Characterization and stabilization of the α-L-fucosidase set from Lacticaseibacillus rhamnosus INIA P603.

This study describes the molecular identification, biochemical characterization, and stabilization of three recombinant AlfA, AlfB, and AlfC fucosidases from Lacticaseibacillus rhamnosus INIA P603. Even though previous studies revealed the presence of fucosidase activity in L. rhamnosus extracts, the identification of the fucosidases, their physicochemical properties, and the substrate spectrum remained unknown. Although the presence of alfB is not common in strains of L. rhamnosus, fucosidases from L. rhamnosus INIA P603 were selected because this strain exhibited higher fucosidase activity in culture and the complete set of fucosidases. A high yield of purified recombinant AlfA, AlfB, and AlfC fucosidases was obtained (8, 12, and 18 mg, respectively). AlfA, AlfB, and AlfC showed their optimal activities at pH 5.0 and 4.0 at 60 °C, 40 °C, and 50 °C, respectively. Unlike 3-fucosyllactose, all three recombinant fucosidases were able to hydrolyze 2'-fucosyllactose (2'-FL), and their activities were improved through their immobilization on agarose supports. Nevertheless, immobilized AlfB exhibited the highest hydrolysis, releasing 39.6 µmol of fucose mg enzyme-1 min-1. Only the immobilized AlfB was able to synthetize 2'-FL. In conclusion, the enzymatic properties elucidated in this study support the potential ability of fucosidases from L. rhamnosus INIA P603 to hydrolyze fucosylated substrates as well as justifying interest for further research into AlfB for its application to catalyze the synthesis of fucosylated prebiotics. KEY POINTS: • Few strains of L. rhamnosus exhibited alfB on their chromosomes. • Fucosidases from L. rhamnosus INIA P603 were characterized and stabilized. • Although all the fucosidases hydrolyzed 2'-FL, only AlfB transfucosylated lactose.

SOLTI-1503 PROMETEO TRIAL: combination of talimogene laherparepvec with atezolizumab in early breast cancer.

New treatment strategies such as immune checkpoint inhibitors and oncolytic viruses are opening new possibilities in cancer therapy. Preliminary results in melanoma and other tumors showed that the combination of talimogene laherparepvec with an anti-PD-1/PD-L1 or anti-CTLA4 has greater efficacy than either therapy alone, without additional safety concerns beyond those expected for each agent. The presence of residual cancer after neoadjuvant chemotherapy in early breast cancer patients is an unmet medical need. SOLTI-1503 PROMETEO is a window of opportunity trial, which evaluates the combination of talimogene laherparepvec in combination with atezolizumab in women with operable HER2-negative breast cancer who present residual disease after neoadjuvant chemotherapy. The primary end point is the rate of residual cancer burden 0/1. Clinical Trial Registration: NCT03802604.

Production of recombinant glycosidases fused with Usp45 and SpaX to avoid the purification and immobilization stages.

The elucidation of the physicochemical properties of glycosidases is essential for their subsequent technological application, which may include saccharide hydrolysis processes and oligosaccharide synthesis. As the application of cloning, purification and enzymatic immobilization methods can be time consuming and require a heavy financial investment, this study has validated the recombinant production of the set of Lacticaseibacillus rhamnosus fucosidases fused with Usp45 and SpaX anchored to the cell wall of Lacticaseibacillus cremoris subsp cremoris MG1363, with the aim of avoiding the purification and stabilization steps. The cell debris harboring the anchored AlfA, AlfB and AlfC fucosidases showed activity against p-nitrophenyl α-L-fucopyranoside of 6.11 ±â€¯0.36, 5.81 ±â€¯0.29 and 9.90 ±â€¯0.58 U/mL, respectively, and exhibited better thermal stability at 50 °C than the same enzymes in their soluble state. Furthermore, the anchored AlfC fucosidase transfucosylated different acceptor sugars, achieving fucose equivalent concentrations of 0.94 ±â€¯0.09 mg/mL, 4.11 ±â€¯0.21 mg/mL, and 4.08 ±â€¯0.15 mg/mL of fucosylgalatose, fucosylglucose and fucosylsucrose, respectively.

