In the diagnostic setting of head and neck squamous cell carcinoma (HNSCC), there is an unmet need for robust histological prognosticators, since grading alone is considered to be of disputed clinical relevance. In this context, the present study assessed site-specific and tumor compartment characteristics as potential histological risk factors in HNSCC. Morphological and immunophenotypic (such as CD8 and PD-L1) characteristics of tumor cells, the immune microenvironment and the invasive margin (IM) were assessed in 248 patients with HNSCC who were followed up for >20 years, to determine their site specificity and impact on the overall survival of patients. Laryngeal and hypopharyngeal carcinomas were characterized by keratinization, cell cannibalism and anaplastic features; oropharyngeal carcinoma was characterized by a high grade and necrosis; and oral carcinomas was characterized by keratin pearls, eosinophil infiltrates and perineural invasion (P<0.05). CD8 distribution homogeneity, tumor center and/or IM perineural invasion, and sparse lymphocytic host response in the IM (LHR-IM) were unfavorable prognosticators (P<0.05). Among these parameters, only LHR-IM and perineural invasion throughout the tumor independently predicted an unfavorable prognosis, along with disease stage. Grade, keratinization, anaplastic features, cell cannibalism, necrosis-related features, eosinophils, worst pattern of invasion (the most aggressive pattern of tumor cell proliferation found at the invasive front), tumor lymphocytic and CD8+ T-cell infiltrates, and primary site were not associated with prognosis in the present study. In conclusion, perineural invasion and LHR-IM were confirmed as histological risk factors in HNSCC, which should be included in pathology reports. To revise the assessment of HNSCC tumor grade, the aforementioned site-specific histological characteristics may be used in deep learning algorithms.

Background: Dose-dense sequential (dds) chemotherapy has changed the clinical outcome of patients with early breast cancer (BC). To investigate the impact of dose intensity (DI) in the adjuvant setting of BC, this observational trial (HE 10/10) was conducted assessing the long-term survival outcome, safety and toxicity of a currently widely used chemotherapeutic regimen. In addition, the prognostic significance of tumor infiltrating lymphocytes (TILs) and infiltrating CD8+ lymphocytes were also evaluated in the same cohort. Patients and methods: Totally, 1054 patients were prospectively enrolled in the current study with 1024 patients being eligible, while adequate tissue was available for 596 of them. TILs, CD8+ lymphocytes in intratumoral areas in contact with malignant cells (iCD8), CD8+ lymphocytes in tumor stroma (sCD8) as well as the total number of CD8+ lymphocytes within the tumor area (total CD8) were assessed by immunohistochemistry. Results: Within a median follow-up of 125.18 months, a total of 200 disease-free survival (DFS) events (19.5%) were reported. Importantly, the 10-year DFS and OS rates were 78.4% (95% CI 75.0–81.5) and 81.7% (95% CI 79.0–84.1), respectively. Interestingly, higher CD8+ T cells as well as TILs in the tumor microenvironment were associated with an improved long-term survival outcome. Conclusions: In conclusion, this study confirms the significance of dds adjuvant chemotherapeutic regimen in terms of long-term survival outcome, safety and toxicity as well as the prognostic significance of TILs and infiltrating CD8+ lymphocytes in BC patients with early-stage disease.

