Beatrice Casini

Epigenetic silencing of miR-145-5p contributes to brain metastasis.

Brain metastasis is a major cause of morbidity and mortality of lung cancer patients. We assessed whether aberrant expression of specific microRNAs could contribute to brain metastasis. Comparison of primary lung tumors and their matched metastatic brain disseminations identified shared patterns of several microRNAs, including common down-regulation of miR-145-5p. Down-regulation was attributed to methylation of miR-145s promoter and affiliated elevation of several protein targets, such as EGFR, OCT-4, MUC-1, c-MYC and, interestingly, tumor protein D52 (TPD52). In line with these observations, restored expression of miR-145-5p and selective depletion of individual targets markedly reduced in vitro and in vivo cancer cell migration. In aggregate, our results attribute to miR-145-5p and its direct targets pivotal roles in malignancy progression and in metastasis.

Quantitative Molecular Analysis of Sentinel Lymph Node May Be Predictive of Axillary Node Status in Breast Cancer Classified by Molecular Subtypes

To determine the performance of intraoperative one-step nucleic acid amplification (OSNA) assay in detecting sentinel lymph node metastases compared to postoperative histology taking into account breast cancer molecular classification and to evaluate whether the level of cytokeratin 19 mRNA copy number may be useful in predicting the likelihood of a positive axillary lymph node dissection. OSNA assay was performed in a prospective series of 903 consecutive sentinel lymph nodes from 709 breast cancer patients using 2 alternate slices of each sentinel lymph node. The remaining 2 slices were investigated by histology. Cytokeratin 19 mRNA copy number, which distinguishes negative cases (<250 copies), micrometastases (+, ≥250≤5000 copies) and macrometastases (++, >5000 copies), was compared to axillary lymph node dissection status and to the biological tumor profile. Concordance between OSNA and histopathology was 95%, specificity 95% and sensitivity 93%. Multiple Corresponce Analysis and logistic regression evidenced that positive axillary lymph node dissection was significantly associated with a higher cytokeratin 19 mRNA copy number (>5000; p<0.0001), HER2 subtype (p = 0.007) and lymphovascular invasion (p<0.0001). Conversely, breast cancer patients with cytokeratin 19 mRNA copy number <2000 mostly presented a luminal subtype and a negative axillary lymph node dissection. We confirmed that OSNA assay can provide standardized and reproducible results and that it represents a fast and quantitative tool for intraoperative evaluation of sentinel lymph node. Omission of axillary lymph node dissection could be proposed in patients presenting a sentinel lymph node with a cytokeratin 19 mRNA copy number <2000 and a Luminal tumor phenotype.

DNA damage repair and survival outcomes in advanced gastric cancer patients treated with first‐line chemotherapy

The DNA damage response (DDR) network is exploited by cancer cells to withstand chemotherapy. Gastric cancer (GC) carries deregulation of the DDR and harbors genetic defects that fuel its activation. The ATM‐Chk2 and ATR‐Chk1‐Wee1 axes are deputed to initiate DNA repair. Overactivation of these pathways in cancer cells may represent an adaptive response for compensating genetic defects deregulating G1‐S transition (e.g., TP53) and ATM/ATR‐initiated DNA repair (e.g., ARID1A). We hypothesized that DDR‐linked biomarkers may predict clinical outcomes in GC patients treated with chemotherapy. Immunohistochemical assessment of DDR kinases (pATM, pChk2, pChk1 and pWee1) and DNA damage markers (γ‐H2AX and pRPA32) was performed in biological samples from 110 advanced GC patients treated with first‐line chemotherapy, either in phase II trials or in routine clinical practice. In 90 patients, this characterization was integrated with targeted ultra‐deep sequencing for evaluating the mutational status of TP53 and ARID1A. We recorded a positive association between the investigated biomarkers. The combination of two biomarkers (γ‐H2AXhigh/pATMhigh) was an adverse factor for both progression‐free survival (multivariate Cox: HR 2.23, 95%CI: 1.47–3.40) and overall survival (multivariate Cox: HR: 2.07, 95%CI: 1.20–3.58). The relationship between the γ‐H2AXhigh/pATMhigh model and progression‐free survival was consistent across the different TP53 backgrounds and was maintained in the ARID1A wild‐type setting. Conversely, this association was no longer observed in an ARID1A‐mutated subgroup. The γ‐H2AXhigh/pATMhigh model negatively impacted survival outcomes in GC patients treated with chemotherapy. The mutational status of ARID1A, but apparently not TP53 mutations, affects its predictive significance.