Primary Chemoradiotherapy Treatment (PCRT) for HER2+ and Triple Negative Breast Cancer Patients: A Feasible Combination.

Primary systemic treatment (PST) downsizes the tumor and improves pathological response. The aim of this study is to analyze the feasibility and tolerance of primary concurrent radio−chemotherapy (PCRT) in breast cancer patients. Patients with localized TN/HER2+ tumors were enrolled in this prospective study. Radiation was delivered concomitantly during the first 3 weeks of chemotherapy, and it was based on a 15 fractions scheme, 40.5 Gy/2.7 Gy per fraction to whole breast and nodal levels I-IV. Chemotherapy (CT) was based on Pertuzumab−Trastuzumab−Paclitaxel followed by anthracyclines in HER2+ and CBDCA-Paclitaxel followed by anthracyclines in TN breast cancers patients. A total of 58 patients were enrolled; 25 patients (43%) were TN and 33 patients HER2+ (57%). With a median follow-up of 24.2 months, 56 patients completed PCRT and surgery. A total of 35 patients (87.5%) achieved >90% loss of invasive carcinoma cells in the surgical specimen. The 70.8% and the 53.1% of patients with TN and HER-2+ subtype, respectively, achieved complete pathological response (pCR). This is the first study of concurrent neoadjuvant treatment in breast cancer in which three strategies were applied simultaneously: fractionation of RT (radiotherapy) in 15 sessions, adjustment of CT to tumor phenotype and local planning by PET. The pCR rates are encouraging.

A Randomized Phase II Study of Anti-CSF1 Monoclonal Antibody Lacnotuzumab (MCS110) Combined with Gemcitabine and Carboplatin in Advanced Triple-Negative Breast Cancer.

PURPOSE: This phase II study determined the efficacy of lacnotuzumab added to gemcitabine plus carboplatin (gem-carbo) in patients with advanced triple-negative breast cancer (TNBC). PATIENTS AND METHODS: Female patients with advanced TNBC, with high levels of tumor-associated macrophages not amenable to curative treatment by surgery or radiotherapy were enrolled. Lacnotuzumab was dosed at 10 mg/kg every 3 weeks, ± a dose on cycle 1, day 8. Gemcitabine (1,000 mg/m2) and carboplatin (dose in mg calculated by area under the curve [mg/mL/min] × (glomerular filtration rate [mL/min] + 25 [mL/min]) were dosed every 3 weeks. Treatment continued until unacceptable toxicity, disease progression, or discontinuation by physician/patient. RESULTS: Patients received lacnotuzumab + gem-carbo (n = 34) or gem-carbo (n = 15). Enrollment was halted due to recruitment challenges owing to rapid evolution of the therapeutic landscape; formal hypothesis testing of the primary endpoint was therefore not performed. Median progression-free survival was 5.6 months [90% confidence interval (CI), 4.47-8.64] in the lacnotuzumab + gem-carbo arm and 5.5 months (90% CI, 3.45-7.46) in the gem-carbo arm. Hematologic adverse events were common in both treatment arms; however, patients treated with lacnotuzumab experienced more frequent aspartate aminotransferase, alanine aminotransferase, and creatine kinase elevations. Pharmacokinetic results showed that free lacnotuzumab at 10 mg/kg exhibited a typical IgG pharmacokinetic profile and target engagement of circulating colony-stimulating factor 1 ligand. CONCLUSIONS: Despite successful target engagement and anticipated pharmacokinetic profile, lacnotuzumab + gem-carbo showed comparable antitumor activity to gem-carbo alone, with slightly poorer tolerability. However, the data presented in this article would be informative for future studies testing agents targeting the CSF1-CSF1 receptor pathway in TNBC.

Palbociclib and Trastuzumab in HER2-Positive Advanced Breast Cancer: Results from the Phase II SOLTI-1303 PATRICIA Trial.

