Independent genomic polymorphisms in the PknH serine threonine kinase locus during evolution of the Mycobacterium tuberculosis Complex affect virulence and host preference.

Species belonging to the Mycobacterium tuberculosis Complex (MTBC) show more than 99% genetic identity but exhibit distinct host preference and virulence. The molecular genetic changes that underly host specificity and infection phenotype within MTBC members have not been fully elucidated. Here, we analysed RD900 genomic region across MTBC members using whole genome sequences from 60 different MTBC strains so as to determine its role in the context of MTBC evolutionary history. The RD900 region comprises two homologous genes, pknH1 and pknH2, encoding a serine/threonine protein kinase PknH flanking the tbd2 gene. Our analysis revealed that RD900 has been independently lost in different MTBC lineages and different strains, resulting in the generation of a single pknH gene. Importantly, all the analysed M. bovis and M. caprae strains carry a conserved deletion within a proline rich-region of pknH, independent of the presence or absence of RD900. We hypothesized that deletion of pknH proline rich-region in M. bovis may affect PknH function, having a potential role in its virulence and evolutionary adaptation. To explore this hypothesis, we constructed two M. bovis 'knock-in' strains containing the M. tuberculosis pknH gene. Evaluation of their virulence phenotype in mice revealed a reduced virulence of both M. bovis knock-in strains compared to the wild type, suggesting that PknH plays an important role in the differential virulence phenotype of M. bovis vs M. tuberculosis.

Impact of the COVID-19 pandemic during Spain's state of emergency on the diagnosis of colorectal cancer.

INTRODUCTION: We evaluate the impact of COVID-epidemic in colorectal cancer (CRC) diagnosis during Spain's state of emergency. METHODS: We compared newly diagnosed patients with patients diagnosed in the same period of 2019. RESULTS: A new diagnosis of CRC decreased 48% with a higher rate of patients diagnosed in the emergency setting (12.1% vs. 3.6%; p = .048) and a lower rate diagnosed in the screening program (5.2% vs. 33.3%; p = .000). CONCLUSIONS: Fewer patients have been diagnosed with CRC, with a higher rate of patients diagnosed in an emergency setting.

Is aflibercept an optimal treatment for wt RAS mCRC patients after progression to first line containing anti-EGFR?

PURPOSE: To determine the efficacy and safety data of aflibercept + FOLFIRI in wt RAS mCRC patients after progression to standard chemotherapy + anti-EGFR treatment. METHODS: Retrospective, observational study in real life conducted in wt RAS mCRC patients treated with FOLFIRI-aflibercept after progression to standard first line chemotherapy + anti-EGFR treatment. RESULTS: A total of 120 patients from 12 Spanish hospitals were enrolled. Median age is 60 years (62.5%/37.5%male/female). Primary tumor site is 24.1%/75.9% right/left-side colon, and 40.8% of patients had a prior resection. All patients had wild-type RAS tumors including 5% of patients with BRAF mutations and received anti-EGFR treatment. At the time aflibercept was initiated, ECOG PS is 0/1 in 96% of patients. Median number of FOLFIRI-aflibercept cycles is 12. Efficacy results: Overall response rate is 33%; progression-free survival (PFS) is 6.9 months (95%CI: 6.1-7.8). Primary tumor resection was the only significant variable related to PFS in the multivariate analysis. Median overall survival (OS) is 14.5 months (95%CI: 9.7-19.3). ECOG and number of metastatic sites were related to OS in multivariate analysis. About 54.1% of patients received a third-line therapy including TAS-102 (23%), regorafenib (18.5%), and capecitabine (9.2%). TOXICITY: Grade 3-4 toxicities were observed in 37.5% of the patients (hematologic 16.6%, hypertension 7.5%, asthenia 5.9%, and perforation 2.5%). Aflibercept dose was reduced in 18.3% of patients. CONCLUSIONS: The results show that patients with wt RAS mCRC who received an anti-EGFR as part of the first-line treatment achieved similar RR, PFS, OS, and toxicities to those reported in VELOUR trial. These results suggest that FOLFIRI-aflibercept after first-line treatment with anti-EGFR is an appropriated option for RAS wt mCRC.