Dose-dense sequential (dds) chemotherapy has tremendously changed the clinical outcome of patients with early breast cancer (BC). To investigate the impact of dose intensity (DI) in the adjuvant setting of BC, this observational trial (HE 10/10) was conducted assessing the long-term survival outcome, safety and toxicity of a now widely used chemotherapeutic regimen consisting of 4 cycles of cyclophosphamide and epirubicin q2 weeks, followed by 4 cycles of docetaxel q3 weeks. In addition, the prognostic significance of tumor infiltrating lymphocytes (TILs) and infiltrating CD8+ lymphocytes were also evaluated in the same cohort. Totally, 1,054 patients were prospectively enrolled in the current study with 1,024 patients being eligible, while adequate tissue was available for 659 of them. TILs, CD8+ lymphocytes in intratumoral areas in contact with malignant cells (iCD8), CD8+ lymphocytes in tumor stroma (sCD8) as well as the total number of CD8+ lymphocytes within the tumor area (total CD8) were assessed by immunohistochemistry. Within a median follow-up of 125.18 months, a total of 200 disease-free survival (DFS) events (19.5%) were reported. The median DFS and overall survival (OS) had not been reached yet at the time of data cut-off for the analysis, while the 10-year DFS and OS rates were 78.4% (95% CI 75.0-81.5) and 81.7% (95% CI 79.0-84.1), respectively. The studied regimen was well tolerated with 94.7% of the patients completing the full course of adjuvant chemotherapy (8 cycles), while the rest of them discontinued the treatment, mainly due to non-fatal reasons. Only a very small percentage of patients faced clinically significant myelotoxicity. Interestingly, higher CD8+ T cells in the tumor microenvironment were associated with an improved long-term survival outcome. In conclusion, this study confirms the significance of dds adjuvant chemotherapeutic regimen in terms of long-term survival outcome, safety and toxicity as well as the prognostic significance of infiltrating CD8+ lymphocytes in BC patients with early stage disease.

Prognostic and predictive biomarkers are being studied for the diagnosis and treatment of breast cancer. The present study retrospectively assessed the mRNA expression of HER family receptor ligands and of other potential prognostic biomarkers and their association with time to progression (TTP), survival and clinicopathological characteristics in patients with metastatic breast cancer (MBC) treated with trastuzumab. A total of 145 tumour tissue samples were analysed. mRNA expression analysis of the transcripts of interest was performed and the association of these markers with selected clinicopathological parameters was examined. HER2 status was centrally re-evaluated. Only 67.6% of patients were truly HER2-positive according to the central HER2 re-evaluation. Heparin binding epidermal growth factor (EGF)-like growth factor, transforming growth factor β1 (TGFB1) and thyroid hormone receptor α (THRA) mRNA expression was higher in HER2-positive patients (P=0.026, P<0.001 and P<0.001). Insulin-like growth factor binding protein 4 was correlated with retinoic acid receptor α, TGFB1 and THRA (rho=0.45, rho=0.60 and rho=0.45). In HER2-positive patients, high neuregulin 1 and high betacellulin were unfavourable factors for TTP [hazard ratio (HR) = 1.78, P=0.040 and HR=2.00, P=0.043, respectively]. In patients with de novo MBC, high EGF expression was associated with a non-significant prolongation of TTP (HR=0.52, P=0.080) and significantly longer survival (HR=0.40, P=0.020). The present study examined clinical and biological implications of specific genes and it was concluded that their expression has an impact on the outcome of trastuzumab-treated patients with MBC.

Tumor-infiltrating lymphocytes (TILs) contribute to breast cancer (BC) prognosis. We investigated the prognostic impact of CD8+ TILs in patients with early breast cancer treated with adjuvant chemotherapy in a large observational clinical trial. Along with a 10 year follow-up, considering the efficacy and safety, we report the results of the translational part of our study. We examined the patients' tumors for total (t), stromal (s), and intratumoral (i) CD8 lymphocyte density (counts/mm2) on tissue-microarray cores. The impact of CD8+ TILs counts on DFS and OS, and its correlation with breast cancer subtypes and standard clinicopathological parameters, were investigated, along with efficacy and safety data. Among the 928 eligible patients, 627 had available CD8+ data. Of which, 24.9% had a high expression of sCD8, iCD8, and total CD8, which were correlated with higher Ki67, TILs density, ER/PgR negativity, and higher histological grade. The 5year DFS and OS rates were 86.1% and 91.4%, respectively. Patients with high iCD8 and tCD8 had longer DFS and OS compared to those with low counts/mm2 (DFS: HR = 0.58, p = 0.011 and HR = 0.65, p = 0.034 and OS: HR = 0.63, p = 0.043 and HR = 0.58, p = 0.020, respectively). Upon adjustment for clinicopathological parameters, iCD8 and tCD8 retained their favorable prognostic significance for DFS and OS, whereas high sCD8 was only prognostic for DFS. Menopausal status, tumor size, and nodal status retained their prognostic significance in all examined multivariate models. CD8+ TILs, and especially their intratumoral subset, represent a potential favorable prognostic factor in early BC.

