Dewi Vernerey

MicroRNAs as non-invasive biomarkers of heart transplant rejection

AIMnRejection is one of the major causes of late cardiac allograft failure and at present can only be diagnosed by invasive endomyocardial biopsies. We sought to determine whether microRNA profiling could serve as a non-invasive biomarker of cardiac allograft rejection.nnnMETHODSnWe included 113 heart transplant recipients from four referral French institutions (test cohort, n = 60, validation cohort, n = 53). In the test cohort, we compared patients with acute biopsy-proven allograft rejection (n = 30) to matched control patients without rejection (n = 30), by assessing microRNAs expression in the heart allograft tissue and patients concomitant serum using RNA extraction and qPCR analysis. Fourteen miRNAs were selected on the basis of their implication in allograft rejection, endothelial activation, and inflammation and tissue specificity.nnnRESULTSnWe identified seven miRNAs that were differentially expressed between normal and rejecting heart allografts: miR-10a, miR-21, miR-31, miR-92a, miR-142-3p miR-155, and miR-451 (P < 0.0001 for all comparisons). Four out of seven miRNAs also showed differential serological expression (miR-10a, miR-31, miR-92a, and miR-155) with strong correlation with their tissular expression. The receiver-operating characteristic analysis showed that these four circulating miRNAs strongly discriminated patients with allograft rejection from patients without rejection: miR-10a (AUC = 0.975), miR-31 (AUC = 0.932), miR-92a (AUC = 0.989), and miR-155 (AUC = 0.998, P < 0.0001 for all comparisons). We confirmed in the external validation set that these four miRNAs highly discriminated patients with rejection from those without. The discrimination capability of the four miRNAs remained significant when stratified by rejection diagnosis (T-cell-mediated rejection or antibody-mediated rejection) and time post-transplant.nnnCONCLUSIONnThis study demonstrates that a differential expression of miRNA occurs in rejecting allograft patients, not only at the tissue level but also in the serum, suggesting their potential relevance as non-invasive biomarkers in heart transplant rejection.

Molecular Microscope Strategy to Improve Risk Stratification in Early Antibody-Mediated Kidney Allograft Rejection

Antibody-mediated rejection (ABMR) is the leading cause of kidney allograft loss. We investigated whether the addition of gene expression measurements to conventional methods could serve as a molecular microscope to identify kidneys with ABMR that are at high risk for failure. We studied 939 consecutive kidney recipients at Necker Hospital (2004-2010; principal cohort) and 321 kidney recipients at Saint Louis Hospital (2006-2010; validation cohort) and assessed patients with ABMR in the first 1 year post-transplant. In addition to conventional features, we assessed microarray-based gene expression in transplant biopsy specimens using relevant molecular measurements: the ABMR Molecular Score and endothelial donor-specific antibody-selective transcript set. The main outcomes were kidney transplant loss and progression to chronic transplant injury. We identified 74 patients with ABMR in the principal cohort and 54 patients with ABMR in the validation cohort. Conventional features independently associated with failure were donor age and humoral histologic score (g+ptc+v+cg+C4d). Adjusting for conventional features, ABMR Molecular Score (hazard ratio [HR], 2.22; 95% confidence interval [95% CI], 1.37 to 3.58; P=0.001) and endothelial donor-specific antibody-selective transcripts (HR, 3.02; 95% CI, 1.00 to 9.16; P<0.05) independently associated with an increased risk of graft loss. The results were replicated in the independent validation group. Adding a gene expression assessment to a traditional risk model improved the stratification of patients at risk for graft failure (continuous net reclassification improvement, 1.01; 95% CI, 0.57 to 1.46; P<0.001; integrated discrimination improvement, 0.16; P<0.001). Compared with conventional assessment, the addition of gene expression measurement in kidney transplants with ABMR improves stratification of patients at high risk for graft loss.

Pathologic classification of antibody-mediated rejection correlates with donor-specific antibodies and endothelial cell activation