PURPOSE: To assess palbociclib in combination with trastuzumab with or without endocrine therapy in patients with HER2-positive advanced breast cancer. PATIENTS AND METHODS: PATRICIA is a prospective, open-label, multicenter phase II trial. Patients had received 2-4 prior lines of anti-HER2-based regimens. Treatment consisted of palbociclib 200 mg daily for 2 weeks and 1 week off plus trastuzumab. The study was based on a Simon two-stage design comprising three cohorts: estrogen receptor (ER)-negative (cohort A), ER-positive (cohort B1), and ER-positive with letrozole (cohort B2). ER-positive patients were randomized to cohorts B1 or B2. Primary endpoint was progression-free survival rate at 6 months (PFS6). Secondary objectives included safety and evaluation of the PAM50 intrinsic subtypes. RESULTS: Seventy-one patients were recruited (n = 15 in cohort A and 28 in each cohort B). The PFS6 rate in cohorts A, B1, and B2 was 33.3% (5/15), 42.8% (12/28), and 46.4% (13/28), respectively. Regarding safety, grade 1-2 and 3-4 toxicities occurred in 97.7% and 84.4% of patients, respectively. The most common grade 3-4 toxicities were neutropenia (66.4%) and thrombocytopenia (11.3%). Regarding PAM50, 59 (83.1%) tumors were profiled. Luminal disease defined by PAM50 was found independently associated with longer PFS compared with non-luminal disease (10.6 vs. 4.2 months median PFS; adjusted hazard ratio = 0.40; P = 0.003). CONCLUSIONS: Palbociclib in combination with trastuzumab is safe and exhibits promising survival outcomes in trastuzumab pretreated ER-positive/HER2-positive advanced breast cancer with a PAM50 Luminal A or B subtype. The enrollment was stopped prematurely, and a new randomized cohort was opened in this population.

Palbociclib combined with endocrine therapy in heavily pretreated HR+/HER2- advanced breast cancer patients: Results from the compassionate use program in Spain (PALBOCOMP).

BACKGROUND: This study evaluated efficacy and safety of palbociclib, a CDK4/6 inhibitor, in heavily-pretreated hormone receptor-positive and human epidermal growth factor receptor 2-negative (HR+/HER2-) metastatic breast cancer (mBC) patients during the compassionate use program in Spain from February 2015 to November 2017. PATIENTS AND METHODS: Patient data were collected retrospectively from 35 hospitals in Spain. Patients with HR+/HER2- mBC who had progressed on ≥4 treatments for advanced disease were eligible. RESULTS: A total of 219 patients received palbociclib in combination with aromatase inhibitors (110; 50.2%), fulvestrant (87; 39.7%), tamoxifen (8; 3.6%) or as single agent (10; 4.6%). Mean age of the patients was 58 years; 31 patients (16.1%) were premenopausal and 162 (83.9%) were postmenopausal at the beginning of treatment with palbociclib. Patients had received a median of 3 previous lines of endocrine therapy (ET) for advanced disease. Real-world tumor response (rwTR) and clinical benefit rate were 5.9% (n = 13) and 46.2% (n = 101), respectively. The median real world progression-free survival (rwPFS) was 6.0 months (95% CI 5.7-7.0) and the median overall survival was 19.0 months (95% CI 16.4-21.7). Subgroup analysis revealed a significant difference in median rwPFS in patients treated with palbociclib plus fulvestrant depending on the duration of prior treatment with fulvestrant monotherapy (>6 versus ≤6 months; HR 1.93, 95% CI 1.37-2.73, p < 0.001). The most frequently reported toxicities were neutropenia, asthenia, thrombopenia and anemia. CONCLUSIONS: Palbociclib can be an effective and safe treatment option in patients with heavily pretreated endocrine-sensitive mBC, especially in those with longer PFS to previous ET.

[Evaluation of the validity of Diagnostic Interview Schedule for Children IV (DISC IV) in the city of Buenos Aires]. / Evaluación de la validez de los diagnósticos DSM IV realizados con un Instrumento Estructurado, el NIMH DISC-IV, en la Ciudad de Buenos Aires.