Purpose The genomic status of non-malignant tissues from carriers of pathogenic germline BRCA1/2 (gBRCA1/2) variants may reveal information towards individualized prophylaxis. We performed spatiotemporal tissue genotype comparisons in a real-life cohort of gBRCA1/2 carriers of Greek origin, who underwent multiple risk-reducing/prophylactic surgeries at various time points. Methods Fifty-three women (median age 36 years) within cancer families were observed for up to 37.5 years; 43 were cancer carriers and 10 were healthy carriers. Histology review and genotyping were performed for 187 paraffin tissues (average: 3.5 per carrier) including 46 carcinomas (40 breast) and 141 non-malignant breast and gynecological samples. Results High allelic imbalance (AI) and somatic pathogenic TP53 variants were present in cancer carriers only (p values < 0.0001). High AI was associated with gBRCA1/2 indels (p < 0.0001) and gBRCA2 alterations (p = 0.0109). Somatic (pathogenic) variants were infrequently shared between non-malignant tissues and matched carcinomas. Aberrations of gBRCA1 variant heterozygosity were noticed in tissues from cancer carriers only (13/43, 30.2%). These pertained to classic LOH (neoplastic lesions in 9/43 carriers, 20.9%) and under-representation of the germline variants (5 samples, 4 non-malignant, all in the breast). Both aberrations coexisted in matched samples in one case. Over time, germline variant heterozygosity prevailed in non-malignant tissues; intra-carrier genomic alterations were aggravated (21.1%), ameliorated (26.3%) or remained stable. Conclusion This real-life case study supports the need to address tissue genotypes from prophylactic surgeries in combination with polygenic scores towards personalized prophylaxis. To this end, knowing the traditionally classified pathogenic potential of a gBRCA1/2 variant may not be enough.

Background/Aim: Human epidermal growth factor receptor 2 (HER2) P95-isoform could be involved in trastuzumab resistance in HER2 metastatic breast cancer. Materials and Methods: A total of 114 metastatic breast cancer patients treated with trastuzumab were evaluated retrospectively. HER2 was centrally reviewed. P95 was evaluated along with other markers possibly affecting trastuzumab efficacy in regards to progression-free survival and overall survival. Results: HER2 was centrally negative in 54 cases. P95 expression was significantly higher in HER2-positive tumors. High p95 was associated with gain of HER2 copy number variations (CNVs), high pHER2Tyr877, Ki67 and HER2 mRNA. P95 as a continuous variable was positively correlated with mRNA expression of HER2 and negatively correlated with HER4 and IGF1. HER2-negative p95-high patients had a marginally higher risk for death (HR=2.15, p=0.055). Conclusion: p95 was associated with higher HER2 CNVs and mRNA expression, pHER2Tyr877 expression and high Ki67, indicating a more aggressive phenotype.

Our hypothesis was that the predictive accuracy of pathogenic variants in genes participating in the homologous recombination repair (HRR) system in patients with epithelial ovarian cancer (EOC) could be improved by considering additional next-generation sequencing (NGS) metrics. NGS genotyping was performed in tumor tissue, retrospectively and prospectively collected from patients with EOC, diagnosed from 8/1998 to 10/2016. Variants were considered clonal when variant allele frequencies corresponded to >25%. The primary endpoint was overall survival (OS). This study included 501 patients with EOC, predominantly with high-grade serous (75.2%) and advanced stage tumors (81.7%); median age was 58 years (22-84). Pathogenic and clonal pathogenic variants in HRR and/or TP53 genes were identified in 72.8% and 66.5% tumors, respectively. With a median follow-up of 123.9 months, the presence of either pathogenic or clonal pathogenic HRR-only variants was associated with longer OS compared to HRR/TP53 co-mutation (HR=0.54; 95% CI, 0.34-0.87, Wald’s p=0.012 and HR=0.45; 95% CI, 0.27-0.78, Wald’s p=0.004, respectively). However, only the presence of clonal HRR-only variants was independently associated with improved OS (HR=0.55; 95% CI, 0.32-0.94, p=0.030). Variant clonality and co-occuring TP53 variants affect the predictive value of HRR pathogenic variants for platinum agents in patients with EOC.Clinical Trial Registration[ClinicalTrials.gov], identifier [NCT04716374].

