Clinical relevance of circulating ESR1 mutations during endocrine therapy for advanced hormone-dependent endometrial carcinoma.

OBJECTIVE: Endocrine therapy is frequently administered in patients with hormone dependent (HR+) metastatic endometrial cancer. ESR1 mutations have emerged as a key mechanism of aromatase inhibitor (AI) resistance in HR + metastatic breast cancer and can be monitored using circulating tumor DNA (ctDNA). The aim of this study was to explore the incidence and clinical relevance of circulating ESR1 mutations in patients treated by AI or megestrol acetate (M) for advanced endometrial carcinoma. METHODOLOGY: This single-center retrospective study was performed at the Henri Becquerel Center (Rouen) and looked for circulating ESR1 gene mutations by droplet digital PCR (E380Q, L536R, Y537S, Y537N, Y537C, D538G, S463P) in patients with advanced HR + endometrial carcinoma treated between 2008 and 2020 for at least 30 days by AI or M. Analyses were performed before exposure and at progression/during endocrine therapy. RESULTS: Twenty-two patients were included: 13 were treated with AI, 12 of whom progressed; 9 patients were treated with M, 8 of whom progressed. 68.1% of the patients had low-grade endometrial carcinoma and 54.5% had received chemotherapy in the metastatic setting. The median duration of treatment was 152 days (min 47 - max 629) with AI and 155 days (min 91-max 1297) with M. Under AI, there was no ESR1 mutation at baseline, and one Y537C mutation at progression with a variant allele frequency (VAF) of 0.14%. Under M, one patient had a Y537C (VAF 0.2%) at baseline that disappeared during treatment. Another patient had a Y537S mutation emergence at progression after 91 days of treatment (VAF 1.83%). There was no significant difference between the circulating DNA concentration before and after hormone therapy (p = 0.16). CONCLUSION: ESR1 mutations do not seem to be involved in the mechanisms of resistance to AI or M in HR+ endometrial cancer. The clinical relevance of their detection is not demonstrated.

Diagnostic and predictive accuracy of anti-mullerian hormone for ovarian function after chemotherapy in premenopausal women with early breast cancer.

PURPOSE: Accurate diagnosis and prediction of loss of ovarian function after chemotherapy for premenopausal women with early breast cancer (eBC) is important for future fertility and clinical decisions regarding the need for subsequent adjuvant ovarian suppression. We have investigated the value of anti-mullerian hormone (AMH) as serum biomarker for this. METHODS: AMH was measured in serial blood samples from 206 premenopausal women aged 40-45 years with eBC, before and at intervals after chemotherapy. The diagnostic accuracy of AMH for loss of ovarian function at 30 months after chemotherapy and the predictive value for that of AMH measurement at 6 months were analysed. RESULTS: Undetectable AMH showed a high diagnostic accuracy for absent ovarian function at 30 months with AUROC 0.89 (96% CI 0.84-0.94, P < 0.0001). PPV of undetectable AMH at 6 months for a menopausal estradiol level at 30 months was 0.77. In multivariate analysis age, pre-treatment AMH and FSH, and taxane treatment were significant predictors, and combined with AMH at 6 months, gave AUROC of 0.90 (95% CI 0.86-0.94), with PPV 0.79 for loss of ovarian function at 30 months. Validation by random forest models with 30% data retained gave similar results. CONCLUSIONS: AMH is a reliable diagnostic test for lack of ovarian function after chemotherapy in women aged 40-45 with eBC. Early analysis of AMH after chemotherapy allows identification of women who will not recover ovarian function with good accuracy. These analyses will help inform treatment decisions regarding adjuvant endocrine therapy in women who were premenopausal before starting chemotherapy.

CEA, CA19-9, circulating DNA and circulating tumour cell kinetics in patients treated for metastatic colorectal cancer (mCRC).

