ESR1 gene amplification and MAP3K mutations are selected during adjuvant endocrine therapies in relapsing Hormone Receptor-positive, HER2-negative breast cancer (HR+ HER2- BC).

BACKGROUND: Previous studies have provided a comprehensive picture of genomic alterations in primary and metastatic Hormone Receptor (HR)-positive, Human Epidermal growth factor Receptor 2 (HER2)-negative breast cancer (HR+ HER2- BC). However, the evolution of the genomic landscape of HR+ HER2- BC during adjuvant endocrine therapies (ETs) remains poorly investigated. METHODS AND FINDINGS: We performed a genomic characterization of surgically resected HR+ HER2- BC patients relapsing during or at the completion of adjuvant ET. Using a customized panel, we comprehensively evaluated gene mutations and copy number variation (CNV) in paired primary and metastatic specimens. After retrieval and quality/quantity check of tumor specimens from an original cohort of 204 cases, 74 matched tumor samples were successfully evaluated for DNA mutations and CNV analysis. Along with previously reported genomic alterations, including PIK3CA, TP53, CDH1, GATA3 and ESR1 mutations/deletions, we found that ESR1 gene amplification (confirmed by FISH) and MAP3K mutations were enriched in metastatic lesions as compared to matched primary tumors. These alterations were exclusively found in patients treated with adjuvant aromatase inhibitors or LHRH analogs plus tamoxifen, but not in patients treated with tamoxifen alone. Patients with tumors bearing MAP3K mutations in metastatic lesions had significantly worse distant relapse-free survival (hazard ratio [HR] 3.4, 95% CI 1.52-7.70, p value 0.003) and worse overall survival (HR 5.2, 95% CI 2.10-12.8, p-value < 0.001) independently of other clinically relevant patient- and tumor-related variables. CONCLUSIONS: ESR1 amplification and activating MAP3K mutations are potential drivers of acquired resistance to adjuvant ETs employing estrogen deprivation in HR+ HER2- BC. MAP3K mutations are associated with worse prognosis in patients with metastatic disease.

LncRNA EPR regulates intestinal mucus production and protects against inflammation and tumorigenesis.

The long non-coding RNA EPR is expressed in epithelial tissues, binds to chromatin and controls distinct biological activities in mouse mammary gland cells. Because of its high expression in the intestine, in this study we have generated a colon-specific conditional targeted deletion (EPR cKO) to evaluate EPR in vivo functions in mice. EPR cKO mice display epithelium hyperproliferation, impaired mucus production and secretion, as well as inflammatory infiltration in the proximal portion of the large intestine. RNA sequencing analysis reveals a rearrangement of the colon crypt transcriptome with strong reduction of goblet cell-specific factors including those involved in the synthesis, assembly, transport and control of mucus proteins. Further, colon mucosa integrity and permeability are impaired in EPR cKO mice, and this results in higher susceptibility to dextran sodium sulfate (DSS)-induced colitis and tumor formation. Human EPR is down-regulated in human cancer cell lines as well as in human cancers, and overexpression of EPR in a colon cancer cell line results in enhanced expression of pro-apoptotic genes. Mechanistically, we show that EPR directly interacts with select genes involved in mucus metabolism whose expression is reduced in EPR cKO mice and that EPR deletion causes tridimensional chromatin organization changes.

Be bold, start cold! cold formalin fixation of colorectal cancer specimens granted superior DNA and RNA quality for downstream molecular analysis.

The use of cold formalin fixation (CFF; i.e., fixating tissue samples with 4 °C precooled formalin) recently attracted further attention owing to its putative improved ability to preserve nucleic acid compared with standard room temperature formalin (SFF). In this study, we aimed to assess the effect of four formalin-based fixation protocols (SFF, CFF, delayed formalin fixation-DFF, and cold formalin hyperfixation; CFH) on both DNA and RNA quality. We collected 97 colorectal cancer (CRC) and analyzed 23 metrics of nucleic acid quantity and quality yield using a multiplatform approach by combining spectrophotometric, fluorimetric, electrophoretic, and polymerase chain reaction (PCR) assays. Following confirmation of fixation-protocol-related different effects via clustering analysis, CFF presented best metrics compared with all protocols, specifically positive coefficients of DV1000-60000, DV2/DV1, DNA λ ratio 260/230, and ABL gene expression absolute copies, and negative coefficient of DV150-1000. The SFF subgroup presented a positive coefficient of DV150-1000 and negative coefficients for DV1000-60000, DV2/DV1, RNA λ ratio 260/230, RNA QuBit concentration, DV100/200, RNA electrophoresis concentration and absolute quantity, and ABL copies. Overall, we confirmed the superior yield performances of CFF preservation for both DNA and RNA compared with the other protocols in our series of CRC samples. Pending further validations and clarification of the specific mechanisms behind these findings, our study supports the implementation of CFF in the pathology unit routine specimen management for tumor tissue molecular profiling.

