Detection of somatic mutations in peritoneal lavages and plasma of endometrial cancer patients: A proof-of-concept study.

Endometrial cancer (EC) is the most common gynecologic malignancy in developed countries. Although most patients are diagnosed at early stages, 15-20% will relapse despite local treatment. Presently, there are no reliable markers to identify patients with worse outcomes who may benefit from adjuvant treatments, such as chemotherapy, and liquid biopsies may be of use in this setting. Peritoneal lavages are systematically performed during endometrial surgery but little data are available about their potential as liquid biopsies. We analyzed KRAS and PIK3CA mutations in paired surgical biopsies, blood and cytology-negative peritoneal lavages in a cohort of 50 EC patients. Surgical biopsies were submitted to next-generation sequencing (NGS) while circulating-free DNA (cfDNA) purified from plasma and peritoneal lavages was analyzed for KRAS and PIK3CA hotspot mutations using a sensitive quantitative polymerase chain reaction (PCR) assay. NGS of biopsies revealed KRAS, PIK3CA or concomitant KRAS + PIK3CA mutations in 33/50 (66%) EC patients. Of those, 19 cases carried hotspot mutations. Quantitative PCR revealed KRAS and/or PIK3CA mutations in the lavages of 9/19 (47.4%) hotspot EC patients. In contrast, only 2/19 (10.5%) blood samples from hotspot EC patients were positive. Mutations found in cfDNA consistently matched those in paired biopsies. One of the two patients positive in plasma and lavage died in less than 6 months. In conclusion, mutational analysis in peritoneal lavages and blood from early stage EC is feasible. Further studies are warranted to determine if it might help to identify patients with worse prognosis. Human genes discussed: KRAS, KRAS proto-oncogene, GTPase; PIK3CA, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha.

Vaccine antibodies against a synthetic epidermal growth factor variant enhance the antitumor effects of inhibitors targeting the MAPK/ERK and PI3K/Akt pathways.

BACKGROUND: The EGFR pathway is involved in intrinsic and acquired resistance to a wide variety of targeted therapies in cancer. Vaccination represents an alternative to the administration of anti-EGFR monoclonal antibodies, such as cetuximab or panitumumab. Here, we tested if anti-EGF antibodies generated by vaccination (anti-EGF VacAbs) could potentiate the activity of drugs targeting the ERK/MAPK and PI3K/Akt pathways. METHODS: Non-small cell lung cancer (NSCLC), colorectal cancer (CRC) and melanoma cell lines harboring KRAS, NRAS, BRAF and PIK3CA mutations were used. Anti-EGF VacAbs were obtained by immunizing rabbits with a fusion protein containing a synthetic, highly mutated variant of human EGF. Cell viability was determined by MTT, total and phosphorylated proteins by Western blotting, cell cycle distribution and cell death by flow cytometry and emergence of resistance by microscopic examination in low density cultures. RESULTS: Anti-EGF VacAbs potentiated the antiproliferative effects of MEK, KRAS G12C, BRAF, PI3K and Akt inhibitors in KRAS, NRAS, BRAF and PIK3CA mutant cells and delayed the appearance of resistant clones in vitro. The effects of anti-EGF VacAbs were comparable or superior to those of panitumumab and cetuximab. The combination of anti-EGF VacAbs with the targeted inhibitors effectively suppressed EGFR downstream pathways and sera from patients immunized with an anti-EGF vaccine also blocked activation of EGFR effectors. CONCLUSIONS: Anti-EGF VacAbs enhance the antiproliferative effects of drugs targeting the ERK/MAPK and PIK3CA/Akt pathways. Our data provide a rationale for clinical trials testing anti-EGF vaccination combined with inhibitors selected according to the patient's genetic profile.

Bone health and predictors of 15-year mortality in a physically active population.

OBJECTIVE: To analyse determinants of mortality at 15 years in a population over 60 years of age and physically active. METHODS: This is a prospective longitudinal study. After 15 years of participating in an active ageing programme, participants were contacted by telephone to verify their state of health and to determine whether in that time they had had any fractures. RESULTS: 561 individuals over 60 years of age were included, 82% of whom were women. Only differences in densitometric data, FRAX values and history of previous fracture at baseline characteristics were found between the group that died at 15 years and the group that remained alive. The only variables that were related to mortality risk were the basal data of the densitometric t-score (OR = .50, P < .001) and history of fracture in any location (OR = 2.44, P < .033). CONCLUSIONS: The value of bone mineral density could be considered as a useful biomarker to calculate the risk of mortality in people over 60 years old with a physically active lifestyle.