BACKGROUND: We previously reported that CEA kinetics are a marker of progressive disease (PD) in metastatic colorectal cancer (mCRC). This study was specifically designed to confirm CEA kinetics for predicting PD and to evaluate CA19-9, cell-free DNA (cfDNA), circulating tumour DNA (ctDNA) and circulating tumour cell (CTC) kinetics. METHODS: Patients starting a chemotherapy (CT) with pre-treatment CEA > 5 ng/mL and/or CA19.9 > 30 UI/mL were prospectively included. Samples were collected from baseline to cycle 4 for CEA and CA19-9 and at baseline and the sixth week for other markers. CEA kinetics were calculated from the first to the third or fourth CT cycle. RESULTS: A total of 192 mCRC patients were included. CEA kinetics based on the previously identified >0.05 threshold was significantly associated with PD (p < 0.0001). By dichotomising by the median value, cfDNA, ctDNA and CA19-9 were associated with PD, PFS and OS in multivariate analysis. A circulating scoring system (CSS) combining CEA kinetics and baseline CA19-9 and cfDNA values classified patients based on high (n = 58) and low risk (n = 113) of PD and was independently associated with PD (ORa = 4.6, p < 0.0001), PFS (HRa = 2.07, p < 0.0001) and OS (HRa = 2.55, p < 0.0001). CONCLUSIONS: CEA kinetics alone or combined with baseline CA19-9 and cfDNA are clinically relevant for predicting outcomes in mCRC. TRIAL REGISTRATION NUMBER: NCT01212510.

Risk of early progression according to circulating ESR1 mutation, CA-15.3 and cfDNA increases under first-line anti-aromatase treatment in metastatic breast cancer.

BACKGROUND: Endocrine therapy is recommended as a first-line treatment for hormone receptor-positive metastatic breast cancer (HR+MBC) patients. No biomarker has been validated to predict tumor progression in that setting. We aimed to prospectively compare the risk of early progression according to circulating ESR1 mutations, CA-15.3, and circulating cell-free DNA in MBC patients treated with a first-line aromatase inhibitor (AI). METHODS: Patients with MBC treated with a first-line AI were prospectively included. Circulating biomarker assessment was performed every 3 months. The primary objective was to determine the risk of progression or death at the next follow-up visit (after 3 months) in case of circulating ESR1 mutation detection among patients treated with a first-line AI for HR+MBC. RESULTS: Overall, 103 patients were included, and 70 (68%) had progressive disease (PD). Circulating ESR1 mutations were detected in 22/70 patients with PD and in 0/33 patients without progression (p < 0.001). Among the ESR1-mutated patients, 18/22 had a detectable mutation prior to progression, with a median delay of 110 days from first detection to PD. The detection of circulating ESR1 mutations was associated with a 4.9-fold (95% CI 3.0-8.0) increase in the risk of PD at 3 months. Using a threshold value of 25% or 100%, a CA-15.3 increase was also correlated with progression (p < 0.001 and p = 0.003, respectively). In contrast to ESR1, the CA-15.3 increase occurred concomitantly with PD in most cases, in 27/47 (57%) with a 25% threshold and in 21/25 (84%) with a 100% threshold. Using a threshold value of either 25% or 100%, cfDNA increase was not correlated with progression. CONCLUSION: The emergence of circulating ESR1 mutations is associated with a 4.9-fold increase in the risk of early PD during AI treatment in HR+MBC. Our results also highlighted that tracking circulating ESR1 mutations is more relevant than tracking CA-15.3 or cfDNA increase to predict progression in this setting. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02473120. Registered 16 June 2015-retrospectively registered after one inclusion (first inclusion 1 June 2015).

Postoperative hunger after outpatient surgery in patients anesthetized with propofol vs sevoflurane: a randomized-controlled trial.

PURPOSE: Previous preclinical and preliminary clinical data suggest an appetite-stimulating effect of propofol compared with halogenated drugs. This study compared the effects of propofol with those of sevoflurane on recovery of hunger during the postoperative period. METHODS: Patients undergoing outpatient transvaginal oocyte retrieval were randomized to propofol-remifentanil (propofol group) or sevoflurane-remifentanil (sevoflurane group) anesthesia. The primary endpoint was the time before feeling hungry (≥ 50/100 mm on a visual analogue scale). Secondary endpoints included plasma levels of ghrelin, leptin, and insulin (ten minutes, one hour, and two hours after anesthesia), caloric intake at first feed, and discharge readiness time. RESULTS: In the 58 patients allocated to either the propofol or sevoflurane group, there was no difference in the median [interquartile range] recovery time of hunger (97 [75-138] vs 97 [80-140] min, respectively; median difference, 1; 95% confidence interval [CI], - 15 to 14; P = 0.91); caloric intake (245 [200-343] vs 260 [171-314] kcal; P = 0.39); or discharge readiness time (125 [85-153] vs 125 [95-174] min, P = 0.29). The groups showed no difference in crude plasma levels of ghrelin, leptin, and insulin at any time-point. When peptide plasma levels were expressed as a % change from baseline, there was a higher insulin plasma level one hour after anesthesia in the sevoflurane group (median difference, 4.9%; 95% CI, - 16.2 to 43.4) compared with the propofol group (median difference, - 21.2%; 95% CI, - 35.7 to 9.1; adjusted P = 0.01). CONCLUSION: Propofol did not accelerate the recovery of hunger compared with sevoflurane after outpatient minor surgery. Moreover, propofol did not have distinguishable effects on other clinical or biological parameters associated with food intake. TRIAL REGISTRATION: www.ClinicalTrials.gov (NCT02272166); registered 22 October, 2014.