Targeting PIK3CA Actionable Mutations in the Circulome: A Proof of Concept in Metastatic Breast Cancer.

The study of circulating cancer-derived components (circulome) is considered the new frontier of liquid biopsy. Despite the recognized role of circulome biomarkers, their comparative molecular profiling is not yet routine. In advanced breast cancer (BC), approximately 40% of hormone-receptor-positive, HER2-negative BC cases harbor druggable PIK3CA mutations suitable for combined alpelisib/fulvestrant treatment. This pilot study investigates PIK3CA mutations in circulating tumor DNA (ctDNA), tumor cells (CTCs), and extracellular vesicles (EVs) with the aim of determining which information on molecular targetable profiling could be recollected in each of them. The in-depth molecular analysis of four BC patients demonstrated, as a proof-of-concept study, that it is possible to retrieve mutational information in the three components. Patient-specific PIK3CA mutations were found in both tissue and ctDNA and in 3/4 cases, as well as in CTCs, in the classical population (large-sized CD45-/EpCAM+/- cells), and/or in the "non-conventional" sub-population (smaller-sized CD44+/EpCAM-/CD45- cells). Consistent mutational profiles of EVs with CTCs suggest that they may have been released by CTCs. This preliminary evidence on the molecular content of the different circulating biomaterials suggests their possible function as a mirror of the intrinsic heterogeneity of BC. Moreover, this study demonstrates, through mutational assessment, the tumor origin of the different CTC sub-populations sustaining the translational value of the circulome for a more comprehensive picture of the disease.

Optimization of a WGA-Free Molecular Tagging-Based NGS Protocol for CTCs Mutational Profiling.

Molecular characterization of Circulating Tumor Cells (CTCs) is still challenging, despite attempts to minimize the drawbacks of Whole Genome Amplification (WGA). In this paper, we propose a Next-Generation Sequencing (NGS) optimized protocol based on molecular tagging technology, in order to detect CTCs mutations while skipping the WGA step. MDA-MB-231 and MCF-7 cell lines, as well as leukocytes, were sorted into pools (2-5 cells) using a DEPArray™ system and were employed to set up the overall NGS procedure. A substantial reduction of reagent volume for the preparation of libraries was performed, in order to fit the limited DNA templates directly derived from cell lysates. Known variants in TP53, KRAS, and PIK3CA genes were detected in almost all the cell line pools (35/37 pools, 94.6%). No additional alterations, other than those which were expected, were found in all tested pools and no mutations were detected in leukocytes. The translational value of the optimized NGS workflow is confirmed by sequencing CTCs pools isolated from eight breast cancer patients and through the successful detection of variants. In conclusion, this study shows that the proposed NGS molecular tagging approach is technically feasible and, compared to traditional NGS approaches, has the advantage of filtering out the artifacts generated during library amplification, allowing for the reliable detection of mutations and, thus, making it highly promising for clinical use.

Clinico-pathological associations and concomitant mutations of the RAS/RAF pathway in metastatic colorectal cancer.