Precision diagnostics of Ewing’s sarcoma by liquid biopsy: circulating EWS-FLI1 fusion transcripts

Background: Limited information is available on the applicative value of liquid biopsy (LB) in rare tumors, including Ewing’s sarcoma (ES). The accepted precision diagnostics standards would greatly benefit from a non-invasive LB test monitoring pathognomonic gene rearrangements in the bloodstream. Methods: Tissue and blood samples were collected from six and four ES patients, respectively. Plasma was cleared by two successive rounds of centrifugation and stored frozen until RNA extraction by the QIAmp CNA kit. RNA was retro-transcribed and subjected to real-time quantitative polymerase chain reaction (RT-qPCR) and digital polymerase chain reaction (dPCR). Reactions were set up using two custom primer sets identifying types 1 and 2 EWS-FLI1 fusion transcripts. Results: The two prevalent types of EWS-FLI1 rearrangements could be identified using only two sets of polymerase chain reaction primers, regardless of patient-specific EWS-FLI1 DNA breakpoints. RT-qPCR and dPCR discriminated the two variants in five tumor tissue RNAs and in four circulating tumor RNAs (ctRNAs). Of note, EWS-FLI1 molecular diagnosis was possible using blood samples even when tumor tissue was not available. ctRNA levels correlated (p < 0.05) with volume-based positron emission tomography (PET) parameters (metabolic tumor volume and total lesion glycolysis), and allowed the fine tracking of disease course after surgery, during adjuvant as well as neoadjuvant chemotherapy, and at follow up in one patient. Conclusions: To our knowledge, this is one of the few single-marker LB assays in solid tumors specifically designed to detect rearranged RNAs in blood, and the first study describing EWS circulating tumor RNAs in ES patients. Altogether, our results support the idea that LB may have a considerable impact on ES patient monitoring and management.

A cut-off of 2150 cytokeratin 19 mRNA copy number in sentinel lymph node may be a powerful predictor of non-sentinel lymph node status in breast cancer patients

Since 2007, one-step nucleic acid amplification (OSNA) has been used as a diagnostic system for sentinel lymph node (SLN) examination in patients with breast cancer. This study aimed to define a new clinical cut-off of CK19 mRNA copy number based on the calculation of the risk that an axillary lymph node dissection (ALND) will be positive. We analyzed 1529 SLNs from 1140 patients with the OSNA assay and 318 patients with positive SLNs for micrometastasis (250 copies) and macrometastasis (5000 copies) underwent ALND. Axillary non–SLNs were routinely examined. ROC curves and Youden’s index were performed in order to identify a new cut-off value. Logistic regression models were performed in order to compare OSNA categorical variables created on the basis of our and traditional cut-off to better identify patients who really need an axillary dissection. 69% and 31% of OSNA positive patients had a negative and positive ALND, respectively. ROC analysis identified a cut-off of 2150 CK19 mRNA copies with 95% sensitivity and 51% specificity. Positive and negative predictive values of this new cut-off were 47% and 96%, respectively. Logistic regression models indicated that the cut-off of 2150 copies better discriminates patients with node negative or positive in comparison with the conventional OSNA cut-off (p<0.0001). This cut-off identifies false positive and false negative cases and true-positive and true negative cases very efficiently, and therefore better identifies which patients really need an ALND and which patients can avoid one. This is why we suggest that the negative cut-off should be raised from 250 to 2150. Furthermore, we propose that for patients with a copy number that ranges between 2150 and 5000, there should be a multidisciplinary discussion concerning the clinical and bio-morphological features of primary breast cancer before any decision is taken on whether to perform an ALND or not.

Deep sequencing and pathway-focused analysis revealed multigene oncodriver signatures predicting survival outcomes in advanced colorectal cancer

Genomic technologies are reshaping the molecular landscape of colorectal cancer (CRC), revealing that oncogenic driver mutations (APC and TP53) coexist with still underappreciated genetic events. We hypothesized that mutational analysis of CRC-linked genes may provide novel information on the connection between genetically-deregulated pathways and clinical outcomes. We performed next-generation sequencing (NGS) analysis of 16 recurrently mutated genes in CRC exploiting tissue specimens from 98 advanced CRC patients. Multiple correspondence analysis (MCA) was used to identify gene sets characterizing negative and positive outliers (patients in the lowest and highest quartile of progression-free survival, PFS). Variables potentially affecting PFS and overall survival (OS) were tested in univariate and multivariate Cox proportional hazard models. Sensitivity analyses and resampling were used to assess the robustness of genomic predictors. MCA revealed that APC and TP53 mutations were close to the negative outlier group, whereas mutations in other WNT pathway genes were in proximity of the positive outliers. Reasoning that genetic alterations interact epistatically, producing greater or weaker consequences in combination than when individually considered, we tested whether patients whose tumors carried a genetic background characterized by APC and TP53 mutations without coexisting mutations in other WNT genes (AMER1, FBXW7, TCF7L2, CTNNB1, SOX9) had adverse survival outcomes. With this approach, we identified two oncodriver signatures (ODS1 and ODS2) associated with shorter PFS (ODS1 multivariate Cox for PFS: HR 2.16, 95%CI: 1.28–3.64, p = 0.004; ODS2 multivariate Cox for PFS: HR 2.61, 95%CI: 1.49–4.58, p = 0.001). Clinically-focused and molecularly-focused sensitivity analyses, resampling, and reclassification of mutations confirmed the stability of ODS1/2. Moreover, ODS1/2 negatively impacted OS. Collectively, our results point to co-occurring driver mutations as an adverse molecular factor in advanced CRC. This relationship depends on a broader genetic context highlighting the importance of genetic interactions.