RéSUMé: OBJECTIF: Des données précliniques et cliniques préliminaires suggèrent un effet de stimulation de l'appétit du propofol par rapport aux gaz halogénés. Cette étude a comparé les effets du propofol à ceux du sévoflurane sur le rétablissement de la faim en période postopératoire. MéTHODE: Des patientes subissant un prélèvement des ovocytes par voie transvaginale ont été randomisées à recevoir une anesthésie à base de propofol et rémifentanil (groupe propofol) ou de sévoflurane et rémifentanil (groupe sévoflurane). Le critère d'évaluation principal était la période de temps avant de ressentir de la faim (≥ 50/100 mm sur une échelle visuelle analogique). Les critères d'évaluation secondaires comprenaient les taux plasmatiques de ghréline, de leptine et d'insuline (à dix minutes, une heure et deux heures après l'anesthésie), l'apport calorique lors du premier repas, et le moment où les patientes étaient prêtes à recevoir leur congé. RéSULTATS: Chez les 58 patientes allouées aux groupes propofol ou sévoflurane, aucune différence n'a été observée dans le temps médian [écart interquartile (ÉIQ)] jusqu'à retour de la faim (97 [75­138] vs 97 [80­140] min, respectivement; différence médiane, 1; intervalle de confiance [IC] 95 %, − 15 à 14; P = 0,91), ni dans l'apport calorique (245 [200­343] vs 260 [171­314] kcal; P = 0,39) ou le moment où elles étaient prêtes à recevoir leur congé (125 [85-153] vs 125 [95-174] min, P = 0,29). Les groupes n'ont démontré aucune différence en matière de taux plasmatiques de ghréline, de leptine et d'insuline à quelque point dans le temps que ce soit. Lorsque les taux plasmatiques de peptides étaient exprimés en % de changement par rapport aux taux de base, on a observé un taux plasmatique plus élevé d'insuline une heure après l'anesthésie dans le groupe sévoflurane (différence médiane, 4,9 %; IC 95 %, − 16,2 à 43,4) par rapport au groupe propofol (différence médiane, − 21,2 %, IC 95 %, − 35,7 à 9,1; P ajusté = 0,01). CONCLUSION: Le propofol n'a pas accéléré le retour de la faim par rapport au sévoflurane après une chirurgie ambulatoire mineure. De plus, le propofol n'a pas démontré d'effets distinctifs sur d'autres paramètres cliniques ou biologiques associés à l'ingestion de nourriture. ENREGISTREMENT DE L'éTUDE: www.ClinicalTrials.gov (NCT02272166); enregistrée le 22 octobre 2014.

Circulating ESR1 mutations at the end of aromatase inhibitor adjuvant treatment and after relapse in breast cancer patients.

