The EPICAL trial, a phase Ib study combining first line afatinib with anti-EGF vaccination in EGFR-mutant metastatic NSCLC.

INTRODUCTION: Combination of anti-EGFR monoclonal antibodies or immune checkpoint inhibitors with TKIs has shown minimal benefit in EGFR mutant (EGFR-mut) NSCLC patients. Consequently, new combination approaches are needed. PATIENTS AND METHODS: The EPICAL was a single arm, phase 1b study to evaluate safety, tolerability and anti-tumor activity of first line afatinib combined with anti-EGF vaccination in advanced EGFR-mut patients. EGFR status and mutations in liquid biopsies were determined by reverse transcriptase-polymerase chain reaction; serum biomarkers by ELISA and Western blotting analysis. RESULTS: The assay enrolled 23 patients, 21 completed the anti-EGF immunization phase. Treatment was well tolerated and no serious adverse events (SAEs) related to the anti-EGF vaccine were reported. Objective response and disease control rates were 78.3% (95%CI = 53.6-92.5) and 95.7% (95%CI = 78.1-99.9), respectively. After a median follow-up of 24.2 months, median progression-free survival (PFS) was 14.8 months (95% CI = 9.5-20.1) and median overall survival (OS) 26.9 months (95% CI = 23.0-30.8). Among the 21 patients completing the immunization phase, PFS was 17.5 months (95% CI = 12.0-23.0) and OS 26.9 months (95% CI = 24.6-NR). At the end of the immunization phase, all 21 patients showed high serum titers of anti-EGF antibodies, while EGF levels had decreased significantly. Finally, treatment with fully immunized patient's sera inhibited the EGFR pathway in tumor cells growing in vitro. CONCLUSIONS: Combination treatment with an anti-EGF vaccine is well tolerated; induces a sustained immunogenic effect and might enhance the clinical efficacy of EGFR TKIs.

Large scale, prospective screening of EGFR mutations in the blood of advanced NSCLC patients to guide treatment decisions.

BACKGROUND: In a significant percentage of advanced non-small-cell lung cancer (NSCLC) patients, tumor tissue is unavailable or insufficient for genetic analyses. We prospectively analyzed if circulating-free DNA (cfDNA) purified from blood can be used as a surrogate in this setting to select patients for treatment with epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs). PATIENTS AND METHODS: Blood samples were collected in 119 hospitals from 1138 advanced NSCLC patients at presentation (n = 1033) or at progression to EGFR-TKIs (n = 105) with no biopsy or insufficient tumor tissue. Serum and plasma were sent to a central laboratory, cfDNA purified and EGFR mutations analyzed and quantified using a real-time PCR assay. Response data from a subset of patients (n = 18) were retrospectively collected. RESULTS: Of 1033 NSCLC patients at presentation, 1026 were assessable; with a prevalence of males and former or current smokers. Sensitizing mutations were found in the cfDNA of 113 patients (11%); with a majority of females, never smokers and exon 19 deletions. Thirty-one patients were positive only in plasma and 11 in serum alone and mutation load was higher in plasma and in cases with exon 19 deletions. More than 50% of samples had <10 pg mutated genomes/µl with allelic fractions below 0.25%. Patients treated first line with TKIs based exclusively on EGFR positivity in blood had an ORR of 72% and a median PFS of 11 months. Of 105 patients screened after progression to EGFR-TKIs, sensitizing mutations were found in 56.2% and the p.T790M resistance mutation in 35.2%. CONCLUSIONS: Large-scale EGFR testing in the blood of unselected advanced NSCLC patients is feasible and can be used to select patients for targeted therapy when testing cannot be done in tissue. The characteristics and clinical outcomes to TKI treatment of the EGFR-mutated patients identified are undistinguishable from those positive in tumor.