Myo-Spain: Spanish Registry of patients with idiopathic inflammatory myopathy. Methodology.

OBJECTIVES: To describe the methods of the Spanish Registry of patients with idiopathic inflammatory myopathy (IIM) (Myo-Spain), as well as its strengths and limitations. The main objective of the project is to analyse the evolution and clinical management of a cohort of patients with IIM. METHODS: Observational, longitudinal, ambispective and multicentre study of a cohort of patients with IIM seen in rheumatology units in Spain. All patients with a diagnosis of IMM will be included in the regular follow-up of the participating centres, regardless of age on initiation of the process. Incident cases will be all patients who at the beginning of the study have been diagnosed for less than 12 months and prevalent cases for more than 12 months. The registry will include data from the visit at baseline, one year and two years. Socio-demographic, clinical, analytical variables, complications, comorbidities, association with other rheumatic diseases, hospital admissions, mortality and treatments will be collected. In addition, indices, scales and questionnaires of activity, muscle involvement, damage, disability, and quality of life will be determined. The recruitment period will be 23 months. The purpose is to obtain a cohort of 400 patients with IMM. CONCLUSIONS: Myo-Spain registry provides the opportunity to develop a cohort of incident and prevalent patients with IMM in Spain. Myo-Spain will be able to assess in detail the clinical characteristics of the disease at different times. The comprehensive information collected during the visits is expected to provide a broad source of data for future analysis.

Bone Health and Predictors of 15-Year Mortality in a Physically Active Population. / Salud ósea y predictores de mortalidad a 15 años en una población físicamente activa.

OBJECTIVE: To analyse determinants of mortality at 15years in a population over 60years of age and physically active. METHODS: This is a prospective longitudinal study. After 15years of participating in an active aging programme, participants were contacted by telephone to verify their state of health and to determine whether in that time they had had any fractures. RESULTS: A total of 561 individuals over 60years of age were included, 82% of whom were women. Only differences in densitometric data, FRAX values and history of previous fracture at baseline characteristics were found between the group that died at 15years and the group that remained alive. The only variables that were related to mortality risk were the basal data of the densitometric T-score (OR=.50, P<.001) and history of fracture in any location (OR=2.44, P<.033). CONCLUSIONS: The value of bone mineral density could be considered as a useful biomarker to calculate the risk of mortality in people over 60years old with a physically active lifestyle.

Myo-Spain: Spanish Registry of Patients with Idiopathic Inflammatory Myopathy. Methodology. / Myo-Spain: Registro de pacientes con miopatía inflamatoria idiopática de España. Metodología.

OBJECTIVES: To describe the methods of the Spanish Registry of patients with idiopathic inflammatory myopathy (IIM) (Myo-Spain), as well as its strengths and limitations. The main objective of the project is to analyse the evolution and clinical management of a cohort of patients with IIM. METHODS: Observational, longitudinal, ambispective and multicentre study of a cohort of patients with IIM seen in rheumatology units in Spain. All patients with a diagnosis of IMM will be included in the regular follow-up of the participating centres, regardless of age on initiation of the process. Incident cases will be all patients who at the beginning of the study have been diagnosed for less than 12 months and prevalent cases for more than 12 months. The registry will include data from the visit at baseline, one year and two years. Socio-demographic, clinical, analytical variables, complications, comorbidities, association with other rheumatic diseases, hospital admissions, mortality and treatments will be collected. In addition, indices, scales and questionnaires of activity, muscle involvement, damage, disability, and quality of life will be determined. The recruitment period will be 23 months. The purpose is to obtain a cohort of 400 patients with IMM. CONCLUSIONS: Myo-Spain registry provides the opportunity to develop a cohort of incident and prevalent patients with IMM in Spain. Myo-Spain will be able to assess in detail the clinical characteristics of the disease at different times. The comprehensive information collected during the visits is expected to provide a broad source of data for future analysis.