Circulating Tumor Cells in Patients Undergoing Resection of Colorectal Cancer Liver Metastases. Clinical Utility for Long-Term Outcome: A Prospective Trial.

BACKGROUND: Patients with resected colorectal cancer liver metastases display heterogeneous clinical behavior. The identification of new prognostic factors would help in making more accurate decisions. OBJECTIVE: The aim of this study was to evaluate the survival impact of circulating tumor cells (CTCs) in this setting. METHODS: We conducted a prospective study of patients with resected liver metastases of colorectal cancer. Patients were included in the study from February 2009 to January 2013. The CellSearch System™ was employed for the detection of pre- and postsurgery CTCs. A positive test was defined as two or more CTCs/7.5 mL of blood. Recurrence rate, disease-free survival, and overall survival were calculated, and univariate and multivariate analyses were performed. RESULTS: Forty-four patients were included in our study. After a median follow-up of 60 months (range 28-74), 32 patients experienced recurrence (72.7%). The CTCs number was determined and the test was positive in 8 patients (18.6%) before surgery and 13 patients (29.5%) after surgery. The postoperative detection of CTCs was not related to any clinical outcome; however, the preoperative detection of CTCs was significantly related to behavior. All patients in the preoperative CTC-positive group relapsed, versus 65% in the CTC-negative group (p = 0.051). Disease-free survival was 19 months in the preoperative CTC-negative group versus 7 months in the CTC-positive group (p = 0.01). Additionally, overall survival was 69 months in the preoperative CTC-negative group versus 17 months in the CTC-positive group (p = 0.004). Preoperative CTC count remained significant in multivariate analysis. CONCLUSIONS: In this cohort of colorectal cancer liver metastases patients, the presence of two or more preoperative CTCs was associated with disease progression and poor survival despite complete resection.

Complications from the primary tumour are not related with survival in patients with synchronous stage IV colorectal cancer receiving chemotherapy without primary tumour resection.

BACKGROUND: The aim of this study was to evaluate the rate of complications from the primary tumour (CPT) requiring surgical or endoscopic intervention during chemotherapy treatment in patients with incurable synchronous stage IV colorectal cancer, the possibility of predicting such complications and their influence on survival. METHODS: One hundred and twenty-five patients were initially treated with chemotherapy. Patients were grouped on the basis of appearance or not of CPT. We assessed the relation between age, gender, carcinoembryonic antigen (CEA) level, primary tumour location, alkaline phosphatase level, unilobar or bilobar liver involvement, presence of peritoneal carcinomatosis, the number of sites of metastatic disease, the addition of target therapies to chemotherapy, the ability to traverse the tumour with an endoscope and the appearance of complications due to the primary tumour and overall survival. RESULTS: Mean age was 64.9 years, and 89 patients were men. Over a mean of 234 days, 25 patients (20 %) developed a CPT. Eighteen patients required surgery, and seven were treated exclusively by an endoscopic procedure. Mean survival was 15.8 months. We found a statistically relevant correlation between the inability to traverse the tumour with an endoscope and the occurrence of a CPT. There was no statistical differences in survival between both groups, but patients receiving target therapies had better survival. CONCLUSION: Twenty percent of patients will suffer a CPT during chemotherapy treatment. The inability to pass the tumour with an endoscope can predict the CPT. Survival was only related to the addition of target therapies to chemotherapy.

Building on the clinical applicability of ctDNA analysis in non-metastatic pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma represents one of the solid tumors showing the worst prognosis worldwide, with a high recurrence rate after adjuvant or neoadjuvant therapy. Circulating tumor DNA analysis raised as a promising non-invasive tool to characterize tumor genomics and to assess treatment response. In this study, surgical tumor tissue and sequential blood samples were analyzed by next-generation sequencing and were correlated with clinical and pathological characteristics. Thirty resectable/borderline pancreatic ductal adenocarcinoma patients treated at the Hospital Universitario de Navarra were included. Circulating tumoral DNA sequencing identified pathogenic variants in KRAS and TP53, and in other cancer-associated genes. Pathogenic variants at diagnosis were detected in patients with a poorer outcome, and were correlated with response to neoadjuvant therapy in borderline pancreatic ductal adneocarcinoma patients. Higher variant allele frequency at diagnosis was associated with worse prognosis, and thesum of variant allele frequency was greater in samples at progression. Our results build on the potential value of circulating tumor DNA for non-metastatic pancreatic ductal adenocarcinoma patients, by complementing tissue genetic information and as a non-invasive tool for treatment decision. Confirmatory studies are needed to corroborate these findings.