The clinical significance of PD-L1 in advanced gastric cancer is dependent on ARID1A mutations and ATM expression

ABSTRACT Whether PD-L1 expression is associated with survival outcomes in gastric cancer (GC) is controversial. The inhibition of the PD-1/PD-L1 pathway is effective against genomically unstable tumors. Hypothesizing that also the clinical significance of PD-L1 might be dependent on the activation of molecular circuits ensuring genomic stability, we evaluated PD-L1 expression in tissue samples from 72 advanced GC patients treated with first-line chemotherapy. Samples were already characterized for DNA damage repair (DDR) component expression (pATM, pChk1, pWee1, γ-H2AX and pRPA2) along with mutations in DDR-linked genes (TP53 and ARID1A). Overall, PD-L1 expression was not associated with progression-free survival (PFS) and overall survival (OS), independently on whether we considered its expression in tumor cells (PD-L1-TCs) or in the immune infiltrate (PD-L1-TILs). In subgroup analysis, positive PD-L1-TC immunostaining was associated with better PFS in patients whose tumors did not carry DDR activation (multivariate Cox: HR 0.34, 95%CI: 0.15–0.76, p = 0.008). This subset (DDRoff) was characterized by negative pATM expression or the presence of ARID1A mutations. Conversely, the relationship between PD-L1-TC expression and PFS was lost in a molecular scenario denoting DDR activation (DDRon), as defined by concomitant pATM expression and ARID1A wild-type form. Surprisingly, while PD-L1-TC expression was associated with better OS in the DDRoff subset (multivariate Cox: HR 0.41, 95% CI: 0.17–0.96, p = 0.039), in the DDRon subgroup we observed an opposite impact on OS (multivariate Cox: HR 2.56, 95%CI: 1.06–6.16, p = 0.036). Thus, PD-L1-TC expression may impact survival outcomes in GC on the basis of the activation/inactivation of genome-safeguarding pathways.

Coexisting YAP expression and TP53 missense mutations delineates a molecular scenario unexpectedly associated with better survival outcomes in advanced gastric cancer

We have previously reported that nuclear expression of the Hippo transducer TAZ in association with Wnt pathway mutations negatively impacts survival outcomes in advanced gastric cancer (GC) patients. Here, we extended these previous findings by investigating another oncogenic cooperation, namely, the interplay between YAP, the TAZ paralogue, and p53. The molecular output of the YAP-p53 cooperation is dependent on TP53 mutational status. In the absence of mutations, the YAP-p53 crosstalk elicits a pro-apoptotic response, whereas in the presence of TP53 mutations it activates a pro-proliferative transcriptional program. In order to study this phenomenon, we re-analyzed data from 83 advanced GC patients treated with chemotherapy whose tissue samples had been characterized for YAP expression (immunohistochemistry, IHC) and TP53 mutations (deep sequencing). In doing so, we generated a molecular model combining nuclear YAP expression in association with TP53 missense variants (YAP+/TP53mut(mv)). Surprisingly, this signature was associated with a decreased risk of disease progression (multivariate Cox for progression-free survival: HR 0.53, 95% CI 0.30–0.91, p = 0.022). The YAP+/TP53mut(mv) model was also associated with better OS in the subgroup of patients who received chemotherapy beyond the first-line setting (multivariate Cox: HR 0.36, 95% CI 0.16–0.81, p = 0.013). Collectively, our findings suggest that the oncogenic cooperation between YAP and mutant p53 may translate into better survival outcomes. This apparent paradox can be explained by the pro-proliferative program triggered by YAP and mutant p53, that supposedly renders cancer cells more vulnerable to cytotoxic therapies.