UNLABELLED: The epidemiology of psychiatric disorders in children and adolescents has received little attention in Argentina. One of the problems related to the scarcity of such epidemiological research is linked to the lack of availability of diagnostic interview instruments which have been locally validated. OBJECTIVES: The object of the study was to conduct a validation study of the DISC IV (Spanish version), administered by lay interviewers in the City of Buenos Aires. METHODS: The sample was obtained from the Hospital de Niños "Ricardo Gutiérrez" in the City of Buenos Aires. Lay interviewers administered the DISC IV to 116 youngsters. Then psychiatrists re-administered the DISC IV a week later and immediately afterwards conducted a semi-structured diagnostic clinical interview. Participant in the sample ranged in age from 9 to 17. RESULTS: The sensitivity was 81.5% and the specificity 66.1%. The test-retest reliability was reasonable (Kappa 0.46 standard error 0.09). CONCLUSIONS: In general, the DISC administered by the non-professional interviewer was demonstrated to have the ability to discriminate between youngsters who suffer from psychiatric disorders and healthy youngsters. The confidence level was from moderate to good for the presence of a general psychiatric disorder as well as for disorders of specific states of mind, but for anxiety disorders and behaviour disorders the confidence level was poor.

Dynamic Angiogenic Switch as Predictor of Response to Chemotherapy-Bevacizumab in Patients With Metastatic Colorectal Cancer.

BACKGROUND: Previous studies have shown that metastatic colorectal carcinoma (mCRC) patients treated with bevacizumab, experience variation in the plasma levels of angiogenesis growth factors and related cytokines, called angiogenic switch (AS). The aim of the present study was to analyze the relationship between AS and the clinical response during standard chemotherapy-bevacizumab treatment. PATIENTS AND METHODS: Patients with Eastern Cooperative Oncology Group 0-1 mCRC were eligible. Patients received treatment with standard dose capecitabine plus either oxaliplatin or irinotecan and bevacizumab for 6 cycles. Initial treatment was followed by maintenance therapy with bevacizumab plus capecitabine until progression. Plasma levels of angiogenic-related cytokines (hepatocyte growth factor, placental growth factor, macrophage chemoattractant protein-3, MM-9, eotaxin, basic fibroblast growth factor, and interleukin 18) were prospectively analyzed at baseline and every 8 weeks. Progression-free survival (PFS) was calculated using the Kaplan-Meier method. RESULTS: A total of 71 patients were enrolled. AS was observed in 45 patients (63.4%), 28 of whom experienced AS at the first evaluation after treatment start. Disease control, which includes partial/complete response and stable disease, was seen in 96% of AS patients (43/45), but only in 15/26 (58%) for the remaining patients without evidence of AS (P<0.001). The median PFS of AS patients was 11.4 months (95% confidence interval, 8.6-15.8) versus 8.3 months for patients without AS (95% confidence interval, 5.6-16.4; P=0.04). CONCLUSIONS: Chemotherapy plus Bevacizumab combination in mCRC patients results in dynamic changes in plasma cytokines, which is associated with better disease control and longer PFS. These new findings support continuing studying AS as a potential marker of angiogenesis inhibitor effectiveness.

Exome Sequencing of Plasma DNA Portrays the Mutation Landscape of Colorectal Cancer and Discovers Mutated VEGFR2 Receptors as Modulators of Antiangiogenic Therapies.

Purpose: Despite the wide use of antiangiogenic drugs in the clinical setting, predictive biomarkers of response to these drugs are still unknown.Experimental Design: We applied whole-exome sequencing of matched germline and basal plasma cell-free DNA samples (WES-cfDNA) on a RAS/BRAF/PIK3CA wild-type metastatic colorectal cancer patient with primary resistance to standard treatment regimens, including inhibitors to the VEGF:VEGFR2 pathway. We performed extensive functional experiments, including ectopic expression of VEGFR2 mutants in different cell lines, kinase and drug sensitivity assays, and cell- and patient-derived xenografts.Results: WES-cfDNA yielded a 77% concordance rate with tumor exome sequencing and enabled the identification of the KDR/VEGFR2 L840F clonal, somatic mutation as the cause of therapy refractoriness in our patient. In addition, we found that 1% to 3% of samples from cancer sequencing projects harbor KDR somatic mutations located in protein residues frequently mutated in other cancer-relevant kinases, such as EGFR, ABL1, and ALK. Our in vitro and in vivo functional assays confirmed that L840F causes strong resistance to antiangiogenic drugs, whereas the KDR hot-spot mutant R1032Q confers sensitivity to strong VEGFR2 inhibitors. Moreover, we showed that the D717V, G800D, G800R, L840F, G843D, S925F, R1022Q, R1032Q, and S1100F VEGFR2 mutants promote tumor growth in mice.Conclusions: Our study supports WES-cfDNA as a powerful platform for portraying the somatic mutation landscape of cancer and discovery of new resistance mechanisms to cancer therapies. Importantly, we discovered that VEGFR2 is somatically mutated across tumor types and that VEGFR2 mutants can be oncogenic and control sensitivity/resistance to antiangiogenic drugs. Clin Cancer Res; 24(15); 3550-9. ©2018 AACR.