State of the Art: ctDNA in Upper Gastrointestinal Malignancies.

Circulating tumor DNA (ctDNA) has emerged as a promising non-invasive source to characterize genetic alterations related to the tumor. Upper gastrointestinal cancers, including gastroesophageal adenocarcinoma (GEC), biliary tract cancer (BTC) and pancreatic ductal adenocarcinoma (PADC) are poor prognostic malignancies, usually diagnosed at advanced stages when no longer amenable to surgical resection and show a poor prognosis even for resected patients. In this sense, ctDNA has emerged as a promising non-invasive tool with different applications, from early diagnosis to molecular characterization and follow-up of tumor genomic evolution. In this manuscript, novel advances in the field of ctDNA analysis in upper gastrointestinal tumors are presented and discussed. Overall, ctDNA analyses can help in early diagnosis, outperforming current diagnostic approaches. Detection of ctDNA prior to surgery or active treatment is also a prognostic marker that associates with worse survival, while ctDNA detection after surgery is indicative of minimal residual disease, anticipating in some cases the imaging-based detection of progression. In the advanced setting, ctDNA analyses characterize the genetic landscape of the tumor and identify patients for targeted-therapy approaches, and studies show variable concordance levels with tissue-based genetic testing. In this line, several studies also show that ctDNA serves to follow responses to active therapy, especially in targeted approaches, where it can detect multiple resistance mechanisms. Unfortunately, current studies are still limited and observational. Future prospective multi-center and interventional studies, carefully designed to assess the value of ctDNA to help clinical decision-making, will shed light on the real applicability of ctDNA in upper gastrointestinal tumor management. This manuscript presents a review of the evidence available in this field up to date.

Therapeutic efficacy of pulmonary live tuberculosis vaccines against established asthma by subverting local immune environment.

BACKGROUND: Substantial recent advances in the comprehension of the molecular and cellular mechanisms behind asthma have evidenced the importance of the lung immune environment for disease outcome, making modulation of local immune responses an attractive therapeutic target against this pathology. Live attenuated mycobacteria, such as the tuberculosis vaccine BCG, have been classically linked with a type 1 response, and proposed as possible modulators of the type 2 response usually associated with asthma. METHODS: In this study we used different acute and chronic murine models of asthma to investigate the therapeutic efficacy of intranasal delivery of the live tuberculosis vaccines BCG and MTBVAC by regulating the lung immune environment associated with airway hyperresponsiveness (AHR). FINDINGS: Intranasal administration of BCG, or the novel tuberculosis vaccine candidate MTBVAC, abrogated AHR-associated hallmarks, including eosinophilia and lung remodeling. This correlated with the re-polarization of allergen-induced M2 macrophages towards an M1 phenotype, as well as with the induction of a strong allergen-specific Th1 response. Importantly, vaccine treatment was effective in a scenario of established chronic asthma where a strong eosinophil infiltration was already present prior to immunization. We finally compared the nebulization efficiency of clinical formulations of MTBVAC and BCG using a standard commercial nebulizer for potential aerosol application. INTERPRETATION: Our results demonstrate that pulmonary live tuberculosis vaccines efficiently revert established asthma in mice. These data support the further exploration of this approach as potential therapy against asthma. FUNDING: Spanish Ministry of Science [grant numbers: BIO2014-5258P, RTI2018-097625-B-I00], Instituto de Salud Carlos III, Gobierno de Aragón/Fondo Social Europeo, University of Zaragoza [grant number: JIUZ-2018-BIO-01].

Pulmonary BCG induces lung-resident macrophage activation and confers long-term protection against tuberculosis.