Usefulness of conventional transbronchial needle aspiration in the diagnosis, staging and molecular characterization of pulmonary neoplasias by thin-prep based cytology: experience of a single oncological institute

BACKGROUND Conventional transbronchial needle aspiration (c-TBNA) contributed to improve the bronchoscopic examination, allowing to sample lesions located even outside the tracheo-bronchial tree and in the hilo-mediastinal district, both for diagnostic and staging purposes. METHODS We have evaluated the sensitivity, accuracy, positive predictive value (PPV) and negative predictive value (NPV) of the c-TBNA performed during the 2005-2015 period for suspicious lung neoplasia and/or hilar and mediastinal lymphadenopathy at the Thoracic endoscopy of the Thoracic Surgery Department of the Regina Elena National Cancer Institute, Rome. Data from 273 consecutive patients (205 males and 68 females) were analyzed. RESULTS Among 158 (58%) adequate specimens, 112 (41%) were neoplastic or contained atypical cells, 46 (17%) were negative or not diagnostic. We considered in the analysis first the overall period; then we compared the findings of the first [2005-2011] and second period [2012-2015] and, finally, only those of adequate specimens. During the overall period, sensibility and accuracy values were respectively of 53% and 63%, in the first period they reached 41% and 53% respectively; in the second period sensibility and accuracy reached 60% and 68%. Considering only the adequate specimens, sensibility and accuracy during the overall period were respectively of 80% and 82%; the values obtained for the first period were 68% and 72%. Finally, in the second period, sensibility reached 86% and accuracy 89%. Carcinoma-subtyping was possible in 112 cases, adenocarcinomas being diagnosed in 50 cases; further, in 30 cases molecular predictive data could be obtained. CONCLUSIONS The c-TBNA proved to be an efficient method for the diagnosis/staging of lung neoplasms and for the diagnosis of mediastinal lymphadenopathy. Endoscopists skill and technical development, associated to thin-prep cytology and to a rapid on site examination (ROSE), were able to provide by c-TBNA a high diagnostic yield and molecular predictive data in advanced lung carcinomas.

P3-07-02: Prediction of Non-Sentinel Lymph Node Status in Breast Cancer Patients with a Micrometastatic Sentinel Lymph Node Determined by the One Step Nucleic Acid Amplification (OSNA) Assay.

Background Axillary lymph node dissection (ALND) may not be necessary in women with breast cancer (BC) who have micrometastasis in a sentinel lymph node (SLN), owing to the low risk of non-SLN (NSLN) involvement. In our Institute we validated and adopted the new molecular diagnostic tool OSNA (One Step Nucleic Acid Amplification) based on the quantitative measurement of Cytokeratin 19 (CK19) mRNA. The aims of our work in a subgroup of women with micrometastatic SLN, were: 1) to correlate the copy numbers of CK19 mRNA with the risk of additional positive NSLNs; 2) to assess the relationships between the molecular subtype classification based on the immunohistochemistry phenotypic patterns and the probability of a positive ALND; 3) to verify whether a combination of the new above mentioned parameters is able to identify a subgroup of patients with a micrometastatic SLN and a negligible risk of positive NSLNs in whom ALND may be avoided. Material and Methods: The intraoperative clinical study was conducted on 901 fresh SLNs from 709 consecutive patients with clinically node negative BC. The SLN lysates were analyzed by OSNA assay. If the CK19 mRNA copy number/mL lysate was less than 250 copies/mL, the result was regarded as negative (−); copy numbers between 250 and 5000/mL were regarded as micrometastasis (+), and copy numbers greater than 5000/mL as macrometastasis (++). We analyzed only patients with a micrometastatic SLN. Complementary ALND was performed concurrently in case of OSNA assay positivity and the probability of having a positive lymph node axillary dissection was calculated by the unconditional logistic regression model. This series of BC patients were divided into four main subtypes taking in account the BC classification as defined by a combination of estrogen, progesteron receptors and HER2 status evaluated by immunohistochemistry (IHC) and confirmed by FISH in case of IHC-HER2 2+. Results : OSNA positivity for micrometastasis was reported in 91/709 cases (12,8%).The number of patients with positive ALND was 20 (22%). The statistical analyses showed that the metastatic involvement of NSLNs is associated with SLNs with a high copy numbers (>2000) of CK19 mRNA together with luminal B subtype. Otherwise none of the luminal A patients with a positive SLN but presenting a copy number Conclusions : We showed that biologically-driven analyses may be able to build new models with higher performance in terms of breast cancer axillary status prediction after positive SLN biopsy for micrometastasis. The copy numbers of CK19 mRNA and the molecular subtypes are more advantageous than traditional parameters because they are not pathologist-dependent and therefore they are more reliable and reproducible. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-07-02.