KCNA5, KCNK3, KLF2, SMAD4, SMAD9 and TBX4. These were extended by the three genes ATP13A3, AQP1 and SOX17 from March 2018 onwards following the genes' discovery. RESULTS: A total of 79 mutations were identified in 74 patients (23%). Of the variants 51 (65%) were located in the gene BMPR2 while the other 28 variants were found in ten further PAH genes. We identified disease-causing variants in the genes AQP1, KCNK3 and SOX17 in families with at least two PAH patients. Mutations were not only detected in patients with heritable and idiopathic but also with associated PAH. CONCLUSIONS: Genetic defects were identified in 23% of the patients in a total of 11 PAH genes. This illustrates the benefit of the specific gene panel containing all known PAH genes.

Utility of immunohistochemical staining for the diagnosis of Extra-adrenal mediastinal paraganglioma.

Extra-adrenal, mediastinal paraganglioma are rare tumors that origin from sympathetic ganglia. Common diagnostic steps include CT, MRI and PET-Scan. We present a case where immunohistochemical staining was an essential step for final diagnosis in a patient without symptoms of endocrine activity and an uncommon location of this tumor entity. In combination with clinical particularities on the origin of the tumor and characteristic morphology, the immunohistochemical staining of tumor tissue is a necessary diagnostic tool for paraganglioma.

Nocardiosis mimicking lung cancer in a heart transplant patient with end-stage renal disease.

Nocardiosis is a rare bacterial opportunistic infection that most commonly manifests as lung disease. However, disseminated infection and abscess formation can occur. Due to diverse radiographic findings and difficult cultivation it is not an easy diagnosis to make. Antibiotics such as Trimethoprimsulfamethoxazole alone or in combination with imipenem or imipenem in combination with amikacin need to be administered over a period of at least six to twelve weeks. We report a case of a 64-year old female heart transplant recipient requiring dialysis who suffered from dyspnea and a productive cough among other symptoms. Computed tomography revealed a tumor in the left upper lobe suggesting lung cancer. Both transbronchial and transthoracic biopsy could not confirm a malignant disease. Finally, Nocardia nova was isolated from a bronchoalveolar lavage and specific antibiotic treatment was initiated. As a result, the mass in the left upper lobe significantly regressed after a few weeks.

Tobacco-smoking induced GPR15-expressing T cells in blood do not indicate pulmonary damage.

BACKGROUND: Recently, it was shown that chronic tobacco smoking evokes specific cellular and molecular changes in white blood cells by an excess of G protein-coupled receptor 15 (GPR15)-expressing T cells as well as a hypomethylation at DNA CpG site cg05575921 in granulocytes. In the present study, we aimed to clarify the general usefulness of these two biomarkers as putative signs of non-cancerous change in homeostasis of the lungs. METHODS: In a clinical cohort consisting of 42 patients with chronic obstructive pulmonary disease (COPD), interstitial lung disease (ILD) and pneumonia and a control cohort of 123 volunteers, the content of GPR15-expressing blood cells as well as the degree of methylation at cg05575921 were analysed by flow-cytometry and pyrosequencing, respectively. Smoking behaviour was estimated by questionnaire and cotinine level in plasma. RESULTS: Never-smoking patients could be distinguished from former and current smokers by both the proportion of GPR15-expressing T cells as well as cg05575921 methylation in granulocytes, with 100% and 97% specificity and 100% sensitivity, respectively. However, both parameters were not affected by lung diseases. The degrees of both parameters were not changed neither in non-smoking nor smoking patients, compared to appropriate control cohorts of volunteers. CONCLUSIONS: The degree of GPR15-expressing cells among T cells as well as the methylation at cg05575921 in granulocytes in blood are both rather signs of tobacco-smoking induced systemic inflammation because they don't indicate specifically non-cancerous pathological changes in the lungs.

Perioperative management of patients with severe pulmonary hypertension in major orthopedic surgery: experience-based recommendations.

INTRODUCTION: It is known that pulmonary hypertension is associated with worse outcome in both cardiac and non-cardiac surgery. The aims of our retrospective analysis were to evaluate the outcomes of our patients with pulmonary hypertension undergoing major orthopedic surgery and to give experience-based recommendations for the perioperative management. MATERIAL AND METHODS: From 92 patients with pulmonary hypertension undergoing different kinds of surgical procedures from 2011-2014 in a tertiary academic hospital we evaluated 16 patients with major orthopedic surgery for perioperative morbidity and mortality. RESULTS: Regarding the in-hospital morbidity and mortality, one patient died postoperatively due to pulmonary infection and right heart failure (6.25%) and 6 patients suffered significant postoperative complications (37.5%; bleeding = 1, infection = 1, wound healing deficits = 3; dysrhythmia = 1). CONCLUSION: Our data show that major orthopedic surgery is feasible with satisfactory outcome even in cases of severe pulmonary hypertension by an individualized, disease-adapted interdisciplinary treatment concept.

