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.

Determination of microsatellite instability (MSI)/mismatch repair (MMR) status in cancer has several clinical implications. Our aim was to integrate MSI/MMR status from patients tested in Greece to assess the prevalence of MSI-high (MSI-H)/deficient MMR (dMMR) per tumor type, testing patterns over time and concordance between MSI and MMR status. We retrospectively recorded MSI/MMR testing data of patients with diverse tumor types performed in pathology and molecular diagnostics laboratories across Greece. Overall, 18 of 22 pathology and/or molecular diagnostics laboratories accepted our invitation to participate. In the 18 laboratories located across the country, 7916 tumor samples were evaluated for MSI/MMR status. MSI/MMR testing significantly increased in patients with colorectal cancer (CRC) and other tumor types overtime (p < 0.05). The highest prevalence was reported in endometrial cancer (47 of 225 patients, 20.9%). MSI-H/dMMR was observed in most tumor types, even in low proportions. Among 904 tumors assessed both for MSI and MMR status, 21 had discordant results (overall discordance rate, 2.3%). We reported MSI-H/dMMR prevalence rates in patients with diverse cancers, while demonstrating increasing referral patterns from medical oncologists in the country overtime. The anticipated high rate of concordance between MSI and MMR status in paired analysis was confirmed.

BACKGROUND/AIM:Adenoid cystic carcinoma (ACC) is an aggressive neoplasm even though it has low-grade histological appearance and slow growth. The aim of this study was to identify the immunohistochemical and molecular characteristics of ACC, as well as their correlation with the clinical course of patients.PATIENTS AND METHODS:This is a retrospective multicenter analysis. We included 50 patients diagnosed with ACC in the head and neck between 2000 and 2021. The expression of MYB proto-oncogene transcription factor (MYB), neurotrophic tyrosine kinase receptor (NTRK), human epidermal receptor-2 (HER-2), and Ki-67 was examined through immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). We also performed a clinical follow-up of the patients.RESULTS:The median age of the patients was 58.5 years; moreover, 54% of the patients were male. Compared with female patients, male patients were at a higher risk of both recurrence and death. No HER-2-positive cases were revealed. MYB expression was positive in 28 (56%) cases. However, MYB expression did not significantly affect survival. NTRK expression was positive in eight (16%) cases. NTRK-positive patients had worse overall survival (OS) than NTRK-negative patients (p=0.0246). Additionally, the percentage of NTRK-stained cells was negatively correlated with disease-free survival (p=0.0016) and OS (p=0.0027).CONCLUSION:There was no correlation between MYB positivity and survival. Contrarily, NTRK-positive patients had worse survival, indicating that NTRK is a negative prognostic factor. Tropomyosin receptor kinase inhibitors can be used to treat these patients. Furthermore, MYB-targeted inhibitors are promising therapeutic agents.

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.

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.

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.

Background The shift towards an earlier diagnosis of breast cancer (BC) highlights the need for biomarkers that would identify patients at risk for relapse and metastatic spread and indicate the potential value of additional treatment strategies. Osteopontin (OPN) is a matricellular protein that has been suggested to be a potential biomarker in BC. In the present study, we used archived BC patient samples to assess the clinical utility of OPN. Methods Formalin-fixed paraffin-embedded tumor tissue samples from 975 patients were collected from two large phase III randomized adjuvant chemotherapy trials (HE10/97 and HE10/00) that included patients with high risk BC. All tissue samples were assessed for ER, PgR, Ki67 and HER2 protein expression. OPN protein and mRNA expression was evaluated using immunohistochemistry and quantitative reverse transcription-polymerase chain reaction, respectively. Results OPN mRNA expression data were available for 814 patients, whereas OPN protein expression data were available for 546 patients. The majority of patients were ER/PgR-positive (78.3%), HER2-negative (76.5%) and Ki67-positive (55.2%) and had received adjuvant radiation therapy (76.8%) and hormonal therapy (81.1%). OPN mRNA expression was significantly associated with age (60.9% in high OPN tumors vs. 54.1% in low OPN tumors, p = 0.047), ER/PgR-negative status (25.7 vs. 17.2%, p = 0.004) and BC subtypes (p = 0.021). In addition, high OPN mRNA expression was significantly associated with reduced DFS (HR 1.26, 95% CI 1.00–1.59, Wald’s p = 0.050) and OS (HR 1.37, 95% CI 1.05–1.78, p = 0.019), while it retained its prognostic significance for both DFS (HR 1.39, 95% CI 1.10–1.77, p = 0.007) and OS (HR 1.54, 95% CI 1.61–2.05, p = 0.003) in the multivariate analysis. Conclusions We showed that high OPN mRNA expression is associated with decreased DFS and OS in a large cohort of BC patients treated with adjuvant chemotherapy in a clinical trial setting. Our results suggest that OPN may serve as a prognostic factor and a potential target for therapy. Trial registration Australian New Zealand Clinical Trials Registry; HE10/97 ACTRN12611000506998; HE10/00 ACTRN12609001036202

cancer in females worldwide. Advances in diagnosis and treatment have not decreased the incidence of endometrial cancer. Lately, research has been focused on revealing the molecular and genetic characteristics of endometrial cancer in order to provide new insights in the biology of this entity, leading hopefully to innovating therapies. Research has revealed that epigenetic modifications govern endometrial carcinogenesis. In this review, the epigenetic mechanisms that are involved in endometrial cancer as well as the differences between the different types of endometrial cancer are discussed. The review also refers to the putative therapeutic benefits that hopefully can arise.