La hibridación in situ fluorescente en la predicción de recurrencia del carcinoma urotelial / Fluorescent in situ Hybridization as a Predictor of Relapse in Urotelial Carcinoma

Objetivo: Evaluar la utilidad del estudio de alteraciones cromosómicas mediante hibridación in situ fluorescente en una serie de pacientes diagnosticados de carcinoma urotelial con un seguimiento mínimo de 24 meses, y analizar su posible efecto anticipador. Material y métodos: La serie global incluye 338 muestras procedentes de 98 pacientes con 84 episodios de carcinoma urotelial. Para evaluar la capacidad de predicción del test se estudió un subgrupo de 24 pacientes que presentaron como mínimo una recurrencia a lo largo del seguimiento (serie de recurrencia). Se consideraron 3 categorías (episodio coherente positivo, episodio coherente negativo y episodio no coherente) en función de la relación entre los resultados de la hibridación in situ fluorescente del estudio concomitante al nuevo episodio y las muestras precedentes. Resultados: La hibridación in situ fluorescente presentó mayor sensibilidad independientemente del grado, valor predictivo negativo y exactitud, mientras que la especificidad y el valor predictivo positivo fueron superiores para la citología convencional. En la serie de recurrencia 19/29 episodios resultaron coherentes, 11/19 fueron coherentes positivos, todos con carcinoma urotelial de alto grado, y 8/19 coherentes negativos, la mayoría de bajo grado. Conclusiones: La hibridación in situ fluorescente muestra una alta sensibilidad en un seguimiento de 24 meses, y es capaz de predecir recurrencias, especialmente en casos de alto grado. Nuestros datos demuestran también la existencia de carcinomas uroteliales sin alteraciones cromosómicas detectables con la metodología actualmente disponible. Los resultados apoyan un seguimiento multidisciplinar con la utilización combinada de la hibridación in situ fluorescente, citología y cistoscopia (AU)

Objective: To assess the value of the study of chromosomal alterations by fluorescent in situ hybridization in a series of patients diagnosed of urothelial carcinoma and a minimum follow up of twenty four months, as well as evaluate its putative predictive potential. Material and methods: The overall series includes 338 samples from 98 patients with 84 episodes of urothelial carcinoma. A subgroup of 24 patients who had at least one recurrence during the follow up was used to evaluate the predictive potential of the test. Three categories were considered (positive coherent episode, negative coherent episode, and incoherent episode) depending on the relationship between the fluorescent in situ hybridization result in the concomitant study of the new episode and those of the preceding samples. Results: Fluorescent in situ hybridization showed higher sensitivity regardless of grade, negative predictive value and accuracy, while specificity and positive predictive value were superior with conventional cytology. In the recurrence, series 19/29 episodes were coherent, 11/19 were positive coherent with urothelial carcinoma all high grade and 8/19 negative coherent, most low grade. Conclusions: Fluorescent in situ hybridization test shows good sensitivity during a follow up of twenty four months and is able to predict recurrence, especially in cases of high grade. Our data demonstrate the existence of urothelial carcinomas without detectable chromosomal abnormalities by currently available methodology. The results support a multidisciplinary follow up combining fluorescent in situ hybridization, cytology and cystoscopy (AU)

Fluorescent in situ hybridization as a predictor of relapse in urothelial carcinoma.

OBJECTIVE: To assess the value of the study of chromosomal alterations by fluorescent in situ hybridization in a series of patients diagnosed of urothelial carcinoma and a minimum follow up of twenty four months, as well as evaluate its putative predictive potential. MATERIAL AND METHODS: The overall series includes 338 samples from 98 patients with 84 episodes of urothelial carcinoma. A subgroup of 24 patients who had at least one recurrence during the follow up was used to evaluate the predictive potential of the test. Three categories were considered (positive coherent episode, negative coherent episode, and incoherent episode) depending on the relationship between the fluorescent in situ hybridization result in the concomitant study of the new episode and those of the preceding samples. RESULTS: Fluorescent in situ hybridization showed higher sensitivity regardless of grade, negative predictive value and accuracy, while specificity and positive predictive value were superior with conventional cytology. In the recurrence, series 19/29 episodes were coherent, 11/19 were positive coherent with urothelial carcinoma all high grade and 8/19 negative coherent, most low grade. CONCLUSIONS: Fluorescent in situ hybridization test shows good sensitivity during a follow up of twenty four months and is able to predict recurrence, especially in cases of high grade. Our data demonstrate the existence of urothelial carcinomas without detectable chromosomal abnormalities by currently available methodology. The results support a multidisciplinary follow up combining fluorescent in situ hybridization, cytology and cystoscopy.