Diffuse Interstitial Pulmonary Infiltrates in Malignant Melanoma.

Only a few cases of sarcoidosis-like reaction to a pharmacologic compound have been reported in patients with melanoma in the literature. Long-term treatment with interferon alpha may be assumed. We report a case of a 25-y-old man who presented to our department with diffuse interstitial pulmonary infiltrates and slightly enlarged mediastinal lymph nodes as determined by computed tomography. He had a history of malignant melanoma with 18 months of interferon alpha-2 therapy. Histological analysis of a transbronchial biopsy revealed sarcoidosis-like reactions.

Tobacco smoke exposure in pulmonary arterial and thromboembolic pulmonary hypertension.

BACKGROUND: Animal studies and data from a single-center study suggest that tobacco smoke exposure may be a risk factor for precapillary pulmonary hypertension (PH). OBJECTIVE: We aimed to survey tobacco smoke exposure in a large PH collective and to compare it with epidemiological data from healthy subjects. METHODS: This is an international, multicenter, case-control study including patients with pulmonary arterial and chronic thromboembolic PH. All patients were asked specific questions about tobacco smoke exposure. Healthy controls were retrieved from the Swiss Health Survey (n = 18,747). RESULTS: Overall (n = 472), 49% of PH patients were smokers and there was a clear sex difference (women 37%, men 71%). Significantly more PH men were smokers compared with healthy controls, whereas less PH women were ever active smokers. However, 50% of the non-smoking PH women were exposed to secondhand smoke, leading to a significantly higher number of tobacco smoke-exposed individuals compared to healthy controls. PH smokers were significantly younger compared to those not exposed. CONCLUSION: Active and environmental tobacco smoke exposure is common in PH. The higher prevalence of male PH smokers, the higher exposure to environmental tobacco smoke in PH women compared to healthy controls and the lower age at PH diagnosis in smokers may indicate a pathogenic role of tobacco smoke exposure in PH.

Effects of nasal high flow on ventilation in volunteers, COPD and idiopathic pulmonary fibrosis patients.

BACKGROUND: A high flow of air applied by large bore nasal cannulae has been suggested to improve symptoms of chronic respiratory insufficiency. In pediatric patients, nasal high-flow (nHF) ventilation was similarly effective compared to noninvasive ventilation with a face mask. OBJECTIVES: The aim of this study was to describe changes in respiratory parameters. METHODS: We measured pressure amplitudes during the respiratory cycle and mean pressures in patients with idiopathic pulmonary fibrosis (IPF) and COPD. In order to achieve tidal volume and minute volume measurements, we used a polysomnography device. Capillary blood was taken for blood gas analysis before and after nHF breathing (8 h). RESULTS: nHF led to an increase in pressure amplitude and mean pressure in healthy volunteers and in patients with COPD and IPF in comparison with spontaneous breathing. In COPD, nHF increased tidal volume, while no difference in tidal volume was observed in patients with IPF. Interestingly, tidal volume decreased in healthy volunteers. Breathing rates and minute volumes were reduced in all groups. Capillary pCO2 decreased in patients with IPF and COPD. CONCLUSIONS: nHF resulted in significant effects on respiratory parameters in patients with obstructive and restrictive pulmonary diseases. The rise in pressure amplitude and mean pressure and the decrease in breathing rate and minute volume will support inspiratory efforts, helps to increase effectiveness of ventilation and will contribute to a reduction in the work of breathing. A CO2 wash-out effect in the upper airway part of the anatomical dead space may contribute to the beneficial effects of the nHF instrument.

Acute improved hemodynamics following inhaled iloprost in chronic thromboembolic pulmonary hypertension.