Bacteremia in Systemic Lupus Erythematosus in Patients from a Spanish Registry: Risk Factors, Clinical and Microbiological Characteristics, and Outcomes.

OBJECTIVE: To describe the incidence of bacteremia in a large multicentric cohort of patients with systemic lupus erythematosus (SLE) and their clinical characteristics and to identify risk factors. METHODS: All bacteremic episodes from the Spanish RELESSER registry were included. Clinical and laboratory characteristics concerning bacteremia and SLE status, as well as comorbidities at the time of infection, were retrospectively collected. A comparison with sex- and age-matched SLE controls without bacteremia was made. A logistic regression was conducted. RESULTS: The study included 114 episodes of bacteremia in 83 patients. The incidence rate was 2.7/1000 patient-years. At the time of bacteremia, the median age was 40.5 (range: 8-90) years, and 88.6% of patients were female. The Safety of Estrogens in Lupus Erythematosus National Assessment-Systemic Lupus Erythematosus Disease Activity Index was 4 [interquartile range (IQR) 8]; 41% had an SLE flare (66% severe); Systemic Lupus International Collaborating Clinics/American College of Rheumatology Damage Index was 3 (IQR 4). A comorbidity was recorded in 64% of cases. At the time of bacteremia, 88.6% received corticosteroids (68.6% > 10 mg/day) and 57% immunosuppressors. Gram-negative bacilli, most frequently Escherichia coli (29.8%), caused 52.6% of the episodes. The bacteremia-related mortality was 14% and bacteremia was recurrent in 27.2% of cases. A dose-response relationship was found between corticosteroids and bacteremia risk. In the multivariate analysis, these factors were associated with bacteremia: elevated creatinine (OR 1.31, 95% CI 1.01-1.70; p = 0.045), diabetes (OR 6.01, 95% CI 2.26-15.95; p < 0.001), cancer (OR 5.32, 95% CI 2.23-12.70; p < 0.001), immunosuppressors (OR 6.35, 95% CI 3.42-11.77; p < 0.001), and damage (OR 1.65, 95% CI 1.31-2.09; p < 0.001). CONCLUSION: Bacteremia occurred mostly in patients with active SLE and was frequently associated with severe flares and corticosteroid use. Recurrence and mortality were high. Immunosuppressors, comorbidities, and disease-related damage were associated with bacteremia.

Evolution and Clinical Impact of EGFR Mutations in Circulating Free DNA in the BELIEF Trial.

INTRODUCTION: Longitudinal evaluation of mutations in blood samples was a prespecified secondary objective in the BELIEF trial of erlotinib and bevacizumab in advanced EGFR-positive NSCLC. Here, we report the testing results and explore the correlation of EGFR status in blood with clinical outcomes. METHODS: Blood samples were prospectively collected from patients at baseline, at response evaluation, and at progression and sent to a central laboratory. Circulating free DNA was purified and EGFR mutations were analyzed with a validated real-time quantitative polymerase chain reaction assay. RESULTS: EGFR exon 19/21 mutations were detected in 55 of 91 baseline blood samples (60.4%) and correlated with a significantly worse progression-free survival: 11.4 months (95% confidence interval [CI]: 9.0-14.8 mo) for the patients who were positive versus 22.9 months (95% CI: 9.5-33.9 mo) for those who were negative (log-rank p = 0.0020). Among the 74 samples at response, exon 19/21 mutations were detected only in three samples (4.1%). In contrast, 29 of 58 patients (50.0%) were exon 19/21 positive at progression and showed a significantly worse median overall survival of 21.7 months (95% CI: 17.0-30.9 mo) compared with 37.4 months (95% CI: 22.6-53.1 mo) for those who were negative (log-rank p = 0.011). Blood samples at the three time points were available for 48 patients. Of those, among 14 exon 19/21 EGFR-negative at presentation, 13 (93%) were persistently negative for the sensitizing mutations after progression and the p.T790M could only be detected in the blood of two patients. CONCLUSIONS: Longitudinal testing of EGFR mutations in blood can offer valuable clinical information. In patients of the BELIEF study, detection of EGFR mutations in circulating free DNA at presentation was associated with shorter progression-free survival, whereas positivity at progression correlated with shorter overall survival. Finally, patients negative in blood at presentation were almost invariably negative at relapse.