Bacillus Calmette-Guerin (BCG) is an attenuated bacterial vaccine used to protect against Mycobacterium tuberculosis (Mtb) in regions where infections are highly prevalent. BCG is currently delivered by the intradermal route, but alternative routes of administration are of great interest, including intrapulmonary delivery to more closely mimic respiratory Mtb infection. In this study, mice subjected to pulmonary delivery of green fluorescent protein­tagged strains of virulent (Mtb) and attenuated (BCG) mycobacteria were studied to better characterize infected lung cell subsets. Profound differences in dissemination patterns were detected between Mtb and BCG, with a strong tendency of Mtb to disseminate from alveolar macrophages (AMs) to other myeloid subsets, mainly neutrophils and recruited macrophages. BCG mostly remained in AMs, which promoted their activation. These preactivated macrophages were highly efficient in containing Mtb bacilli upon challenge and disrupting early bacterial dissemination, which suggests a potential mechanism of protection associated with pulmonary BCG vaccination. Respiratory BCG also protected mice against a lethal Streptococcus pneumoniae challenge, suggesting that BCG-induced innate activation could confer heterologous protection against respiratory pathogens different from Mtb. BCG drove long-term activation of AMs, even after vaccine clearance, and these AMs reacted efficiently upon subsequent challenge. These results suggest the generation of a trained innate memory-like response in AMs induced by pulmonary BCG vaccination.

Respiratory Immunization With a Whole Cell Inactivated Vaccine Induces Functional Mucosal Immunoglobulins Against Tuberculosis in Mice and Non-human Primates.

Vaccination through the natural route of infection represents an attractive immunization strategy in vaccinology. In the case of tuberculosis, vaccine delivery by the respiratory route has regained interest in recent years, showing efficacy in different animal models. In this context, respiratory vaccination triggers lung immunological mechanisms which are omitted when vaccines are administered by parenteral route. However, contribution of mucosal antibodies to vaccine- induced protection has been poorly studied. In the present study, we evaluated in mice and non-human primates (NHP) a novel whole cell inactivated vaccine (MTBVAC HK), by mucosal administration. MTBVAC HK given by intranasal route to BCG-primed mice substantially improved the protective efficacy conferred by subcutaneous BCG only. Interestingly, this improved protection was absent in mice lacking polymeric Ig receptor (pIgR), suggesting a crucial role of mucosal secretory immunoglobulins in protective immunity. Our study in NHP confirmed the ability of MTBVAC HK to trigger mucosal immunoglobulins. Importantly, in vitro assays demonstrated the functionality of these immunoglobulins to induce M. tuberculosis opsonization in the presence of human macrophages. Altogether, our results suggest that mucosal immunoglobulins can be induced by vaccination to improve protection against tuberculosis and therefore, they represent a promising target for next generation tuberculosis vaccines.

Local recurrence after five years is associated with preoperative chemoradiotherapy treatment in patients diagnosed with stage II and III rectal cancer.

AIM: To asses the moment of local recurrence and its influence on the appraisal of the results of neoadjuvant chemoradiotherapy (CRT). METHODS: We evaluated 317 patients with a preoperative diagnosis of stage II or III rectal cancer who underwent rectal resection. Gender, age, neoadjuvant treatment, circumferencial resection margin, adjuvant treatment, pretreatment carcinoembryonic antigen level, tumor location, TNM stage, lymph node retrieval, abdominoperineal resection, and lymphatic or vascular infiltration were registered prospectively. Follow-up was performed to detect local or systemic recurrences. Timing of local recurrence (LR) in regard to analyzed variables was performed by using analysis of variance. To evaluate the influence of late local recurrence (LLR) on the results of neoadjuvant CRT, we performed a log-rank test censoring all observations at 60 and at 120 months. RESULTS: After a mean follow-up of 73.6 months (range, 1-171), 68 patients developed a recurrence. Twenty-three patients developed LRs (6.9%), and 5 developed LLRs. The earliest relapse was diagnosed 4 months after rectal surgery, and the latest was diagnosed 120 months after surgery. Patients who underwent neoadjuvant CRT developed LR significantly later than patients without neoadjuvant CRT (51.8 vs 13.5 months; P = 0.002). LR rates in patients who underwent preoperative CRT and those who did not were 9.2% and 3.5% (difference, 5.7%), respectively, after censoring all observations at 60 months and 9.2% and 6.1% (difference, 3.1%) after censoring all observations at 120 months. CONCLUSION: Local recurrence was diagnosed significantly later in patients treated with neoadjuvant CRT. Follow-up longer than 5 years is needed to evaluate definitive results in patients treated with neoadjuvant CRT.