Clinical validation of prospective liquid biopsy monitoring in patients with wild-type RAS metastatic colorectal cancer treated with FOLFIRI-cetuximab.

Cancer genomics and translational medicine rely on the molecular profiling of patient's tumor obtained during surgery or biopsy. Alternatively, blood is a less invasive source of tumor DNA shed, amongst other ways, as cell-free DNA (cfDNA). Highly-sensitive assays capable to detect cancer genetic events from patient's blood plasma became popularly known as liquid biopsy (LqB). Importantly, retrospective studies including small number of selected patients with metastatic colorectal cancer (mCRC) patients treated with anti-EGFR therapy have shown LqB capable to detect the acquired clonal mutations in RAS genes leading to therapy resistance. However, the usefulness of LqB in the real-life clinical monitoring of these patients still lack additional validation on controlled studies. In this context, we designed a prospective LqB clinical trial to monitor newly diagnosed KRAS wild-type (wt) mCRC patients who received a standard FOLFIRI-cetuximab regimen. We used BEAMing technique for evaluate cfDNA mutations in KRAS, NRAS, BRAF, and PIK3CA in twenty-five patients during a 2-y period. A total of 2,178 cfDNA mutation analyses were performed and we observed that: a) continued wt circulating status was correlated with a prolonged response; b) smoldering increases in mutant cfDNA were correlated with acquired resistance; while c) mutation upsurge/explosion anticipated a remarkable clinical deterioration. The current study provides evidences, obtained for the first time in an unbiased and prospective manner, that reinforces the utility of LqB for monitoring mCRC patients.

The therapeutic role of fulvestrant in the management of patients with hormone receptor-positive breast cancer.

Although selective estrogen receptor modulators (SERMs), such as tamoxifen, or aromatase inhibitors (AIs), such as anastrozole, are the preferred endocrine treatment approach for most patients with hormone receptor-positive breast cancer, many patients progress despite this therapy or become resistant. Fulvestrant is a selective estrogen receptor down-regulator (SERD) that has demonstrated activity and efficacy in patients with hormone receptor-positive breast cancer previously untreated or treated with hormonal therapy. The efficacy of fulvestrant has been demonstrated in the neoadjuvant and metastatic settings, either alone or in combination with other therapies such as anastrozole or targeted drugs. Additionally, 500 mg of fulvestrant have been shown to be more effective than 250 mg, without significant differences in the toxicity profile. In this review, the unique mode of action of fulvestrant and the clinical data for different dosing regimens both alone or in combination with other drugs is critically assessed.

Novel approach for the determination of volatile compounds in processed onion by headspace gas chromatography-mass spectrometry (HS GC-MS).