BACKGROUND: Over the past few years, next-generation sequencing (NGS) has become reliable and cost-effective, and its use in clinical practice has become a reality. A relevant role for NGS is the prediction of response to anti-EGFR agents in metastatic colorectal cancer (mCRC), where multiple exons from KRAS, NRAS, and BRAF must be sequenced simultaneously. METHODS: We optimized a 14-amplicon NGS panel to assess, in a consecutive cohort of 219 patients affected by mCRC, the presence and clinico-pathological associations of mutations in the KRAS, NRAS, BRAF, and PIK3CA genes from formalin-fixed, paraffin-embedded specimens collected for diagnostics and research at the time of diagnosis. RESULTS: We observed a statistically significant association of RAS mutations with sex, young age, and tumor site. We demonstrated that concomitant mutations in the RAS/RAF pathway are not infrequent in mCRC, and as anticipated by whole-genome studies, RAS and PIK3CA tend to be concurrently mutated. We corroborated the association of BRAF mutations in right mCRC tumors with microsatellite instability. We established tumor side as prognostic parameter independently of mutational status. CONCLUSIONS: To our knowledge, this is the first monocentric, consecutively accrued clinical mCRC cancer cohort tested by NGS in a real-world context for KRAS, NRAS, BRAF, and PIK3CA. Our study has highlighted in clinical practice findings such as the concomitance of mutations in the RAS/RAF pathway, the presence of multiple mutations in single gene, the co-occurrence of RAS and PIK3CA mutations, the prognostic value of tumor side and possible associations of sex with specific mutations.

Her2 assessment using quantitative reverse transcriptase polymerase chain reaction reliably identifies Her2 overexpression without amplification in breast cancer cases.

BACKGROUND: Immunohistochemistry (IHC) and fluorescent-in situ hybridization (FISH) are standard methods to assess human epidermal growth factor receptor 2 (HER2) status in breast cancer (BC) patients. Real-time quantitative polymerase-chain-reaction (qRT-PCR) is able to detect HER2 overexpression. Here we compared FISH, IHC, quantitative PCR (qPCR), and qRT-PCR to determine the concordance rates and evaluate their relative roles in HER2 determination. PATIENTS AND METHODS: We determined HER2 status in 153 BC patients, using IHC, FISH, Q-PCR and qRT-PCR. In discordant cases, we directly measured HER2 protein levels using Western blotting. RESULTS: The overall agreement (OA) between FISH and Q-PCR was 94.1, with a k value of 0.87. Assuming FISH as the standard reference, Q-PCR showed an 86.1% sensitivity and a 99.0% specificity with a global accuracy of 91.6%. OA between FISH and qRT-PCR was 90.8% with a k value of 0.81. Of interest, the disagreement between FISH and qRT-PCR was mostly restricted to equivocal cases. HER2 protein analysis suggested that qRT-PCR correlates better than FISH with HER2 protein levels, particularly where FISH fails to provide conclusive results. SIGNIFICANCE: qRT-PCR may outperform FISH in identifying patients overexpressing HER2 protein. Q-PCR cannot be used for HER2 status assessment, due to its suboptimal level of agreement with FISH. Both FISH and Q-PCR may be less accurate than qRT-PCR as surrogates of HER2 protein determination.

Uncovering the genomic heterogeneity of multifocal breast cancer.

Multifocal breast cancer (MFBC), defined as multiple synchronous unilateral lesions of invasive breast cancer, is relatively frequent and has been associated with more aggressive features than unifocal cancer. Here, we aimed to investigate the genomic heterogeneity between MFBC lesions sharing similar histopathological parameters. Characterization of different lesions from 36 patients with ductal MFBC involved the identification of non-silent coding mutations in 360 protein-coding genes (171 tumour and 36 matched normal samples). We selected only patients with lesions presenting the same grade, ER, and HER2 status. Mutations were classified as 'oncogenic' in the case of recurrent substitutions reported in COSMIC or truncating mutations affecting tumour suppressor genes. All mutations identified in a given patient were further interrogated in all samples from that patient through deep resequencing using an orthogonal platform. Whole-genome rearrangement screen was further conducted in 8/36 patients. Twenty-four patients (67%) had substitutions/indels shared by all their lesions, of which 11 carried the same mutations in all lesions, and 13 had lesions with both common and private mutations. Three-quarters of those 24 patients shared oncogenic variants. The remaining 12 patients (33%) did not share any substitution/indels, with inter-lesion heterogeneity observed for oncogenic mutation(s) in genes such as PIK3CA, TP53, GATA3, and PTEN. Genomically heterogeneous lesions tended to be further apart in the mammary gland than homogeneous lesions. Genome-wide analyses of a limited number of patients identified a common somatic background in all studied MFBCs, including those with no mutation in common between the lesions. To conclude, as the number of molecular targeted therapies increases and trials driven by genomic screening are ongoing, our findings highlight the presence of genomic inter-lesion heterogeneity in one-third, despite similar pathological features. This implies that deeper molecular characterization of all MFBC lesions is warranted for the adequate management of those cancers.