Analysis of the mutational landscape of classic Hodgkin lymphoma identifies disease heterogeneity and potential therapeutic targets.

Defining the mutational landscape of classic Hodgkin lymphoma is still a major research goal. New targeted next-generation sequencing (NGS) techniques may identify pathogenic mechanisms and new therapeutic opportunities related to this disease. We describe the mutational profile of a series of 57 cHL cases, enriched in Hodgkin and Reed-Sternberg (HRS) cells. Overall, the results confirm the presence of strong genomic heterogeneity. However, several variants were consistently detected in genes related to relevant signaling pathways, such as GM-CSF/IL-3, CBP/EP300, JAK/STAT, NF-kappaB, and numerous variants of genes affecting the B-cell receptor (BCR) pathway, such as BTK, CARD11, BCL10, among others. This unexpectedly high prevalence of mutations affecting the BCR pathway suggests some requirement for active BCR signaling for cHL cell viability. Additionally, incubation of a panel of cHL cellular models with selective BTK inhibitors in vitro constrains cell proliferation and causes cell death. Our results indicate new pathogenic mechanisms and therapeutic opportunities in this disease.

CSF1R Protein Expression in Reactive Lymphoid Tissues and Lymphoma: Its Relevance in Classical Hodgkin Lymphoma.

Tumour-associated macrophages (TAMs) have been associated with survival in classic Hodgkin lymphoma (cHL) and other lymphoma types. The maturation and differentiation of tissue macrophages depends upon interactions between colony-stimulating factor 1 receptor (CSF1R) and its ligands. There remains, however, a lack of consistent information on CSF1R expression in TAMs. A new monoclonal antibody, FER216, was generated to investigate CSF1R protein distribution in formalin fixed tissue samples from 24 reactive lymphoid tissues and 187 different lymphoma types. We also analysed the distribution of CSF1R+, CD68+ and CD163+ macrophages by double immunostaining, and studied the relationship between CSF1R expression and survival in an independent series of 249 cHL patients. CSF1R+ TAMs were less frequent in B-cell lymphocytic leukaemia and lymphoblastic B-cell lymphoma than in diffuse large B-cell lymphoma, peripheral T-cell lymphoma, angioimmunoblastic T-cell lymphoma and cHL. HRS cells in cHL and, with the exception of three cases of anaplastic large cell lymphoma, the neoplastic cells in NHLs, lacked detectable CSF1R protein. A CSF1R+ enriched microenvironment in cHL was associated with shorter survival in an independent series of 249 cHL patients. CSF1R pathway activation was evident in the cHL and inactivation of this pathway could be a potential therapeutic target in cHL cases.

Malaria vaccine adjuvants: latest update and challenges in preclinical and clinical research.

There is no malaria vaccine currently available, and the most advanced candidate has recently reported a modest 30% efficacy against clinical malaria. Although many efforts have been dedicated to achieve this goal, the research was mainly directed to identify antigenic targets. Nevertheless, the latest progresses on understanding how immune system works and the data recovered from vaccination studies have conferred to the vaccine formulation its deserved relevance. Additionally to the antigen nature, the manner in which it is presented (delivery adjuvants) as well as the immunostimulatory effect of the formulation components (immunostimulants) modulates the immune response elicited. Protective immunity against malaria requires the induction of humoral, antibody-dependent cellular inhibition (ADCI) and effector and memory cell responses. This review summarizes the status of adjuvants that have been or are being employed in the malaria vaccine development, focusing on the pharmaceutical and immunological aspects, as well as on their immunization outcomings at clinical and preclinical stages.

Patterns of care and outcome for patients with glioblastoma diagnosed during 2008-2010 in Spain.