Pulmonary arterial hypertension in adult-onset Still's disease: A case series and systematic review of the literature.

OBJECTIVE: To investigate the prevalence, clinical characteristics and prognosis of pulmonary arterial hypertension (PAH) in adult onset Still's disease (AOSD). METHODS: We retrospectively reviewed all patients with AOSD diagnosed during a 33-year period in 2 referral tertiary care hospitals, selecting for analysis those who presented PAH confirmed as by right heart catheterization. A systematic review of the literature (PubMed 1990 to July 2018) was also performed, in order to determine the prognosis and the most appropriate treatment strategy for this complication. RESULTS: The overall prevalence of PAH in our AOSD population was 4.8% (2/41). Including our 2 cases, 20 well-documented patients have been reported. PAH may complicate AOSD at any time during its course, and usually occurs in patients who have persistent and severe disease, with a considerable frequency (35%) of previous or concomitant severe clinical complications. In all cases, the etiology of pulmonary hypertension was a group 1 PAH based on the 2015 ESC/ERS guidelines. Most patients in this series had advanced WHO functional classes III-IV at the time of PAH diagnosis, reflecting an important diagnostic delay. Thirty-three percent of patients had a poor outcome despite the therapy, with a mortality rate that reached 22%. The therapeutic strategy that achieved the best results was the use of glucocorticoids, immunosuppression and PAH-specific vasodilator therapy. CONCLUSION: HAP is an under-recognized complication of AOSD that should be kept in mind in the differential diagnosis of those patients who experience dyspnea on exertion or a decrease in exercise tolerance.

Prospective detection of mutations in cerebrospinal fluid, pleural effusion, and ascites of advanced cancer patients to guide treatment decisions.

Many advanced cases of cancer show central nervous system, pleural, or peritoneal involvement. In this study, we prospectively analyzed if cerebrospinal fluid (CSF), pleural effusion (PE), and/or ascites (ASC) can be used to detect driver mutations and guide treatment decisions. We collected 42 CSF, PE, and ASC samples from advanced non-small-cell lung cancer and melanoma patients. Cell-free DNA (cfDNA) was purified and driver mutations analyzed and quantified by PNA-Q-PCR or next-generation sequencing. All 42 fluid samples were evaluable; clinically relevant mutations were detected in 41 (97.6%). Twenty-three fluids had paired blood samples, 22 were mutation positive in fluid but only 14 in blood, and the abundance of the mutant alleles was significantly higher in fluids. Of the 34 fluids obtained at progression to different therapies, EGFR resistance mutations were detected in nine and ALK acquired mutations in two. The results of testing of CSF, PE, and ASC were used to guide treatment decisions, such as initiation of osimertinib treatment or selection of specific ALK tyrosine-kinase inhibitors. In conclusion, fluids close to metastatic sites are superior to blood for the detection of relevant mutations and can offer valuable clinical information, particularly in patients progressing to targeted therapies.

Prospective analysis of liquid biopsies of advanced non-small cell lung cancer patients after progression to targeted therapies using GeneReader NGS platform.

BACKGROUND: In a significant percentage of advanced non-small cell lung cancer (NSCLC) patients, tumor tissue is unavailable or insufficient for genetic analyses at time to progression. We prospectively analyzed the appearance of genetic alterations associated with resistance in liquid biopsies of advanced NSCLC patients progressing to targeted therapies using the NGS platform. METHODS: A total of 24 NSCLC patients were included in the study, 22 progressing to tyrosine kinase inhibitors and two to other treatments. Liquid biopsies samples were obtained and analyzed using the GeneReadTM QIAact Lung DNA UMI Panel, designed to enrich specific target regions and containing 550 variant positions in 19 selected genes frequently altered in lung cancer tumors. Previously, a retrospective validation of the panel was performed in clinical samples. RESULTS: Of the 21 patients progressing to tyrosine kinase inhibitors with valid results in liquid biopsy, NGS analysis identified a potential mechanism of resistance in 12 (57%). The most common were acquired mutations in ALK and EGFR, which appeared in 8/21 patients (38%), followed by amplifications in 5/21 patients (24%), and KRAS mutations in one patient (5%). Loss of the p.T790M was also identified in two patients progressing to osimertinib. Three of the 21 (14%) patients presented two or more concomitant alterations associated with resistance. Finally, an EGFR amplification was found in the only patient progressing to immunotherapy included in the study. CONCLUSIONS: NGS analysis in liquid biopsies of patients progressing to targeted therapies using the GeneReader platform is feasible and can help the oncologist to make treatment decisions.