BACKGROUND: Detection of circulating ESR1 mutations is associated with acquired resistance to aromatase inhibitor (AI) in metastatic breast cancer. Until now, the presence of circulating ESR1 mutations at the end of adjuvant treatment by AI in early breast cancer had never been clearly established. In this context, the aim of the present study was to evaluate the circulating ESR1 mutation frequency at the end of adjuvant treatment and after relapse. METHODS: This monocentric retrospective study was based on available stored plasmas and included all early breast cancer patients who completed at least 2 years of AI adjuvant treatment and experienced a documented relapse after the end of their treatment. Circulating ESR1 mutations (D538G, Y537S/N/C) were assessed by droplet digital PCR in plasma samples taken at the end of adjuvant treatment, at time of relapse and at time of progression under first line metastatic treatment. RESULTS: A total of 42 patients were included, with a median adjuvant AI exposure of 60 months (range 41-85). No circulating ESR1 mutation was detectable at the end of AI adjuvant therapy. At first relapse, 5.3% of the patients (2/38) had a detectable circulating ESR1 mutation. At time of progression on first-line metastatic treatment, 33% of the patients (7/21) under AI had a detectable circulating ESR1 mutation compared to none of the patients under chemotherapy (0/10). The two patients with a detectable ESR1 mutation at relapse were treated by AI and had an increase of their variant allele fraction at time of progression on first-line metastatic treatment. CONCLUSIONS: Circulating ESR1 mutation detection at the end of AI-based adjuvant treatment is not clinically useful. Circulating ESR1 mutation could be assessed as soon as first relapse to guide interventional studies.

Diagnostic value of CA19.9, circulating tumour DNA and circulating tumour cells in patients with solid pancreatic tumours.

BACKGROUND: The direct comparison of CA19.9, circulating tumour cells (CTCs) and circulating tumour DNA (ctDNA) using endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) has never been performed for the diagnosis of solid pancreatic tumours (SPTs). METHODS: We included 68 patients with a SPT referred for EUS-FNA. CTCs were analysed using size-based platform and ctDNA using digital PCR. The sensitivity, specificity, negative and positive predictive values were evaluated for each marker and their combination. RESULTS: SPTs corresponded to 58 malignant tumours (52 pancreatic adenocarcinoma (PA) and 6 others) and 10 benign lesions. The sensitivity and specificity for PA diagnosis were 73% and 88% for EUS-FNA, 67% and 80% for CTC, 65% and 75% for ctDNA and 79% and 93% for CA19.9, respectively. The positivity of at least 2 markers was associated with a sensitivity and specificity of 78% and 91%, respectively. CtDNA was the only marker associated with overall survival (median 5.2 months for ctDNA+ vs 11.0 months for ctDNA-, P=0.01). CONCLUSIONS: CA19.9 alone and in combination with ctDNA and/or CTC analysis may represent an efficient method for diagnosing PA in patients with SPTs. Further studies including a larger cohort of patients with both malignant and benign lesions will be necessary to confirm these promising results.

Influence of adjuvant chemotherapy on anti-Müllerian hormone in women below 35 years treated for early breast cancer.

The impact of chemotherapy on fertility appears to be of essential importance for the youngest cancer survivors. The aim of this study was to assess plasma anti-Mullërian hormone (AMH) evolution, using an automated sensitive AMH immunoassay in women younger than 35 years old before and after treatment with adjuvant chemotherapy for early breast cancer. We selected 54 women aged less than 35 years old, at the time of breast cancer diagnosis, who received chemotherapy between 2008 and 2014, and with plasma samples collected from the diagnosis, to 1 year, 3 years and 5 years post-diagnosis. The median AMH decreased markedly in the year after the diagnosis compared with the pretreatment values (P < 0.0001), and slightly increased 2 years later (P = 0.007, comparing 1-year and 3-years post-diagnosis concentrations), without any additional AMH recovery 5 years after diagnosis. This recovery did not reach age-dependent AMH expected values (P < 0.0001, comparing AMH measured values to AMH expected values). Addition of taxanes to an anthracyclines + alkylating-based regimen was associated with a worse AMH decrease (P = 0.007). Ovarian tissue cryopreservation before treatment did not influence the AMH recovery. These results highlight the necessity of fertility counselling before treatment, especially in women wanting children.

Short report: Monitoring ESR1 mutations by circulating tumor DNA in aromatase inhibitor resistant metastatic breast cancer.