BACKGROUNDnHumoral immune responses during heart transplantation may result in antibody-mediated rejection (AMR), which is now taken into account on endomyocardial biopsy (EMB) specimens and ranked according to the pathologic AMR (pAMR) grades of the International Society for Heart and Lung Transplantation classification. This classification might benefit from new immunohistological markers and validation by others biomarkers, namely donor-specific antibodies (DSA).nnnMETHODSnFrom the 293 protocol EMBs performed in 113 patients in our institution during a 1-year period for this prospective study, 280 EMB specimens were available with both histology and immunohistochemistry. C4d and labeling of intravascular cells by cluster of differentiation (CD) 68 were performed on paraffin sections. Available sera (n = 150) concomitant of EMB specimens were tested for the presence of DSA. All of the pAMR+ EMB specimens, along with a set of randomized pAMR0 EMB specimens, were immunolabeled for mammalian target of rapamycin (mTOR) effectors, phosphorylated 70 S6-kinase (p70S6K) and phosphorylated S6 ribosomal protein (pS6RP).nnnRESULTSnAMR was diagnosed in 37 EMB specimens (13.2%): 1 pAMR1(I+), 27 pAMR1(H+), and 9 pAMR2. The proportion of DSA-positive EMB varied according to the pAMR grade, with pAMR0, pAMR1(H+), and pAMR2 EMB presenting 17.6%, 77.3%, and 100% of DSA-positivity, respectively. Among the 30 pAMR+ specimens with available DSA testing and the 30 pAMR0 randomized specimens, mTOR pathway immunohistochemistry showed endothelial cell positivity for p70S6K in 17 pAMR+ EMB specimens (56.7%) and in 1 pAMR0 EMB specimen (3.3%). pS6RP was detected in 8 pAMR+ EMB specimens (26.7%) and in 1 pAMR0 EMB specimen (3.3%).nnnCONCLUSIONSnp70S6K and pS6RP immunohistochemistry afford new markers of AMR on EMB specimens because their expression is correlated with microcirculation inflammation and DSA. The correlation of DSA with pAMR grade suggests that this grading system is valid.

Genetic polymorphisms of matrix metalloproteinase 12 and 13 genes are implicated in endometriosis progression.

BACKGROUNDnMatrix metalloproteinases (MMPs) may contribute to endometriosis. We tested whether eight functional polymorphisms of these genes could modify the risk of endometriosis.nnnMETHODSnIn this case-control study, 227 endometriosis and 241 controls were genotyped for MMP1 -1607 1G/2G, MMP2 -1575 G/A (MMP2.1), -1306 C/T (MMP2.2), MMP3 -1612 5A/6A, MMP7 -153 C/T (MMP7.1), -181 A/G (MMP7.2), MMP12 -82 A/G and MMP13-77 A/G. Association between MMP genotypes and superficial (SUP), deep infiltrating (DIE) and endometriomas (OMA) was tested for each polymorphism separately, using unconditional logistic regression and then for combined genotypes, using the combination test.nnnRESULTSnWhen considering all cases, MMP2 polymorphisms were found to be significant, mainly due to DIE (P = 0.023). A small difference between SUP and controls was found for MMP7.2 (P = 0.032) and MMP12 (P = 0.035), in the absence of correction for multiple testing. Using the combination test, the best association when comparing SUP with controls was obtained for MMP12-MMP13 (P = 0.004) for the combined genotype A/G-A/A (odds ratio = 27.60, 95% confidence interval: 2.80-272.40).nnnCONCLUSIONSnThese data show a potential role for MMP12 -82 A/G and MMP13 -77 A/G combined polymorphisms in superficial endometriosis. As no association was found with deep infiltrating endometriosis, this combination of polymorphisms might protect from a more in-depth penetration of tissues.

Duration of Adjuvant Chemotherapy for Stage III Colon Cancer

BACKGROUND Since 2004, a regimen of 6 months of treatment with oxaliplatin plus a fluoropyrimidine has been standard adjuvant therapy in patients with stage III colon cancer. However, since oxaliplatin is associated with cumulative neurotoxicity, a shorter duration of therapy could spare toxic effects and health expenditures. METHODS We performed a prospective, preplanned, pooled analysis of six randomized, phase 3 trials that were conducted concurrently to evaluate the noninferiority of adjuvant therapy with either FOLFOX (fluorouracil, leucovorin, and oxaliplatin) or CAPOX (capecitabine and oxaliplatin) administered for 3 months, as compared with 6 months. The primary end point was the rate of disease‐free survival at 3 years. Noninferiority of 3 months versus 6 months of therapy could be claimed if the upper limit of the two‐sided 95% confidence interval of the hazard ratio did not exceed 1.12. RESULTS After 3263 events of disease recurrence or death had been reported in 12,834 patients, the noninferiority of 3 months of treatment versus 6 months was not confirmed in the overall study population (hazard ratio, 1.07; 95% confidence interval [CI], 1.00 to 1.15). Noninferiority of the shorter regimen was seen for CAPOX (hazard ratio, 0.95; 95% CI, 0.85 to 1.06) but not for FOLFOX (hazard ratio, 1.16; 95% CI, 1.06 to 1.26). In an exploratory analysis of the combined regimens, among the patients with T1, T2, or T3 and N1 cancers, 3 months of therapy was noninferior to 6 months, with a 3‐year rate of disease‐free survival of 83.1% and 83.3%, respectively (hazard ratio, 1.01; 95% CI, 0.90 to 1.12). Among patients with cancers that were classified as T4, N2, or both, the disease‐free survival rate for a 6‐month duration of therapy was superior to that for a 3‐month duration (64.4% vs. 62.7%) for the combined treatments (hazard ratio, 1.12; 95% CI, 1.03 to 1.23; P = 0.01 for superiority). CONCLUSIONS Among patients with stage III colon cancer receiving adjuvant therapy with FOLFOX or CAPOX, noninferiority of 3 months of therapy, as compared with 6 months, was not confirmed in the overall population. However, in patients treated with CAPOX, 3 months of therapy was as effective as 6 months, particularly in the lower‐risk subgroup. (Funded by the National Cancer Institute and others.)