Background: We evaluated the association between pathogenic mutations and overall survival (OS) in patients with cancer referred to Hellenic Cooperative Oncology Group-affiliated Departments. Patients and methods: Patients referred from 12/1980 to 1/2017 had molecular testing (for research) of archival tumor tissue collected at the time of first diagnosis (non-metastatic, 81%; metastatic, 19%). Tumor-specific gene panels (16-101 genes) were used to identify pathogenic mutations in clinically relevant genes. NGS genotyping was performed at the Laboratory of Molecular Oncology, Aristotle University of Thessaloniki. Annotation of mutations was performed at MD Anderson Cancer Center. Results: We analyzed 3,084 patients (median age, 57 years; men, 22%) with sequencing data. Overall, 1,775 (58% of 3,084) patients had pathogenic mutations. The median follow-up was 7.52 years (95% CI, 7.39-7.61). In patients with non-metastatic tumors, after stratification by tumor type, increasing age, higher grade, and histology other than adenocarcinoma were associated with shorter OS. OS was also shorter in patients with pathogenic TP53 (HR=1.36; p<0.001), MLL3 (HR=1.64; p=0.005), and BRCA1 (HR=1.46; p=0.047) mutations compared to wild-type genes. In multivariate analyses, independent prognostic factors predicting shorter OS were pathogenic mutations in TP53 (HR=1.37, p=0.002) and MLL3 (HR=1.50, p=0.027); increasing age (HR=1.02, p<0.001); and increasing grade (HR=1.46, p<0.001). In patients with metastatic cancer, older age and higher grade were associated with shorter OS and maintained their independent prognostic significance (increasing age, HR=1.03, p<0.001 and higher grade, HR=1.73, p<0.001). Conclusions: Analysis of molecular data reveals prognostic biomarkers, regardless of tissue or organ of origin to improve patient management.

Genotypic and phenotypic comparisons of tumors in multiple tissue samples from the same patient are important for understanding disease evolution and treatment possibilities. Panel NGS genotyping is currently widely used in this context, whereby NGS variant filtering and final evaluation constitute the basis for meaningful comparisons. Here, we present the genotype data used for genotype / phenotype comparisons between matched primary / metastatic colorectal tumors in the work by Chatzopoulos et al (doi: 10.1016/j.humpath.2020.10.009), as well as the process followed for obtaining these data. We describe key issues while processing routinely formalin-fixed paraffin-embedded (FFPE) tumors for genotyping, NGS application (Ion Torrent), a stringent variant filtering algorithm for genotype analyses in FFPE tissues and particularly in matched tumor samples, and provide the respective datasets. Apart from research, tumor NGS genotyping is currently applied for clinical diagnostic purposes in Oncology. The datasets and method description provided herein (a) are important for comprehending the peculiarities of FFPE tumor genotyping, which is still mostly based on principles of germline DNA genotyping; (b) can be used in pooled analyses, e.g., of primary / metastatic tumors for the investigation of tumor evolution.