The administration of oleoyl-estrone to lactating dams induces selective changes in the normal growth pattern of their pups.

To determine the effects of oleoyl-estrone treatment on the lactating dams and on the growth pattern of developing rats, female Wistar rats were randomly divided into two groups after delivery. One group received a daily gavage of 0.2 mL sunflower oil containing 10 micromol oleoyl-estrone/kg (treated group) and the other received a daily intragastric gavage of 0.2 mL sunflower oil (control group). Treatment was carried out during the first 15 days of lactation. Dams were killed on days 1, 15 or 20 after delivery and pups were sacrificed on days 1, 15, 20 or 30. Treated dams showed a transient decrease in food intake, significant lower lipid content than control dams, with a parallel maintenance of protein content and no appreciative changes in plasmatic parameters. However, a significant increase in brown adipose tissue mass was detected in treated group. Pups from treated dams showed a decrease in their growth rate that was reflected in the lower adipose tissue mass in different locations, except in the case of brown adipose tissue and, that continued after cessation of treatment. Thus, treatment affects dams in a selective way that does not coincide with a simple food restriction model.

Different uptake and handling of oleoyl-estrone by fetuses and neonatal rats.

To determine whether oleoyl-estrone can be transferred from mothers to their offspring either during pregnancy or lactation, a gavage of tracer dose of (3)H-Oleoyl-estrone was given to 21-day pregnant rats and to lactating rats on day 15 after delivery. In pregnant rats, the label was found in maternal blood as well as in liver and fetal serum, the latter showing the highest specific activity observed. In lactating rats, oleoyl-estrone label was found both in the mammary gland and maternal serum; in the pups, label was found in their stomach contents (i.e., clotted milk) and serum. The results suggest that the placenta effectively blocks the passage of oleoyl-estrone to the fetuses probably because of its high esterase activity. On the other hand, oleoyl-estrone is easily transferred from dams to pups, as a component of milk.

Weight loss with long-term intermittent treatment with oral oleoyl-estrone in lean Zucker male rats.

To determine whether or not the long-term intermittent treatment with oleoyl-estrone (OE) in rats induces a cumulative weight loss, adult male rats were treated with OE over three alternating 10-day periods, with a 30-day "recovery" period occurring between each period. At the end of the third treatment period, the rats were allowed to recover and were then mated with females. Each treatment period produced a decrease of ca. 7% in body weight with no rebound during the recovery periods, whereas weight changed at the same pace in controls. The greatest difference in body weight was observed during the last days of treatment. OE-treated rats retained their initial protein pools throughout the treatment. Furthermore, treated and control males remained fertile and were able to procreate. Thus, we can conclude that intermittent OE treatment induces a cumulative weight loss in adult male rats.

Technical note: Measurement of total estrone content in foods. Application to dairy products.

Estrone is a powerful growth-inducing hormone that is present in milk, mainly in the form of fatty acid esters, at concentrations that promote growth in experimental animals. We present here a method useful for the measurement of this natural hormone in foods and applied it to several common dairy products. Samples were frozen, finely powdered, and lyophilized then extracted with trichloromethane/methanol; the dry extract was saponified with potassium hydroxide. The free estrone evolved was extracted with ethyl acetate and was used for the estimation of total estrone content through radioimmunoassay. Application of the method to dairy products showed high relative levels of total estrone (essentially acyl-estrone) in milk, in the range of 1 microM, which were halved in skimmed milk. Free estrone levels were much lower, in the nanomolar range. A large proportion of estrone esters was present in all other dairy products, fairly correlated with their fat content. The amount of estrone carried by milk is well within the range, where its intake may exert a physiological response in the sucklings for which it is provided. These growth-inducing and energy expenditure-lowering effects may affect humans ingesting significant amounts of dairy products.