BACKGROUND: To assess management patterns and outcome in patients with glioblastoma multiforme (GBM) treated during 2008-2010 in Spain. METHODS: Retrospective analysis of clinical, therapeutic, and survival data collected through filled questionnaires from patients with histologically confirmed GBM diagnosed in 19 Spanish hospitals. RESULTS: We identified 834 patients (23% aged >70 years). Surgical resection was achieved in 66% of patients, although the extent of surgery was confirmed by postoperative MRI in only 41%. There were major postoperative complications in 14% of patients, and age was the only independent predictor (Odds ratio [OR], 1.03; 95% confidence interval [CI],1.01-1.05; P = .006). After surgery, 57% received radiotherapy (RT) with concomitant and adjuvant temozolomide, 21% received other regimens, and 22% were not further treated. In patients treated with surgical resection, RT, and chemotherapy (n = 396), initiation of RT ≤42 days was associated with longer progression-free survival (hazard ratio [HR], 0.8; 95% CI, 0.64-0.99; P = .042) but not with overall survival (HR, 0.79; 95% CI, 0.62-1.00; P = .055). Only 32% of patients older than 70 years received RT with concomitant and adjuvant temozolomide. The median survival in this group was 10.8 months (95% CI, 6.8-14.9 months), compared with 17.0 months (95% CI, 15.5-18.4 months; P = .034) among younger patients with GBM treated with the same regimen. CONCLUSIONS: In a community setting, 57% of all patients with GBM and only 32% of older patients received RT with concomitant and adjuvant temozolomide. In patients with surgical resection who were eligible for chemoradiation, initiation of RT ≤42 days was associated with better progression-free survival.

Enhancing immunogenicity to PLGA microparticulate systems by incorporation of alginate and RGD-modified alginate.

Poly-lactide-co-glycolide acid (PLGA) and alginate represent two different families of polymers widely used for microencapsulation application, even more, for vaccination purposes as particulate delivery/adjuvant systems. Combination of these polymers has been previously considered for tissue engineering and drug delivery, however there is currently no report regarding their combination for vaccine application. In the present work, a w/o/w solvent extraction technique was developed to prepare novel 1µm microparticles (MP) composed of PLGA and a small percentage of alginate (PLGA-alg MP). In addition, RGD-modified alginate was also employed as biofunctionalized material favoring MP-cell interaction (PLGA-alg-RGD MP). Two malaria synthetic peptides, SPf66 and S3, were microencapsulated into PLGA, PLGA-alg and PLGA-alg-RGD MP. The diverse MP formulations resulted very similar in terms of size and morphology, although the addition of alginate improved encapsulation efficiency and reduced the amount of surface adsorbed peptide. Immunization studies in Balb/c mice by intradermal route demonstrated that incorporation of alginate elicited higher humoral and cellular immune responses leading to more balanced Th1/Th2 responses. Furthermore, administration of MP containing RGD-modified alginate showed evidence of cell targeting by enhancing immunogenicity of microparticles, in particular with regard to cellular responses such as IFN-γ secretion and lymphoproliferation.

Comparison of the adjuvanticity of two different delivery systems on the induction of humoral and cellular responses to synthetic peptides.

The aim of this work was to test, evaluate, and compare the immunogenicity of the S3 malarial short synthetic model peptide in Balb/c mice when it was delivered with different adjuvants. Specifically, it studied the adjuvanticity of two different particulate delivery systems, human compatible Montanide((R)) ISA 720 w/o emulsion and poly-lactide-co-glycolide acid microparticles, in terms of the enhancement and sub-set type of the immune response elicited following immunization. Aditionally, conventional aluminum hydroxide gel adjuvant was included as a reference. Aluminum adjuvant failed to improve the lack of immunogenicity of this antigenic peptide on its own. On the other hand, Montanide and microparticles given subcutaneously resulted in effective adjuvants and revealed mixed Th1/Th2 immune responses, with moderate antibody and lymphoproliferative responses, and higher IFN-gamma secretion for Montanide. Hence, microparticles administered intradermally (not possible with Montanide) elicited superior and potent antibody levels, including higher cytophilic isotype (IgG2a), and the greatest limphoproliferation and IFN-gamma levels. The results here presented support the capability and suitability of microparticle delivery systems to reach the adequate adjuvanticity necessary for future malaria vaccine development.