ABSTRACT We investigated the expression profile of selected microRNAs (miRs) in serum and tissue samples from patients with sporadic parathyroid adenomas (sPAs). This was a prospective, controlled cohort study. Forty patients with sPAs who had undergone parathyroidectomy (PTX) were included. MiR extraction was performed from (i) 40 formalin‐fixed paraffin‐embedded samples (FFPEs) of sPAs, (ii) 10 FFPEs of normal parathyroid tissue (NPT), (iii) serum samples of the 40 patients with sPAs (t1 = baseline; t2 = 2 months post‐PTX), and (vi) serum samples of 10 healthy individuals (controls; t1 = baseline and t2 = 2 months later). Ten miRs were selected based on their interaction with genes related to parathyroid tumorigenesis (miR‐17‐5p, miR‐24‐3p, miR‐29b‐3p, miR‐31‐5p, miR‐135b‐5p, miR‐186‐5p, miR‐195‐5p, miR‐330‐3p, miR‐483‐3p, and miR‐877‐5p). At tissue level, the relative expression of miR‐17‐5p, miR‐31‐5p, miR‐135b‐5p, miR‐186‐5p, and miR‐330‐3p was significantly decreased (fold change [FC]: 0.17, FC: 0.03, FC: 0.01, FC: 0.10, FC: 0.10, respectively; all p values <0.001), and the expression of miR‐24‐3p and miR‐29b‐3p was significantly increased (FC: 12.4, p < 0.001; FC: 18.5, p = 0.011, respectively) in sPA compared with NPT samples. The relative expression of miR‐135b‐5p was also significantly decreased in the serum samples of patients compared with controls (FC: 0.7, p = 0.035). No significant differences were found in the serum samples of patients before and after PTX. MiRs that regulate genes linked to parathyroid tumors such as menin 1 (miR‐24‐3p, miR‐29b‐3p), cyclin D1 (miR‐17‐5p), calcium sensing receptor (miR‐31‐5p, miR‐135b‐5p), cyclin‐dependent kinase inhibitors (miR‐186‐5p), and β‐catenin (miR‐330‐3p) were significantly deregulated in sPAs compared with NPT samples, suggesting a role for epigenetic changes in parathyroid tumorigenesis. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Background Tumor-infiltrating lymphocytes (TILs) and their subsets contribute to breast cancer prognosis. We investigated the prognostic impact of CD3+, CD8+ and FOXP3+ TILs in patients with early intermediate/high-risk breast cancer treated with adjuvant anthracycline-based chemotherapy within two randomized trials conducted by our Group. Methods We examined 1011 patients (median follow-up 130.9 months) and their tumors for total, stromal (s) and intratumoral (i) CD3, CD8 and FOXP3 lymphocyte density (counts/mm 2 ) on tissue-microarray cores by immunohistochemistry. Morphological sTIL density on whole H&E-stained sections was also evaluated. Results The majority of TILs were CD3+. Total CD3 and CD8, sCD3 and sCD8, iCD3 and iCD8, sFOXP3 and iFOXP3 were strongly correlated (Spearman’s rho values > 0.6). High individual lymphocytic subsets and sTIL density were strongly associated with high tumor grade, higher proliferation and HER2-positive and triple-negative tumors (all p values < 0.001). Higher sTIL density (10% increments), high density of almost each individual marker and all-high profiles conferred favorable prognosis. However, when adjusted for sTIL density, stromal and intratumoral lymphocytic subsets lost their prognostic significance, while higher sTIL density conferred up to 15% lower risk for relapse. Independently of sTIL density, higher total CD3+ and CD8+ TILs conferred 35% and 28% lower risk for relapse, respectively. Conclusions Stromal and intratumoral CD3+, CD8+ and FOXP3+ TIL density do not seem to add prognostic information over the morphologically assessed sTIL density, which is worth introducing in routine histology reports.

Background/Aim: Early-stage gastric cancer has a high risk of recurrence, despite trimodality therapy with surgery, chemotherapy and radiation. To improve patient selection for adjuvant chemoradiotherapy, we evaluated the prognostic significance of immunohistochemical and genetic biomarkers in patients with resected gastric adenocarcinoma. Patients and Methods: Tumors from 119 patients were subjected to immunohistochemistry for 12 protein biomarkers, as well as next-generation sequencing. Clinical and biomarker data were available for 91 patients. Results: EBV- positive tumors and tumors with mutations had higher intratumoral CD8 tumor-infiltrating lymphocyte density (p=0.009 and p=0.017, respectively). PIK3CA mutations were correlated with VEGFA overexpression (p=0.042), while KRAS mutations and HER2 expression were mutually exclusive (p=0.036). PTEN expression univariately confirmed longer overall survival (HR=0.27; p=0.046), while there was a trend between the presence of KRAS mutations and inferior disease free and overall survival. Conclusion: PTEN protein expression and KRAS mutations may predict disease outcome in early-stage gastric cancer. These results need to be further validated in larger cohorts.