Docetaxel, cisplatin, and fluorouracil chemotherapy for metastatic or unresectable locally recurrent anal squamous cell carcinoma (Epitopes-HPV02): a multicentre, single-arm, phase 2 study

BACKGROUNDnThe incidence of anal squamous cell carcinoma has been increasing markedly in the past few decades. Currently, there is no validated treatment for advanced-stage anal squamous cell carcinoma. Therefore, we aimed to validate the clinical activity and safety of docetaxel, cisplatin, and fluorouracil (DCF) chemotherapy in patients with metastatic or unresectable locally recurrent anal squamous cell carcinoma.nnnMETHODSnWe did a multicentre, single-arm, phase 2 study. We recruited patients from 25 academic hospitals, cancer research centres, and community hospitals in France who were aged 18 years or older with histologically confirmed anal squamous cell carcinoma, with metastatic disease or with unresectable local recurrence; an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1; and with at least one evaluable lesion according to the Response Evaluation Criteria in Solid Tumors (version 1.1). Chemotherapy-naive patients received either six cycles of standard DCF (75 mg/m2 docetaxel and 75 mg/m2 cisplatin on day 1 and 750 mg/m2 per day of fluorouracil for 5 days, every 3 weeks) or eight cycles of modified DCF (40 mg/m2 docetaxel and 40 mg/m2 cisplatin on day 1 and 1200 mg/m2 per day of fluorouracil for 2 days, every 2 weeks), which were administered intravenously. The choice between the standard versus modified regimens was recommended based on, but not limited to, age (≤75 years vs >75 years) and ECOG performance status (0 vs 1). The primary endpoint was investigator-assessed progression-free survival at 12 months from the first DCF cycle; for the primary endpoint to be met, at least 11 (17%) of 66 enrolled patients had to be alive without disease progression at 12 months. Efficacy and safety analyses were done in a modified intention-to-treat population, defined as all patients who were evaluable for progression at 12 months who received at least one cycle of DCF. This trial is registered at ClinicalTrials.gov, number NCT02402842, and the final results are presented here.nnnFINDINGSnBetween Sept 17, 2014, and Dec 7, 2016, we enrolled 69 patients. Of these patients, three did not receive DCF. Of the 66 patients who received treatment, 36 received the standard DCF regimen and 30 received modified DCF. The primary endpoint was met: 31 (47%) of 66 patients were alive and progression free at 12 months. 22 (61%) of 36 patients who received the standard DCF regimen and 18 (60%) of 30 patients who received the modified DCF regimen had disease progression at data cutoff. 46 (70%) of 66 patients had at least one grade 3-4 adverse event (30 [83%] of 36 in the standard DCF regimen and 16 [53%] of 30 in the modified DCF regimen). The most common grade 3-4 adverse events were neutropenia (15 [23%]; eight [22%] for standard DCF vs seven [23%] for modified DCF), diarrhoea (12 [18%]; nine [25%] vs three [10%]), asthenia (ten [15%]; eight [22%] vs two [7%]), anaemia (ten [15%]; six [17%] vs four [13%]), lymphopenia (eight [12%]; three [8%] vs five [17%]), mucositis (seven [11%]; seven [19%] vs none), and vomiting (seven [11%]; five [14%] vs two [7%]). No grade 4 non-haematological adverse events and febrile neutropenia were observed with modified DCF, whereas three (8%) grade 4 non-haematological adverse events and five (14%) cases of febrile neutropenia were reported with standard DCF. 97 serious adverse events were reported (69 in patients who received the standard DCF regimen [61 drug-related] and 28 in those given the modified DCF regimen [14 drug-related]). No treatment-related deaths were recorded.nnnINTERPRETATIONnCompared with standard DCF, modified DCF provided long-lasting response with good tolerability in patients with metastatic or unresectable locally recurrent anal squamous cell carcinoma with ECOG performance status of 0-1 in the first-line setting, and therefore could be considered as a new standard of care for these patients. Regarding the elevated risk of high-grade and serious adverse events and febrile neutropenia, standard DCF cannot be recommended in this situation.nnnFUNDINGnBesançon University Hospital and Ligue contre le cancer Grand-Est.