Acquired estrogen receptor gene (ESR1) mutations have been recently reported as a marker of resistance to aromatase inhibitors in hormone receptor positive metastatic breast cancer. We retrospectively considered seven patients treated for metastatic breast cancer with available samples from the primary tumor before any treatment, cryopreserved metastasis removed during progression and concomitant plasmas. All these seven patients were in disease progression after previous exposure to aromatase inhibitors for at least 6 months, and were assessed for ESR1 mutations detection in tumor and circulating DNA. For these patients, Sanger sequencing identified four metastases with clear ESR1 mutation and one possible, whereas digital PCR identified six mutated metastases. Then, under blind conditions and using digital PCR, corresponding circulating ESR1 mutations were successfully detected in four of these six metastatic breast cancer patients. Moreover, in two patients with serial blood samples following treatments exposure, the monitoring of circulating ESR1 mutations clearly predicted disease evolution. In the context of high interest for ESR1 mutations, our results highlight that these acquired recurrent mutations may be tracked in circulating tumor DNA and may be of clinical relevance for metastatic breast cancer patient monitoring.

Cardiovascular outcomes of patients treated for non-Hodgkin lymphoma with first-line doxorubicin-based chemotherapy.

We conducted a single-center retrospective study to assess cardiovascular (CV) toxicity and treatment discontinuation for CV toxicity in diffuse large B-cell lymphoma (DLBCL) or follicular lymphoma (FL) patients treated with immunochemotherapy (R-CHOP-like). Between 2006 and 2017, 433 patients were included (DLBCL: n = 345, FL: n = 88). The median age was 63 years (50-73). We defined three types of CV toxicity: early-onset cardiovascular toxicity (the event occurred within 6 months following treatment start); subacute toxicity (the event occurred between 6 months and 1 year after treatment start) and late toxicity (the event occurred 1 year or more after treatment start). Forty-eight (11.1%) patients experienced at least one anthracycline-related CV event. Seven patients experienced treatment discontinuation due to CV toxicity. Early-onset and subacute cardiac events were primarily acute heart failure (34.3%) and atrial fibrillation (28.6%). History of ischemic heart disease (p = 0.02) and valvular heart disease (p = 0.03) were associated with a higher risk of anthracycline-related CV event occurrence.

Circulating PIK3CA mutation detection at diagnosis in non-metastatic inflammatory breast cancer patients.

Inflammatory breast cancer (IBC) is an aggressive BC subtype with poor outcomes. A targetable somatic PIK3CA mutation is reported in 30% of IBC, allowing for treatment by PI3Kα-specific inhibitors, such as alpelisib. The aim of this study was to evaluate the detection rate of circulating PIK3CA mutation in locally-advanced IBC (LAIBC) patients harbouring a PIK3CA mutation on initial biopsy. This monocentric retrospective study was based on available stored plasma samples and tumour biopsies at diagnosis from all LAIBC patients treated with neo-adjuvant chemotherapy (NCT) between 2008 and 2018 at the Centre Henri Becquerel. PIK3CA mutations (E542K, E545K, H1047R/L) were assessed by droplet digital PCR (ddPCR) in plasma samples and tumoral tissue at diagnosis. A total of 55 patients were included. Overall, 14/55 patients (25%) had a PIK3CA mutation identified on baseline biopsy (H1047R = 8; H1047L = 3; E545K = 2; E542K = 1). Among them, 11 (79%) patients had enough DNA for circulating DNA analyses, and corresponding circulating PIK3CA mutations were found in 6/11 (55%). Among the 41 patients without PIK3CA mutations on biopsy, 32 (78%) had enough DNA for circulating DNA analysis, and no circulating PIK3CA mutation was identified. Our results revealed no prognostic or predictive value of PIK3CA mutations at the diagnosis of non-metastatic IBC but highlighted the prognostic value of the cfDNA rate at diagnosis. Our study showed that a corresponding circulating PIK3CA mutation was identified in 55% of LAIBC patients with PIK3CA-mutated tumours, while no circulating mutation was found among patients with PI3KCA wild-type tumours.

Impact of Taxanes, Endocrine Therapy, and Deleterious Germline BRCA Mutations on Anti-müllerian Hormone Levels in Early Breast Cancer Patients Treated With Anthracycline- and Cyclophosphamide-Based Chemotherapy.