Background/Aim: Ovarian cancer (OVCA) is characterized by genomic/molecular intra-patient heterogeneity (IPH). Tissue histology and morphological features are surrogates of the underlying genomic/molecular contexture. We assessed the morphological IPH of OVCA tumor compartments and of lymphocytic infiltrates in multiple matched samples per patient. Materials and Methods: We examined 294 hematoxylin & eosin (H&E) OVCA tumor whole sections from 70 treatment-naïve patients who had undergone cytoreductive surgery. We assessed morphological subtypes as immunoreactive (IR), solid – proliferative (SD), papilloglandular (PG), and mesenchymal transition (MT); subtype load per patient; stromal tumor-infiltrating lymphocyte (sTIL) density as average per sample; and, as maximal sTIL values (max-TILs) among all samples per patient, ovaries and implants. Results: Among all 294 tumor sections, the most frequent primary morphological subtype was PG (n=150, 51.0%), followed by MT (71, 24.1%), SD (48, 16.3%) and IR (15, 5.1%). Subtype combinations were observed in 67/294 sections (22.8%) and IPH in 48/70 patients (68.6%). PG prevailed in ovaries (p<0.001), SD and MT in implants (p=0.023 and p<0.001, respectively). sTILs were higher in SD compared to non-SD (p=0.019) and lower in PG, respectively (p<0.001). sTIL density was higher in implants than in ovaries (p<0.001). Higher max-TILs were associated with stage IV disease (p=0.043), upper abdominal dissemination (p=0.024), endometrioid histology (p=0.013), and grade 3 tumors (p=0.021). Favorable prognosticators were higher max-TILs per patient (PFS, OS) and higher SD-load (PFS). Conclusion: Clinically relevant morphological and host immune-response IPH appear to be the norm in OVCA. This may complicate efforts to decipher sensitivity of the tumor to certain treatment modalities from a single pre-operative biopsy.

Although primary colorectal carcinomas (CRCs) frequently share genetic alterations with their metastases, morphologic surrogates reflecting the genotype contexture of metastases remain largely unknown. We investigated phenotype/genotype associations in paired primary and metastatic colorectal adenocarcinomas from 75 patients. Thirty-three (44%) metastatic lesions were synchronous and 42 (56%) were metachronous. Tumor budding, micronecrosis, and tumor-infiltrating lymphocyte (TIL) density were compared with matched next-generation sequencing genotypes. Micronecrosis in the primary were significantly associated with nodal status (P = 0.0054) and with micronecrosis in metastatic sites (P = 0.0216), particularly in metachronous metastases (P = 0.0033). With a 57-gene panel, one or more mutations were identified in 64 (85.3%) cases. In metastases, high (brisk) TILs were associated with overall mutational burden (P = 0.0058) and with mutations in EGF (P = 0.0325), RAS genes (P = 0.0043), and MMR genes (P = 0.0069), whereas high-level micronecrosis correlated with mutations in APC (P = 0.0004) and MSH6 (P = 0.0385) genes. Genomic alterations were shared in 90.1% of primary/metastatic pairs, but clonality of the same mutation was shared in only 57.1% of paired lesions. Compared with synchronous, metachronous metastases had more private clonal alterations (P = 0.0291); in this group, clonal alterations coincided with brisk TILs (P = 0.0334) and high micronecrosis (P = 0.0133). High TILs in metastatic lesions were predictive of favorable overall survival (log-rank P = 0.044). The observed phenotype/genotype associations favor the clonal evolution model in CRC metastases that seems accompanied by intense host immune response. If the role of micronecrosis and brisk TILs in metachronous metastases is validated in larger studies, these histologic parameters will be worth adding in the armamentarium for the evaluation of metastatic CRC.

Background/Aim: Early-stage colorectal cancer (CRC) carries a wide range of survival probabilities. Novel biomarkers in this setting are eagerly awaited. Cancer stem cells (CSCs) are considered one of the reasons for treatment failure. This study sought to determine whether activation of pathways governing the function of CSC's could correlate with treatment outcomes. Materials and Methods: Tumor specimens from 325 patients were analyzed with immunohistochemistry (IHC) for Hedgehog and Notch pathway activation and results were correlated with prognosis. Results: Positive Notch3 protein expression was an unfavorable prognostic factor for disease-free survival (DFS) and overall survival (OS) (HR=2.43, p=0.024 and HR=2.56, p=0.028, respectively). Activation of the Shh pathway showed univariately longer DFS (HR=0.49, p=0.032). Possible crosstalk between the two pathways was indicated. No further associations between pathway activation and outcome were evident. Conclusion: Apart from Notch 3, activation of the pathways, as indicated by IHC expression of their components, did not result in differences in terms of DFS or OS.