High ERp5/ADAM10 expression in lymph node microenvironment and impaired NKG2D ligands recognition in Hodgkin lymphomas.

Herein we describe that in classic Hodgkin lymphomas (cHL, n = 25) the lymph node (LN) stroma displayed in situ high levels of transcription and expression of the disulfide-isomerase ERp5 and of the disintegrin-metalloproteinase ADAM10, able to shed the ligands for NKG2D (NKG2D-L) from the cell membrane. These enzymes were detected both in LN mesenchymal stromal cells (MSCs) and in Reed-Sternberg (RS) cells; in addition, MIC-A and ULBP3 were present in culture supernatants of LN MSCs or RS cells. NKG2D-L-negative RS cells could not be killed by CD8(+)αßT or γδT cells; tumor cell killing was partially restored by treating RS cells with valproic acid, which enhanced NKG2D-L surface expression. Upon coculture with LN MSCs, CD8(+)αßT and γδT cells strongly reduced their cytolytic activity against NKG2D-L(+) targets; this seems to be the result of TGF-ß, present at the tumor site, produced in vitro by LN MSCs and able to down-regulate the expression of NKG2D on T lymphocytes. In addition, CD8(+)αßT and γδT cells from the lymph nodes of cHL patients, cocultured in vitro with LN MSCs, underwent TGF-ß-mediated down regulation of NKG2D. Thus, in cHL the tumor microenvironment is prone to inhibit the development of an efficient antitumor response.

Quantitative Real Time PCR assessment of hormonal receptors and HER2 status on fine-needle aspiration pre-operatory specimens from a prospectively accrued cohort of women with suspect breast malignant lesions.

OBJECTIVES: Reliable assessment of estrogen, progesterone (ER and PR), and HER2 receptor status are essential in breast cancer (BC) treatment. Immunohistochemical methods are limited by intra- and inter-laboratory variability. Furthermore, current methods are not the ideal approach for reproducing the biological continuum of ER, PR, and HER2 receptor levels, due to their intrinsic, semi-quantitative nature, relying in part on subjective interpretation. METHODS: In the present study, we tested a molecular approach to define ER, PR, and HER2 status in fine-needle-aspirate (FNA) samples from patients with early BC. We performed flow cytometry analysis on 88 FNA specimens from suspect BC patients to determine cellularity. We used quantitative Real Time PCR (QRT-PCR) to assess ER, PR, HER2 status, and qPCR for HER2 gene copy number (GCN). RESULTS: ER and PR mRNA levels showed a highly significant correlation with IHC data on surgical samples. qPCR showed greater accuracy than IHC in defining HER2 status. QRT-PCR defined better than IHC the continuous spectrum of the expression of the assessed receptors. Moreover, PCR analysis demonstrated a strict correlation between HER2 status and higher levels of its transcript, correctly stratifying HER2+ and HER2- patients. Finally, there was a strongly significant agreement between HER2 GCN assessed on FNA specimens by qPCR and FISH data obtained on pathological tissue specimens. CONCLUSIONS: The present results support a comprehensive approach to determine ER, PR, and HER2 status by PCR (QRT-PCR and qPCR) in FNA specimens, with high relevance for therapeutic strategies like neoadjuvant treatment.

ß-amyloid 1-42 induces physiological transcriptional regulation of BACE1.