Background: Limited evidence exists on the impact of adding a taxane, using endocrine therapy and carrying a deleterious germline BRCA mutation on ovarian reserve measured by anti-müllerian hormone (AMH) levels of young breast cancer patients receiving (neo)adjuvant cyclophosphamide- and anthracycline-based chemotherapy. Methods: This is a biomarker analysis including young (≤ 40 years) early breast cancer patients with known germline BRCA mutational status and available prospectively collected frozen plasma samples before and after chemotherapy. Chemotherapy consisted of either six cycles of FEC (5 fluorouracil 500 mg/m2, epirubicin 100 mg/m2, cyclophosphamide 500 mg/m2) or three cycles of FEC followed by three cycles of docetaxel (D, 100 mg/m2). Endocrine therapy consisted of tamoxifen (±GnRH agonists). AMH levels at baseline, 1 and 3 years after diagnosis were compared according to type of chemotherapy (FEC only vs. FEC-D), use of endocrine therapy (yes vs. no) and deleterious germline BRCA mutations (mutated vs. negative). Results: Out of 148 included patients, 127 (86%) received D following FEC chemotherapy, 90 (61%) underwent endocrine therapy, and 35 (24%) had deleterious germline BRCA mutations. In the whole cohort, AMH levels drastically dropped 1 year after diagnosis (p < 0.0001) with a slight but significant recovery at 3 years (p < 0.0001). One year after diagnosis, patients treated with FEC only had higher median AMH levels than those who received FEC-D (0.22 vs. 0.04 µg/L, p = 0.0006); no difference was observed at 3 years (0.06 and 0.18 µg/L, p = 0.47). Patients under endocrine therapy had significantly higher AMH levels than those who did not receive this treatment 1 year after diagnosis (0.12 vs. 0.02 µg/L; p = 0.008), with no difference at 3 years (0.11 and 0.20 µg/L, p = 0.22). AMH levels were similar between BRCA-mutated and BRCA-negative patients at baseline (1.94 vs. 1.66 µg/L, p = 0.53), 1 year (0.09 vs. 0.06 µg/L, p = 0.39) and 3 years (0.25 vs. 0.16 µg/L; p = 0.43) after diagnosis. Conclusions: In breast cancer patients receiving FEC chemotherapy, adding D appeared to negatively impact on their ovarian reserve in the short-term; no further detrimental effect was observed for endocrine therapy use and presence of a deleterious germline BRCA mutation.

[Clinical relevance of ESR1 circulating mutations detection in hormone receptor positive metastatic breast cancer]. / Intérêt clinique de la détection des mutations circulantes d'ESR1 chez les patientes traitées pour un cancer du sein métastatique exprimant les récepteurs hormonaux.

If hormone therapy is a key treatment for hormone receptor positive advanced breast cancers, secondary resistance occurs as a rule. Recently, acquired alterations of the ESR1 gene have been identified as a mechanism of resistance on aromatase inhibitor (AI) treatment. The selective pressure by AI exposure during the metastatic setting triggers the emergence of ESR1 activating mutations. In that context, the "liquid biopsy" concept has been used to detect this molecular resistance before progression. Thus, the ESR1 circulating mutation detection will soon be used in daily practice to help monitoring patients on AI treatment and provide an early change for specific therapies that still have to be determined in prospective clinical trials. This review will present the acquired ESR1 mutations, as well as the methods used for their detection in blood and the potential clinical impact of this approach for hormone receptor positive breast cancer management.

New Drug Combination Strategies in Melanoma: Current Status and Future Directions.

Melanoma is the deadliest form of skin cancer and one of the most difficult cancers to treat. Overall, melanomas have more mutations than any other cancer type. Oncogenic mutations in c-KIT, NRAS and BRAF components of the MAPK pathway have been identified in nearly 90% of cutaneous melanoma and this information has been used to develop small molecules that inhibit their activity. Highly selective BRAF and MEK inhibitors have demonstrated impressive clinical results. However, the short duration of response, the acquired resistance in most cases and the toxicity issues support the rationale for drug combination approaches to improve the outcome of MAPK inhibitors, increase their efficacy, prevent and/or overcome resistance. This review discusses several promising rational combinatorial strategies investigated or could be investigated in clinical studies.