A novel headspace gas chromatography-mass spectrometry (HS GC-MS) method was developed for analysis of volatile compounds in onion (Allium cepa L. var. cepa, 'Recas'). MS was operated using full scan mode and selective ion monitoring (SIM) mode in order to quantify some specific compounds with increased sensitivity relative to full scan mode. The limits of detection and quantitation ranged from 0.01 to 0.10 µg/g and from 0.02 to 3.83 µg/g fresh weight, respectively, for studied compounds. The procedure allowed the identification of eighteen compounds and quantitation of nine compounds in the volatile fraction of onion, belonging mainly to di-, and trisulfides and aldehydes. These methods were applied to evaluate how high-pressure (HP) as a processing technology affects onion volatile compounds, responsible in part of the onion biological activity. Onion samples were treated at T1: 200 MPa/25°C/5 min, T2: 400 MPa/25°C/5 min and T3: 600 MPa/25°C/5 min (treatments). In addition, the difference among diced, freeze-dried and pulverized onions (groups) was studied, in order to select the process more adequate for better preserving volatile compounds. The results obtained in full scan mode showed that both main factors (group and treatment) had a significant effect (P<0.001). There were also significant differences between groups and treatments for all compounds, being the main effect of group more marked by HS GC-MS using selective ion monitoring (SIM) mode. For 2-methyl 2-pentenal, dimethyl trisulfide, and methyl propyl trisulfide it has been observed an increase in freeze-dried and pulverized onion samples compared with diced samples regardless the HP treatment. However, freeze-drying and pulverization processes affected the stability of propionaldehyde, 1-propanethiol, hexanal, dipropyl disulfide, and dipropyl trisulfide, diminishing their content regardless the HP treatment. HP at 200 and 400 MPa/25°C/5 min were the least detrimental treatments to the total fraction of volatile compounds, not affecting or even increasing the levels of some volatile compounds.

Evaluación de la validez de los diagnósticos DSM IV realizados con un Instrumento Estructurado, el NIMH DISC-IV, en la Ciudad de Buenos Aires / Evaluation of the validity of diagnostic interview schedule for children IV (DISC IV) in the city of Buenos Aires

La epidemiología de los trastornos psiquiátricos de niños y adolescentes ha recibido poca atención en nuestro país, Uno de los problemas relacionados con el escaso desarrollo de investigación epidemiológica en Argentina está ligado a la no disponibilidad de entrevistas diagnósticas estructura das testeadas localmente. Objetivos: el objetivo fue realizar un estudio de validación DISC IV (versión en español), administrada por encuestadores legos en la ciudad de Buenos Aires. Métodos: la muestra fue obtenida del Hospital de Niños "Ricardo Gutiérrez" de la Ciudad de Buenos Aires. Dos grupos de participantes fueron incluidos en el estudio, un grupo con resultado positivo a la administración del DISC y otro grupo equivalente con resultado negativo. Los psiquiatras administraron nuevamente el DISC una semana más tarde y a continuación realizaron una entrevista clínica di agnóstica semiestructurada 116 participantes completaron las evaluaciones y formaron parte de la muestra. Resultados: la sensibilidad fue de 81,5 por ciento y la especificidad de 66,1 por ciento. La reproducibilidad test-retest fue moderada (Kappa 0,46 error standard 0,09). Conclusiones: en líneas generales, el DISC administrado por encuesta dores legos demostró poseer buena capacidad para discriminar entre jóvenes que padecen trastornos psiquiátricos y jóvenes sanos. La confiabilidad fue de moderada a buena, tanto para la presencia de algún trastorno psiquiátrico en forma global como para los trastornos del estado de ánimo en particular, mientras que para los trastornos de ansiedad y los trastornos de conducta resultó muy pobre.

The epidemiology of psychiatric disorders in children and adolescents has received little attention in Argentina. One of the problems related to the scarcity of such epidemiological research is linked to the lack of availability of diagnostic interview instruments which have been locally validated. Objectives: The object of the study was to conduct a validation study of the DISC IV (Spanish version), administered by lay interviewers in the City of Buenos Aires. Methods: The sample was obtained from the Hospital de Niños "Ricardo Gutierrez" in the City of Buenos Aires. Lay interviewers administered the DISC IV to 116 youngsters. Then psychiatrists re-administered the DISC IV a week later and immediately afterwards conducted a semi-structured diagnostic clinical interview. Participant in the sample ranged in age from 9 to 17. Results: The sensitivity was 81.5 percent and the specificity 66, 1 percent. The test-re test reliability was reasonable (Kappa 0.46 standard error 0.09). Conclusions: In general, the DISC administered by the non-professional interviewer was demonstrated to have the ability to discriminate between youngsters who suffer from psychiatric disorders and healthy youngsters. The confidence level was from moderate to good for the presence of a general psychiatric disorder as well as for disorders of specific states of mind, but for anxiety disorders and behaviour disorders the confidence level was poor.