Epigenetic changes, including altered small non-coding RNAs, appear to be implicated in the pathogenesis of sporadic parathyroid adenomas (PAs). In this study, we investigated the circular RNAs (circRNAs) expression profile in sporadic PAs. Sixteen tissue samples of sporadic PAs, and four samples of normal parathyroid tissue (NPT) were investigated. Sample preparation and microarray hybridization were performed based on the Arraystar’s standard protocols, and circRNAs sequences were predicted by bioinformatics tools. We identified 35 circRNAs that were differentially expressed in sporadic PAs compared to NPT; 22 were upregulated, and 13 were downregulated, according to the pre-defined thresholds of fold-change > 2.0 and p < 0.05. In the subgroup analysis of PAs from male patients (n = 7) compared to PAs from female patients (n = 9), we also find a different expression profile. In particular, 19 circRNAs were significantly upregulated, and four circRNAs were significantly downregulated in male patients, compared to female counterparts. We show here for the first time a differential circRNA expression pattern in sporadic PAs compared to NPT, and a different expression profile in PA samples from male compared to female patients, suggesting an epigenetic role in the PA pathogenesis, and also an effect of gender in the epigenetic regulation of PAs.

Abstract Background - aim: Information on the prognostic role of cytotoxic CD8+ T cells in the era of modern adjuvant CT is limited. The primary objective of the present report is to assess the prognostic impact of CD8+ cells in patients with intermediate or high-risk EBC (T1-3N1-2M0) treated with dds-CT. Secondary endpoints are safety, disease-free survival (DFS) and overall survival (OS). Patients and Methods: Patients (N=1,000) were treated with 4 cycles of Epirubicin, 75mg/m2, and Cyclophophamide, 600mg/m2 every 2 weeks followed by 4 cycles of Docetaxel (D), 100mg/m2 every 3 weeks with G-CSF support in all cycles. Trastuzumab was initiated concurrently with D and continued for a total of 1 year. Hormonal and radiation therapy were given post CT, as indicated. Formalin-fixed paraffin-embedded tumors were available for 642 patients (64.2%) and were centrally assessed for immunohistochemical subtypes (IHC4; N=526), stromal TILs density by morphology (N=636), as well as stromal and intratumoral cytotoxic CD8+ T cell numbers (N=554). TILs and CD8+ were assessed as continuous variables for associations and as 10% increments for outcome. Results: In total, 901/1,000 pts (90.1%) completed 8 cycles of CT. Severe (gradeIII-IV) toxicitiesincludedneutropenia (5.6%), leucopenia (3.6%), lymphopenia (2.1%), hand-footsyndrome (2.1%), and hepatotoxicity (1.8%). Febrileneutropenia occurred in 1.6% of the patients. The 5-year DFS and OS rates were 89.5% and 93.1%, respectively. Luminal A tumors were classified in 26.2%, Luminal B in 35.2%, luminal HER2 in 9.5%; HER2-enriched in 7.2%; and, triple-negative (TNBC) in 21.9% of informative patients. Among subtypes, stromal TILs density was higher in HER2-enriched and TNBC (p<0.001); intratumoral CD8+ values were higher in TNBC (p<0.001); and, stromal CD8+ were higher in HER2-enriched (p=0.034). In all patients, TILs density and intratumoral CD8+ cell numbers were not associated with DFS and OS, while increased stromal CD8+ were marginally associated with prolonged DFS (HR=0.98, 95%CI 0.96-1.00, p=0.066).Adjusted for histological grade, menopausal, ER/PgR and nodal status, higher stromal CD8+ were associated with prolonged DFS (HR=0.98, 95% CI 0.96-1.00, p=0.043). In TNBC, higher stromal TILs density conferred prolonged DFS (HR=0.97, 95%CI 0.94-0.99, p=0.029), which retained its prognostic significance in multivariate analysis (HR=0.97, 95% CI 0.94-1.00, p=0.049). Conclusions: In this study, dds-CT was well tolerated and active in patients with EBC. We confirm the presence of morphologically assessed higher TILs density, and of higher cytotoxic CD8+ T cell numbers in hormone receptor negative EBC, as well as the favorable prognostic impact of higher stromal TILs density in TNBC. In comparison to stromal TILs density, higher stromal CD8+ may confer favorable prognosis irrespectively of EBC subtype. Stromal CD8+ seems to be a marker worth further standardizing for reporting on immune cell infiltrates in EBC. Citation Format: Kourea HP, Koletsa T, Kotoula V, Koliou G-A, Batistatou A, Pentheroudakis G, Arapantoni-Dadioti P, Zagouri F, Bobos M, Sotiropoulou M, Papoudou-Bai A, Chrisafi S, Efstratiou I, Aravantinos G, Nicolaou I, Gogas H, Visvikis A, Christodoulou C, Petraki C, Koutras A, Psyrri A, Pectasides D, Fountzilas G. Prognostic significance of CD8+ tumor-infiltrating lymphocytes (TILs) in patients with early breast cancer (EBC) treated with dose-dense sequential adjuvant chemotherapy (dds-CT). An observational study (ACTRN12616001043426) [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-08-13.