PRIMA-1 and PRIMA-1Met (APR-246): From Mutant/Wild Type p53 Reactivation to Unexpected Mechanisms Underlying Their Potent Anti-Tumor Effect in Combinatorial Therapies.

p53 protects cells from genetic assaults by triggering cell-cycle arrest and apoptosis. Inactivation of p53 pathway is found in the vast majority of human cancers often due to somatic missense mutations in TP53 or to an excessive degradation of the protein. Accordingly, reactivation of p53 appears as a quite promising pharmacological approach and, effectively, several attempts have been made in that sense. The most widely investigated compounds for this purpose are PRIMA-1 (p53 reactivation and induction of massive apoptosis )and PRIMA-1Met (APR-246), that are at an advanced stage of development, with several clinical trials in progress. Based on publications referenced in PubMed since 2002, here we review the reported effects of these compounds on cancer cells, with a specific focus on their ability of p53 reactivation, an overview of their unexpected anti-cancer effects, and a presentation of the investigated drug combinations.

Direct circulating tumor DNA detection from unpurified plasma using a digital PCR platform.

BACKGROUND: In standard pre-analytical conditions, an isolation step is required for circulating tumor DNA (ctDNA) analysis. The need for this step remains unclear with the development of ultrasensitive detection technologies such as digital PCR (dPCR). The aim of our study was to evaluate the ctDNA detection by dPCR platform either directly from plasma (plasma group, PG) or after an isolation step (isolation group, IG). METHODS: We included 17 patients corresponding to a selection of 43 blood samples in metastatic colorectal cancer patients. For each sample, ctDNA was analyzed with or without isolation step (IG and PG, respectively) using KRAS, NRAS and BRAF mutations identified from the tumor tissue. ctDNA detection was performed after a preamplication step using dPCR platform (QuantStudio™ 3D Digital PCR System). ctDNA detection rate and mutant allelic frequencies (MAF) were compared between IG and PG. RESULTS: Our results showed a detection rate at 93% in IG vs. 88% in PG. The concordance rate between the two groups was 91% (39/43) for ctDNA detection with the four discordant cases occurring in patients with low MAF (<0.5%). The mean value of MAF were 16.9±18.9 and 18.5±18.9 for IG and PG, respectively (p=0.24). The correlation coefficient r2 for MAF was 0.82 between the two methods (p<0.0001). CONCLUSION: In conclusion, our results show that direct detection of ctDNA from unpurified plasma is a feasible approach, particularly from sample with high MAF (>0.5%).

Heparinase enables reliable quantification of circulating tumor DNA from heparinized plasma samples by droplet digital PCR.

BACKGROUND: Heparin is often used as a blood anticoagulant for tumor marker analysis but results in the inhibition of PCR detection of circulating tumor DNA (ctDNA), which has been deemed a potential "liquid biopsy". We aimed to evaluate the impact of heparinase addition on heparinized plasma samples to allow ctDNA analysis. METHODS: Plasma samples were collected in heparinized (n=194) and EDTA (n=8) tubes from hormone receptor-positive metastatic breast cancer (HR+MBC) (n=144) and pancreatic adenocarcinoma (PA) patients (n=50). Circulating ESR1 and KRAS mutations were detected with or without heparinase by digital PCR in HR+MBC and PA patients, respectively. Patients were classified into 2 subgroups i) inhibition, I+ and ii) no inhibition, I- based on a threshold of 200copies/µL for PCR inhibition by heparin. RESULTS: In the I+ subgroup (91/144 HR+MBC and 26/50 PA), heparinase treatment significantly improved PCR efficacy, enabling ctDNA detection in 22/91 and 13/26 patients. Moreover, comparable results for ctDNA detection (4/8) were obtained with heparinized and EDTA PA samples. In the I- subgroup, heparinase addition did not quantitatively and qualitatively alter ctDNA detection. CONCLUSION: Heparinase addition removes the heparin inhibition and allows accurate ctDNA detection in heparinized samples. These findings could make the samples from heparinized blood suitable for ctDNA analysis.

Kinetics, prognostic and predictive values of ESR1 circulating mutations in metastatic breast cancer patients progressing on aromatase inhibitor.