The pathogenesis of Alzheimer's disease (AD) is only partially understood. ß-amyloid (Aß) is physiologically generated by sequential cleavage of its precursor protein by the ß- and the γ-secretase and it is normally disposed of. In Alzheimer's disease, Aß is excessively produced or less dismissed, but the hypothesis on its physiological and pathological role are heterogeneous and often discordant. It has been described a positive feedback loop from the γ- to the ß-secretase cleavages of Aß precursor protein, which is activated by mutations of Presenilin 1 (PS1), the catalytic core of the γ-secretase. These findings show that Aß precursor protein as well the activity of the γ-secretase are required to obtain the up-regulation of ß-secretase which is induced by Presenilin 1 mutations. Then, Aß 1-42 is the Aß precursor protein derivative that up-regulates the expression of ß-secretase, and c-jun N-terminal kinase (JNK)/c-Jun and ERK1/2 are involved. Here, we describe the activation of ß-secretase and c-jun N-terminal kinase related proteins by monomeric Aß 1-42, defining the conditions that most efficiently strike the described signaling without producing toxicity. Taken together these data imply that monomeric Aß 1-42, at non-toxic concentrations and time frames, are able to induce a signaling pathway that leads to transcriptional activation of ß-secretase.

Plasma HDL pattern, cholesterol efflux and cholesterol loading capacity of serum in carriers of a novel missense variant (Gly176Trp) of endothelial lipase.

BACKGROUND: Loss of function variants of LIPG gene encoding endothelial lipase (EL) are associated with primary hyperalphalipoproteinemia (HALP), a lipid disorder characterized by elevated plasma levels of high density lipoprotein cholesterol (HDL-C). OBJECTIVE: Aim of the study was the phenotypic and genotypic characterization of a family with primary HALP. METHODS: HDL subclasses distribution was determined by polyacrylamide gradient gel electrophoresis. Serum content of preß-HDL was assessed by (2D)-electrophoresis. Cholesterol efflux capacity (CEC) of serum mediated by ABCA1, ABCG1 or SR-BI was assessed using cells expressing these proteins. Cholesterol loading capacity (CLC) of serum was assayed using cultured human macrophages. Next generation sequencing was used for DNA analysis. Plasma EL mass was determined by ELISA. RESULTS: Three family members had elevated plasma HDL-C, apoA-I and total phospholipids, as well as a reduced content of preß-HDL. These subjects were heterozygous carriers of a novel variant of LIPG gene [c.526 G>T, p.(Gly176Trp)] found to be deleterious in silico. Plasma EL mass in carriers was lower than in controls. CEC of sera mediated by ABCA1 and ABCG1 transporters was substantially reduced in the carriers. This effect was maintained after correction for serum HDL concentration. The sera of carriers were found to have a higher CLC in cultured human macrophages than control sera. CONCLUSION: The novel p.(Gly176Trp) variant of endothelial lipase is associated with changes in HDL composition and subclass distribution as well as with functional changes affecting cholesterol efflux capacity of serum which suggest a defect in the early steps of revere cholesterol transport.

Plasma Cell-Free DNA Integrity Assessed by Automated Electrophoresis Predicts the Achievement of Pathologic Complete Response to Neoadjuvant Chemotherapy in Patients With Breast Cancer.

PURPOSE: The study of plasma cell-free DNA integrity (cfDI) has shown potential for providing useful information in neoplastic patients. The aim of this study is to estimate the accuracy of an electrophoresis-based method for cfDI evaluation in the assessment of pathologic complete response (pCR) in patients with breast cancer (BC) undergoing neoadjuvant chemotherapy (NACT). PATIENTS AND METHODS: Fifty-one patients with BC undergoing anthracycline-/taxane-based NACT were recruited. Plasma samples were collected from each patient at diagnosis (t0), after anthracycline administration (t1), and after NACT completion (t2). The concentration of differently sized cell-free DNA fragments was assessed by automated electrophoresis. cfDI, expressed as cfDI index, was calculated as the ratio of 321-1,000 bp sized fragment concentration to 150-220 bp sized fragment concentration assessed at t2. cfDI index was then used to build an exploratory classifier for BC response to NACT, directly comparing its sensitivity and specificity with magnetic resonance imaging (MRI), through bootstrapped logistic regression. RESULTS: cfDI index was assessed on 38 plasma samples collected from as many patients at t2, maintaining a 30/70 ratio between pCR and non-pCR patients. cfDI index showed an area under the receiver operating characteristic curve in predicting the achievement of pCR of 81.6, with a cutoff above 2.71 showing sensitivity = 81.8 and specificity = 81.5. The combination of cfDI index and MRI showed, in case of concordance, an area under the receiver operating characteristic curve of 92.6 with a predictive value of complete response of 87.5 and a predictive value of absence of complete response of 94.7. CONCLUSION: cfDI index measured after NACT completion shows great potential in the assessment of pCR in patients with BC. The evaluation of its use in combination with MRI is strongly warranted in prospective studies.