We investigated the predictive and prognostic significance of tumoral messenger RNA levels of vascular endothelial growth factor A (VEGFS) splice variants in metastatic colorectal cancer (mCRC) patients treated with bevacizumab. VEGFA145b had negative predictive significance predominantly in those patients with right-sided primary tumors. All VEGFAxxxb variants were negative prognosticators for patients with right-sided mCRC, whereas VEGFA165b was of favorable prognostic significance in patients with left-sided tumors. Background: Alternative splicing of vascular endothelial growth factor A (VEGFA) results in VEGFAxxxb anti-angiogenic isoforms that fail to activate angiogenesis. Bevacizumab, widely used in patients with metastatic colorectal cancer (CRC), binds both VEGFA and VEGFAxxxb isoforms. Patients and Methods: Formalin-fixed, paraffin-embedded primary tumors from metastatic CRC patients treated with first-line FOLFIRI (leucovorin, 5-fluorouracil, irinotecan, and oxaliplatin) + bevacizumab (n = 285) or FOLFIRI only (n = 75) were collected. The relative expression of VEGFA121a, 121b, 145a, 145b, 165a, and 165b was assessed with custom TaqMan-MGB assays and quantitative PCR. Results: At a median follow-up of 101.5 months, left-sided primary CRC was a favorable prognosticator (median survival, 29.2 vs. 18.2 months; P = .015). Positive high VEGFA145b was an unfavorable factor for progression-free survival (PFS; hazard ratio [HR] = 1.66; 95% confidence interval [CI], 1.13-2.44; P = .009) in patients who received FOLFIRI + bevacizumab, without prognostic significance in FOLFIRI-only patients (HR = 0.70; 95% CI, 0.34-1.44; P = .33). The adverse effect on PFS of 145b was more pronounced in patients with right-sided colon cancer (HR = 2.62; 95% CI, 1.35-5.12; P = .005), especially in those who received bevacizumab (HR = 2.85; 95% CI, 1.31-6.21; P = .008). In patients with right-sided colon primary tumors, isoform 121b correlated with inferior PFS (HR = 1.73; 95% CI, 0.94-3.18; P = .076) and overall survival (OS; HR = 2.0; 95% CI, 1.08-3.72; P = .028). In patients with left-sided primary tumors, positive high 165b correlated with superior PFS (HR = 0.76; 95% CI, 0.59-0.99; P = .044) and OS (HR = 0.68; 95% CI, 0.52-0.90; P = .006). At multivariate analysis, right-sided primary tumor was associated with inferior PFS (HR = 1.28; 95% CI, 1.00-1.64), while 145b consistently retained predictive significance for lack of benefit in PFS with bevacizumab (HR = 1.71; 95% CI, 1.16-2.53). Multivariate analysis for OS showed that VEGFA165b expression was favorable in patients with left-sided but unfavorable in patients with right-sided primary tumors (P-interaction < .001). Conclusion: The antiangiogenic isoform VEGFA145b messenger RNA may predict resistance to bevacizumab. Differences in biological relevance and prognostic significance of various VEGFA isoforms were found for right- versus left-sided primary tumors. (C) 2019 Elsevier Inc. All rights reserved.