PURPOSE: To assess the prognostic and predictive value of circulating ESR1 mutation and its kinetics before and after progression on aromatase inhibitor (AI) treatment. PATIENTS AND METHODS: ESR1 circulating D538G and Y537S/N/C mutations were retrospectively analyzed by digital droplet PCR after first-line AI failure in patients treated consecutively from 2010 to 2012 for hormone receptor-positive metastatic breast cancer. Progression-free survival (PFS) and overall survival (OS) were analyzed according to circulating mutational status and subsequent lines of treatment. The kinetics of ESR1 mutation before (3 and 6 months) and after (3 months) AI progression were determined in the available archive plasmas. RESULTS: Circulating ESR1 mutations were found at AI progression in 44/144 patients included (30.6%). Median follow-up from AI initiation was 40 months (range 4-94). The median OS was decreased in patients with circulating ESR1 mutation than in patients without mutation (15.5 versus 23.8 months, P=0.0006). The median PFS was also significantly decreased in patients with ESR1 mutation than in patients without mutation (5.9 vs 7 months, P=0.002). After AI failure, there was no difference in outcome for patients receiving chemotherapy (n = 58) versus non-AI endocrine therapy (n=51) in patients with and without ESR1 mutation. ESR1 circulating mutations were detectable in 75% of all cases before AI progression, whereas the kinetics 3 months after progression did not correlate with outcome. CONCLUSION: ESR1 circulating mutations are independent risk factors for poor outcome after AI failure, and are frequently detectable before clinical progression. Interventional studies based on ESR1 circulating status are warranted.

Motile sperm organelle morphology examination (MSOME) and sperm head vacuoles: state of the art in 2013.

BACKGROUND Approximately 10 years after the first publication introducing the motile sperm organelle morphology examination (MSOME), many questions remained about sperm vacuoles: frequency, size, localization, mode of occurrence, biological significance and impact on male fertility potential. Many studies have tried to characterize sperm vacuoles, to determine the sperm abnormalities possibly associated with vacuoles, to test the diagnostic value of MSOME for male infertility or to question the benefits of intracytoplasmic morphologically selected sperm injection (IMSI). METHODS We searched PubMed for articles in the English language published in 2001-2012 regarding human sperm head vacuoles, MSOME and IMSI. RESULTS A bibliographic analysis revealed consensus for the following findings: (i) sperm vacuoles appeared frequently, often multiple and preferentially anterior; (ii) sperm vacuoles and sperm chromatin immaturity have been associated, particularly in the case of large vacuoles; (iii) teratozoospermia was a preferred indication of MSOME and IMSI. CONCLUSION The high-magnification system appears to be a powerful method to improve our understanding of human spermatozoa. However, its clinical use remains unclear in the fields of male infertility diagnosis and assisted reproduction techniques (ARTs).

Effects of vitamin A on in vitro maturation of pre-pubertal mouse spermatogonial stem cells.

Testicular tissue cryopreservation is the only potential option for fertility preservation in pre-pubertal boys exposed to gonadotoxic treatment. Completion of spermatogenesis after in vitro maturation is one of the future uses of harvested testicular tissue. The purpose of the current study was to evaluate the effects of vitamin A on in vitro maturation of fresh and frozen-thawed mouse pre-pubertal spermatogonial stem cells in an organ culture system. Pre-pubertal CD1 mouse fresh testes were cultured for 7 (D7), 9 (D9) and 11 (D11) days using an organ culture system. Basal medium was supplemented with different concentrations of retinol (Re) or retinoic acid (RA) alone or in combination. Seminiferous tubule morphology (tubule diameter, intra-tubular cell type), intra-tubular cell death and proliferation (PCNA antibody) and testosterone level were assessed at D7, D9 and D11. Pre-pubertal mouse testicular tissue were frozen after a soaking temperature performed at -7 °C, -8 °C or -9 °C and after thawing, were cultured for 9 days, using the culture medium preserving the best fresh tissue functionality. Retinoic acid at 10(-6)M and retinol at 3.3.10(-7)M, as well as retinol 10(-6)M are favourable for seminiferous tubule growth, maintenance of intra-tubular cell proliferation and germ cell differentiation of fresh pre-pubertal mouse spermatogonia. Structural and functional integrity of frozen-thawed testicular tissue appeared to be well-preserved after soaking temperature at -8 °C, after 9 days of organotypic culture using 10(-6)M retinol. RA and Re can control in vitro germ cell proliferation and differentiation. Re at a concentration of 10(-6)M maintains intra-tubular cell proliferation and the ability of spermatogonia to initiate spermatogenesis in fresh and frozen pre-pubertal mouse testicular tissue using a soaking temperature at -8 °C. Our data suggested a possible human application for in vitro maturation of cryopreserved pre-pubertal testicular tissue.