An update on liquid biopsy analysis for diagnostic and monitoring applications in non-small cell lung cancer.

INTRODUCTION: Collection of tumor samples is not always feasible in non-small cell lung cancer (NSCLC) patients, and circulating free DNA (cfDNA) extracted from blood represents a viable alternative. Different sensitive platforms have been developed for genetic cfDNA testing, some of which are already in clinical use. However, several difficulties remain, particularly the lack of standardization of these methodologies. Areas covered: Here, the authors present a review of the literature to update the applicability of cfDNA for diagnosis and monitoring of NSCLC patients. Expert commentary: Detection of somatic alterations in cfDNA is already in use in clinical practice and provides valuable information for patient management. Monitoring baseline alterations and emergence of resistance mutations is one of the most important clinical applications and can be used to non-invasively track disease evolution. Today, different technologies are available for cfDNA analysis, including whole-genome or exome sequencing and targeted methods that focus on a selection of genes of interest in a specific disease. In the case of Next Generation Sequencing (NGS) approaches, in depth coverage of candidate mutation loci can be achieved by selecting a limited number of targeted genes.

A rational use of glucocorticoids in patients with early arthritis has a minimal impact on bone mass.

INTRODUCTION: Glucocorticoid (GC)-induced osteoporosis is a frequent complication in patients with rheumatoid arthritis. However, little information exists about the consequences of GC use in patients with early arthritis. Here we describe the variables underlying the use of GC in early arthritis, as well as its effect on bone-mineral density. METHODS: Data from 116 patients in our early arthritis register were analyzed (90 women; median age, 52.5 years, interquartile range (IQR, 38.5-66); 6-month median disease duration at entry (IQR, 4-9)). In this register, the clinical and treatment information was recorded systematically, including the cumulative GC dose. Lumbar spine, hip, and forearm bone-mineral density (BMD) measurements were performed at entry and after a 2-year follow-up. A multivariate analysis was performed to establish the variables associated with the use of GCs, as well as those associated with variations in BMD. RESULTS: Of the patients with early arthritis studied, 67% received GCs during the 2-year follow-up. GCs were more frequently prescribed to elderly patients, those with higher basal disease activity and disability, and patients with positive rheumatoid factor. When adjusted for these variables, GCs were less frequently prescribed to female patients. The use of GCs was associated with an increase of BMD in the ultradistal region of the forearm, although it induced a significant loss of BMD in the medial region of the forearm. No relevant effect of GC was noted on the BMD measured at other locations. CONCLUSIONS: The frequent use of GCs as a "bridge therapy" in patients with early arthritis does not seem to be associated with relevant loss of bone mass. Moreover, cumulative GC administration might be associated with an increase of juxtaarticular BMD.

Dissection of the ascending aorta in a patient with HLA-B27 associated ankylosing spondylitis.

We describe a 38-year-old patient with ankylosing spondylitis complicated by a non-traumatic dissection of the ascending aorta without concomitant Marfan's syndrome.

[Relapsing polychondritis associated with a lymphoplasmocytic lymphoma and erythema nodosum]. / Policondritis recidivante asociada a linfoma linfoplasmocítico y eritema nudoso.

Relapsing polychondritis is a disease of unknown etiology characterized by episodic inflammation of cartilaginous tissues. More rarely, it has been described as a paraneoplastic phenomenon mainly associated with myelodysplastic syndromes or other haematologic diseases. We present a case of relapsing polychondritis associated to low degree lymphoplasmocytic lymphoma whose picture was punctuated by cutaneous erythema nodosum and anterior uveitis. The clinical evolution was satisfactory with glucocorticoids and rituximab treatment.