Clinical and molecular characterisation of hereditary and sporadic metastatic colorectal cancers harbouring microsatellite instability/DNA mismatch repair deficiency

BACKGROUNDnPatients treated with chemotherapy for microsatellite unstable (MSI) and/or mismatch repair deficient (dMMR) cancer metastatic colorectal cancer (mCRC) exhibit poor prognosis. We aimed to evaluate the relevance of distinguishing sporadic from Lynch syndrome (LS)-like mCRCs.nnnPATIENTS AND METHODSnMSI/dMMR mCRC patients were retrospectively identified in six French hospitals. Tumour samples were screened for MSI, dMMR, RAS/RAF mutations and MLH1 methylation. Sporadic cases were molecularly defined as those displaying MLH1/PMS2 loss of expression with BRAFV600E and/or MLH1 hypermethylation and no MMR germline mutation.nnnRESULTSnAmong 129 MSI/dMMR mCRC patients, 81 (63%) were LS-like and 48 (37%) had sporadic tumours; 22% of MLH1/PMS2-negative mCRCs would have been misclassified using an algorithm based on local medical records (age, Amsterdam II criteria, BRAF and MMR statuses when locally tested), compared to a systematical assessment of MMR, BRAF and MLH1 methylation statuses. In univariate analysis, parameters associated with better overall survival were age (Pxa0<xa00.0001), metastatic resection (Pxa0=xa00.001) and LS-like mCRC (Pxa0=xa00.01), but not BRAFV600E. In multivariate analysis, age (hazard ratio (HR)xa0=xa03.19, Pxa0=xa00.01) and metastatic resection (HRxa0=xa04.2, Pxa0=xa00.001) were associated with overall survival, but not LS. LS-like patients were associated with more frequent liver involvement, metastatic resection and better disease-free survival after metastasectomy (HRxa0=xa00.28, Pxa0=xa00.01). Median progression-free survival of first-line chemotherapy was similar between the two groups (4.2 and 4.2 months; Pxa0=xa00.44).nnnCONCLUSIONSnLS-like and sporadic MSI/dMMR mCRCs display distinct natural histories. MMR, BRAF mutation and MLH1 methylation testing should be mandatory to differentiate LS-like and sporadic MSI/dMMR mCRC, to determine in particular whether immune checkpoint inhibitors efficacy differs in these two populations.

A phase 2 randomized trial to evaluate the impact of a supervised exercise program on cardiotoxicity at 3 months in patients with HER2 overexpressing breast cancer undergoing adjuvant treatment by trastuzumab: design of the CARDAPAC study

BackgroundThe overexpression of human epidermal growth factor receptor-2 (HER2) in breast cancer is a poor prognosis. Trastuzumab improves overall survival but is associated with cardiotoxicity, especially a decline in left ventricular ejection fraction (LVEF). In addition, chemotherapy and radiotherapy increase fatigue and pain, decrease physical capacity and health-related quality of life. To date, no study has evaluated the benefits of physical activity on the side effects of treatment in patients with HER2 positive breast cancer. The aim of this study is to evaluate the impact of 3 months’ exercise intervention on myocardial function and in particular on the rate of cardiotoxicity.MethodsThis multicenter, randomized clinical trial will include 112 patients treated by adjuvant trastuzumab for HER2 positive breast cancer to investigate the effects of a 3 months’ supervised exercise program (intermittent exercise, combining moderate and high intensities; 55 minutes duration, 3 times per week), on the rate of cardiotoxicity [defined by either a decrease of the LVEF under 50% or an absolute drop of LVEF of 10%] between baseline and at 3 months and on strength, aerobic capacity, metabolic, inflammatory and hormonal parameters. Health-related quality of life, fatigue, pain and level of physical activity will also be assessed. Participants are randomly allocated to one of the two groups (“training group” vs “standard oncological care”). Performance-based and self-reported outcomes are assessed at baseline, at the end of supervised exercise program and at six months follow-up.DiscussionAlthough physical exercise is recommended to reduce the side effects of adjuvant treatments in breast cancer patients, no randomized study has been conducted to assess the benefits of a physical training program in patients with HER2 overexpressing breast cancer. Cardiac toxicity of trastuzumab may be minimized with an exercise program combining high and moderate intensities. This type of program may be safe, feasible and effective but also increase cardiorespiratory fitness and improve health-related quality of life. If these benefits are confirmed, this exercise intervention could be systematically proposed to patients during the course of treatment by trastuzumab in addition to standard oncological care.Trial registrationNational Clinical Trials Number (NCT02433067); Registration 28 april 2015.