Endothelial colony-forming cells from patients with chronic myeloproliferative disorders lack the disease-specific molecular clonality marker.

Two putative types of circulating endothelial progenitor cells have been recently identified in vitro: (1) endothelial colony-forming cell (ECFC) and (2) colony-forming unit-endothelial cell (CFU-EC). Only the former is now recognized to belong to endothelial lineage. We have used the ECFC and CFU-EC assays to readdress the issue of the clonal relation between endothelial progenitor cells and hematopoietic stem cells in patients with Philadelphia-positive and Philadelphia-negative chronic myeloproliferative disorders. Both ECFCs and CFU-ECs were cultured from peripheral blood mononuclear cells, and either BCR-ABL rearrangement or JAK2-V617F mutation were assessed in both types of endothelial colonies. We found that ECFCs lack the disease-specific markers, which are otherwise present in CFU-ECs, thus reinforcing the concept that the latter belongs to the hematopoietic lineage, and showing that in chronic myeloproliferative disorders the cell that gives rise to circulating ECFC has a distinct origin from the cell of the hematopoietic malignant clone.

Anti-cancer activity of 5-O-alkyl 1,4-imino-1,4-dideoxyribitols.

New derivatives of 1,4-dideoxy-1,4-imino-D-ribitol have been prepared and evaluated for their cytotoxicity on solid and haematological malignancies. 1,4-Dideoxy-5-O-[(9Z)-octadec-9-en-1-yl]-1,4-imino-D-ribitol (13, IC(50) ∼2 µM) and its C(18)-analogues (IC(50) <10 µM) are cytotoxic toward SKBR3 (breast cancer) cells. 13 also inhibits (IC(50) ∼8 µM) growth of JURKAT cells.

SLFN11 captures cancer-immunity interactions associated with platinum sensitivity in high-grade serous ovarian cancer.

Large independent analyses on cancer cell lines followed by functional studies have identified Schlafen 11 (SLFN11), a putative helicase, as the strongest predictor of sensitivity to DNA-damaging agents (DDAs), including platinum. However, its role as a prognostic biomarker is undefined, partially due to the lack of validated methods to score SLFN11 in human tissues. Here, we implemented a pipeline to quantify SLFN11 in human cancer samples. By analyzing a cohort of high-grade serous ovarian carcinoma (HGSOC) specimens before platinum-based chemotherapy treatment, we show, for the first time to our knowledge, that SLFN11 density in both the neoplastic and microenvironmental components was independently associated with favorable outcome. We observed SLFN11 expression in both infiltrating innate and adaptive immune cells, and analyses in a second, independent, cohort revealed that SLFN11 was associated with immune activation in HGSOC. We found that platinum treatments activated immune-related pathways in ovarian cancer cells in an SLFN11-dependent manner, representative of tumor-immune transactivation. Moreover, SLFN11 expression was induced in activated, isolated immune cell subpopulations, hinting that SLFN11 in the immune compartment may be an indicator of immune transactivation. In summary, we propose SLFN11 is a dual biomarker capturing simultaneously interconnected immunological and cancer cell-intrinsic functional dispositions associated with sensitivity to DDA treatment.

Engagement of CD31 delivers an activating signal that contributes to the survival of chronic lymphocytic leukaemia cells.

The present study showed that engagement of CD31 delivers a survival signal in chronic lymphocytic leukaemia (CLL) cells. We describe two groups of CLL, showing different kinetics of apoptosis in vitro and distinct ratios between anti-apoptotic and pro-apoptotic proteins: CLL-I displayed low Bcl-x(L) /Bax and Bcl-2/Bax ratio and underwent rapid apoptosis in vitro; CLL-II had high Bcl-x(L) /Bax and Bcl-2/Bax ratio and were resistant to apoptosis for several days. Nurse-like cells, expressing vimentin, CD68 and CD31 were detected mainly in CLL-II cultures. Of note, CD31 cross-linking, obtained with a specific monoclonal antibody (mAb), induced phosphatidylinositol-3-kinase-dependent Akt phosphorylation and nuclear translocation of the nuclear factor-kBp65 and p52 subunits in both CLL groups, leading to upregulation of Bcl-2 and Bcl-x(L) transcription and increased cell survival. Binding to CD31(+) stable transfectants, could also deliver an anti-apoptotic signal in B cells of both CLL-I and CLL-II, increasing the Bcl-2 and Bcl-x(L) protein content, regardless the expression of CD38. On the other hand, the addition of the F(ab')2 (that is unable to oligomerize the target molecule) of the anti-CD31 mAb prevented these effects. These data suggest that the CD31 adhesion system may play a role also in vivo in maintaining CLL survival.