BACKGROUND: Chronic thromboembolic pulmonary hypertension (CTEPH) is a potential consequence to pulmonary embolism. The histologic picture is similar to idiopathic pulmonary hypertension (IPAH) suggesting that vascular remodeling also contributes to CTEPH. The treatment of choice is pulmonary endarterectomy. However, this treatment option is not adequate for all patients with CTEPH. Currently, no data exist on standard vasodilative therapy for CTEPH. Intravenous and oral prostanoids, both well-known vasodilators in IPAH, have been used with promising results, whereas the same has not been consistently observed for inhaled iloprost. OBJECTIVE: In this study, we examined acute hemodynamic effects of inhaled iloprost in patients with CTEPH. METHODS: In a prospective study, right heart catheterization was performed in 20 patients (mean age 56 years, New York Heart Association class II-IV) at the time of diagnosis of CTEPH. Pulmonary vascular resistance (PVR), mean pulmonary arterial pressure (mPAP), cardiac output (CO), mean systemic arterial pressure (MAP) and oxygen partial pressure (PaO(2)) were obtained before and 20 min after inhaling 5 mug iloprost. Subsequently, all patients were evaluated for pulmonary endarterectomy. Six patients were eligible for surgery. RESULTS: Significant changes in pulmonary and systemic hemodynamics were observed following the inhalation of iloprost (before to after inhalation): PVR: 1,057 +/- 404.3 to 821.3 +/- 294.3 dyn.s.cm(-5), p < 0.0001; mPAP: 50.55 +/- 8.43 to 45.75 +/- 8.09 mm Hg, p = 0.0002; CO: 3.66 +/- 1.05 to 4.05 +/- 0.91 l/min, p < 0.0106. MAP and PaO(2) decreased significantly (MAP: 94.15 +/- 11.58 to 89.45 +/- 14.29 mm Hg, p = 0.0111; PaO(2): 7.33 +/- 1.17 to 6.64 +/- 1.25 kPa, p = 0.0260). CONCLUSIONS: Hemodynamic changes directly following inhalation of iloprost suggest a significant contribution of a reversible component of vasoconstriction to pulmonary arterial hypertension in patients with CTEPH.

Stretch-induced alveolar type II cell apoptosis: role of endogenous bradykinin and PI3K-Akt signaling.

Apoptosis of alveolar type II (ATII) cells in response to high-amplitude mechanical stretch represents an important mechanism of ventilation-induced lung injury. Previously, it was demonstrated in an in vitro model that stretch-induced ATII cell apoptosis was prevented by angiotensin-converting enzyme (ACE) inhibitors. This study investigates the mechanism by which ACE inhibitors prevent stretch-induced apoptosis and elucidates the role of bradykinin as an endogenous anti-apoptotic factor. Rat ATII cells cultured on flexible membranes were subjected to cyclic stretch (40 cycles/min; 30% increase in surface area) and compared with static controls. Angiotensinogen, the bradykinin precursor T-kininogen, and bradykinin receptor expression were measured by RT-PCR; Angiotensin II and phosphoinositol 3 OH-kinase (PI3K) activity (as phospho-Akt) were measured by enzyme-linked immunosorbent assay; and Bcl-2 and Bcl-X(L) were measured by Western blot. Stretch did not influence angiotensinogen expression or induce angiotensin II generation. The angiotensin II receptor antagonist saralasin did not prevent stretch-induced apoptosis, whereas ACE inhibitors did. Stretch reduced ATII cell bradykinin release (T-kininogen expression and bradykinin supernatant concentration), and subsequently led to reduced PI3K activity and decreased concentrations of the anti-apoptotic proteins Bcl-2/Bcl-X(L). Bradykinin substitution or addition of keratinocyte or hepatocyte growth factor prevented stretch-induced decrease in PI3K activity and Bcl-2/Bcl-X(L) and reduced stretch-induced apoptosis. Mechanical stretch impairs a constitutively expressed, autocrine anti-apoptotic ATII cell survival signal involving bradykinin-mediated stimulation of the PI3K-Akt-Bcl-2/Bcl-X(L) pathway. Restoration of this pathway prevents stretch-induced apoptosis. This may be beneficial when mechanical ventilation cannot completely avoid alveolar overdistension to maintain oxygenation.

Diagnosis of aspergilloma in a pleural cavity (persistent pneumothorax) using classic imaging methods.

The diagnosis of pulmonary aspergillosis is based on serum-analysis, as well as histological and microbiological analysis of bronchial lavage and transbronchial biopsies. When Aspergillus develops within a preformed cavity, however, these tests are likely to be negative. In this situation, classic imaging techniques such as chest X-ray and high resolution-computed tomography (HR-CT) can be of great diagnostic use. We here describe the case of a 62-year-old woman with a history of breast cancer and subsequent ablation of the left breast and radiotherapy. The case demonstrates an example of a pleuropulmonary aspergilloma, in which sero- and micro-biological detection failed. Thorax HR-CT exhibited the cavity, a small persistent pneumothorax, partially filled by an oval density. This density clearly dislocated according to gravity following a positional change of the patient from supine to prone. The density thus revealed mobility which was typical of aspergilloma. Following excision, this diagnosis was confirmed. A density within a cavity may be differentiated by its mobility from differential diagnoses such as lung cancer which would not be expected to exhibit mobility.