Prognostic value of baseline seric Syndecan-1 in initially unresectable metastatic colorectal cancer patients: a simple biological score.

In first‐line metastatic colorectal cancer (mCRC), baseline prognostic factors allowing death risk and treatment strategy stratification are lacking. Syndecan‐1 (CD138) soluble form was never described as a prognostic biomarker in mCRC. We investigated its additional prognostic value for overall survival (OS). mCRC patients with unresectable disease at diagnosis were treated with bevacizumab‐based chemotherapy in two independent prospective clinical trials (development set: nu2009=u2009126, validation set: nu2009=u200951, study NCT00489697 and study NCT00544011, respectively). Serums were collected at baseline for CD138 measurement. OS determinants were assessed and, based on the final multivariate model, a prognostic score was proposed. Two independent OS prognostic factors were identified: Lactate Dehydrogenase (LDH) high level (pu2009=u20090.0066) and log‐CD138 high level (pu2009=u20090.0190). The determination of CD138 binary information (cutoff: 75 ng/mL) allowed the assessment of a biological prognostic score with CD138 and LDH values, identifying three risk groups for death (median OS= 38.9, 30.1 and 19.8 months for the low, intermediate and high risk groups, respectively; pu2009<u20090.0001). This score had a good discrimination ability (C‐indexu2009=u20090.63). These results were externally confirmed in the validation set. Our study provides robust evidence in favor of the additional baseline soluble CD138 prognostic value for OS, in mCRC patients. A simple biological scoring system is proposed including LDH and CD138 binary status values.

Development and validation of the FRAGIRE tool for assessment an older person’s risk for frailty

BackgroundFrailty is highly prevalent in elderly people. While significant progress has been made to understand its pathogenesis process, few validated questionnaire exist to assess the multidimensional concept of frailty and to detect people frail or at risk to become frail. The objectives of this study were to construct and validate a new frailty-screening instrument named Frailty Groupe Iso-Ressource Evaluation (FRAGIRE) that accurately predicts the risk for frailty in older adults.MethodsA prospective multicenter recruitment of the elderly patients was undertaken in France. The subjects were classified into financially-helped group (FH, with financial assistance) and non-financially helped group (NFH, without any financial assistance), considering FH subjects are more frail than the NFH group and thus representing an acceptable surrogate population for frailty. Psychometric properties of the FRAGIRE grid were assessed including discrimination between the FH and NFH groups. Items reduction was made according to statistical analyses and experts’ point of view. The association between items response and tests with “help requested status” was assessed in univariate and multivariate unconditional logistic regression analyses and a prognostic score to become frail was finally proposed for each subject.ResultsBetween May 2013 and July 2013, 385 subjects were included: 338 (88%) in the FH group and 47 (12%) in the NFH group. The initial FRAGIRE grid included 65 items. After conducting the item selection, the final grid of the FRAGIRE was reduced to 19 items. The final grid showed fair discrimination ability to predict frailty (area under the curve (AUC)u2009=u20090.85) and good calibration (Hosmer-Lemeshow P-valueu2009=u20090.580), reflecting a good agreement between the prediction by the final model and actual observation. The Cronbachs alpha for the developed tool scored as high as 0.69 (95% Confidence Interval: 0.64 to 0.74). The final prognostic score was excellent, with an AUC of 0.756. Moreover, it facilitated significant separation of patients into individuals requesting for help from others (P-valueu2009<u20090.0001), with sensitivity of 81%, specificity of 61%, positive predictive value of 93%, negative predictive value of 34%, and a global predictive value of 78%.ConclusionsThe FRAGIRE seems to have considerable potential as a reliable and effective tool for identifying frail elderly individuals by a public health social worker without medical training.