Novel 2-[(benzylamino)methyl]pyrrolidine-3,4-diol derivatives as alpha-mannosidase inhibitors and with antitumor activities against hematological and solid malignancies.

Novel alpha-mannosidase inhibitors of the type (2R,3R,4S)-2-({[(1R)-2-hydroxy-1-arylethyl]amino}methyl)pyrrolidine-3,4-diol have been prepared and assayed for their anticancer activities. Compound 30 with the aryl group=4-trifluoromethylbiphenyl inhibits the proliferation of primary cells and cell lines of different origins, irrespective of Bcl-2 expression levels, inducing a G2/Mcell cycle arrest and by modification of genes involved in cell cycle progression and survival.

Circulating Tumor DNA Using Tagged Targeted Deep Sequencing to Assess Minimal Residual Disease in Breast Cancer Patients Undergoing Neoadjuvant Chemotherapy.

In breast cancer patients undergoing neoadjuvant chemotherapy before surgery, there is an unmet need for noninvasive predictive biomarkers of response. The analysis of circulating tumor DNA (ctDNA) in particular has been the object of several reports, but few of them have studied the applicability of tagged targeted deep sequencing (tTDS) to clinical practice and its performance compared with droplet digital PCR (ddPCR). Here, we present the first results from an ongoing study involving a prospectively accrued, monocentric cohort of patients affected by invasive breast cancer, undergoing neoadjuvant chemotherapy followed by surgery with curative intent as per clinical practice. A pretreatment tumor biopsy and plasma samples were collected before and during treatment, after surgery, and every six months henceforth or until relapse, whichever came first. Pretreatment biopsies were sequenced with a 409-gene massive parallel sequencing (MPS) panel, allowing the identification of target mutations and their research in plasma by tTDS and ddPCR as a complementary approach. Using tTDS, we demonstrated the presence of at least one deleterious mutation in all the relapsed cases we studied (n = 4), with an average lead time of six months before clinical relapse. The association with ddPCR was suboptimal, and only one relapsed patient could be identified with such method. tTDS shows potential as an early noninvasive method for the detection of MRD in BC patients.

Development of a long non-coding RNA signature for prediction of response to neoadjuvant chemoradiotherapy in locally advanced rectal adenocarcinoma.

Standard treatment for locally advanced rectal adenocarcinoma (LARC) includes a combination of chemotherapy with pyrimidine analogues, such as capecitabine, and radiation therapy, followed by surgery. Currently no clinically useful genomic predictors of benefit from neoadjuvant chemoradiotherapy (nCRT) exist for LARC. In this study we assessed the expression of 8,127 long noncoding RNAs (lncRNAs), poorly studied in LARC, to infer their ability in classifying patients' pathological complete response (pCR). We collected and analyzed, using lncRNA-specific Agilent microarrays a consecutive series of 61 LARC cases undergoing nCRT. Potential lncRNA predictors in responders and non-responders to nCRT were identified with LASSO regression, and a model was optimized using k-fold cross-validation after selection of the three most informative lncRNA. 11 lncRNAs were differentially expressed with false discovery rate < 0.01 between responders and non-responders to NACT. We identified lnc-KLF7-1, lnc-MAB21L2-1, and LINC00324 as the most promising variable subset for classification building. Overall sensitivity and specificity were 0.91 and 0.94 respectively, with an AUC of our ROC curve = 0.93. Our study shows for the first time that lncRNAs can accurately predict response in LARC undergoing nCRT. Our three-lncRNA based signature must be independently validated and further analyses must be conducted to fully understand the biological role of the identified signature, but our results suggest lncRNAs may be an ideal biomarker for response prediction in the studied setting.