Purpose: Endometrial cancer is a very common type of cancer in females worldwide. Advances in diagnosis and treatment have not decreased the incidence of endometrial cancer. Lately, research has been focused on revealing the molecular and genetic characteristics of endometrial cancer in order to provide new insights in the biology of this entity, leading hopefully to innovating therapies. Research has revealed that epigenetic modifications govern endometrial carcinogenesis. In this review, the epigenetic mechanisms that are involved in endometrial cancer as well as the differences between the different types of endometrial cancer are discussed. The review also refers to the putative therapeutic benefits that hopefully can arise.

Background-Aim Early breast cancer is a heterogeneous disease, and, therefore, prognostic tools have been developed to evaluate the risk for distant recurrence. In the present study, we sought to develop a risk for recurrence score (RRS) based on mRNA expression of three proliferation markers in high-risk early breast cancer patients and evaluate its ability to predict risk for relapse and death. In addition the Adjuvant! Online score (AOS) was also determined for each patient, providing a 10-year estimate of relapse and mortality risk. We then evaluated whether RRS or AOS might possibly improve the prognostic information of the clinical treatment score (CTS), a model derived from clinicopathological variables. Methods A total of 1,681 patients, enrolled in two prospective phase III trials, were treated with anthracycline-based adjuvant chemotherapy. Sufficient RNA was extracted from 875 samples followed by multiplex quantitative reverse transcription-polymerase chain reaction for assessing RACGAP1, TOP2A and Ki67 mRNA expression. The CTS, slightly modified to fit our cohort, integrated the prognostic information from age, nodal status, tumor size, histological grade and treatment. Patients were also classified to breast cancer subtypes defined by immunohistochemistry. Likelihood ratio (LR) tests and concordance indices were used to estimate the relative increase in the amount of information provided when either RRS or AOS is added to CTS. Results The optimal RRS, in terms of disease-free survival (DFS) and overall survival (OS), was based on the co-expression of two of the three evaluated genes (RACGAP1 and TOP2A). CTS was prognostic for DFS (p<0.001), while CTS, AOS and RRS were all prognostic for OS (p<0.001, p<0.001 and p = 0.036, respectively). The use of AOS in addition to CTS added prognostic information regarding DFS (LR-Δχ2 8.7, p = 0.003), however the use of RRS in addition to CTS did not. For estimating OS, the use of either AOS or RRS in addition to CTS added significant prognostic information. Specifically, the use of both CTS and AOS had significantly better prognostic value vs. CTS alone (LR-Δχ2 20.8, p<0.001), as well as the use of CTS and RRS vs. CTS alone (LR-Δχ2 4.8, p = 0.028). Additionally, more patients were scored as high-risk by AOS than CTS. According to immunohistochemical subtypes, prognosis was improved in the Luminal A (LR-Δχ2 7.2, p = 0.007) and Luminal B (LR-Δχ2 8.3, p = 0.004) subtypes, in HER2-negative patients (LR-Δχ2 23.4, p<0.001) and in patients with >3 positive nodes (LR-Δχ2 23.9, p<0.001) when AOS was added to CTS. Conclusions The current study has shown a clear benefit in predicting overall survival of high-risk early breast cancer patients when combining CTS with either AOS or RRS. The combination of CTS and AOS adds significant prognostic information compared to CTS alone for DFS, while the combination of CTS with either AOS or RRS has better prognostic value than CTS alone for OS. These findings could possibly add on the information needed for the best risk prediction strategy in high-risk early breast cancer patients in a rather simple and inexpensive way, especially in Luminal A and B subtypes, HER2-negative patients and those with >3 positive nodes.

Gastrointestinal Stromal tumors (GIST) are the most common sarcomas of the gastrointestinal tract, with transformation typically driven by activating mutations of cKIT and less commonly platelet-derived growth factor receptor alpha (PDGFRA). Successful targeting of tyrosine-protein kinase Kit with imatinib, a tyrosine kinase inhibitor, has had a major impact in the survival of patients with GIST in both the adjuvant and metastatic setting. A recent modification of treatment guidelines for patients with localized, high-risk GIST extended the adjuvant treatment duration from 1 year to 3 years. In this paper, we review the clinical data of patients with GIST treated in the Oncology Outpatient Unit of “Attikon” University Hospital and aim to assess which patients are eligible for prolongation of adjuvant imatinib therapy as currently suggested by treatment recommendations.

Abstract Background-Aim: The aim of the present study was to explore the efficacy of dose-dense sequential adjuvant chemotherapy followed by trastuzumab in HER2-positive patients according to the immunohistochemically (IHC) defined subtypes. Patients and methods: A total of 771 formalin-fixed paraffin-embedded (FFPE) tumor tissue samples, prospectively collected from 990 eligible patients with high-risk N0 or N1 operable breast cancer participating in a phase III trial (HE10/05), were centrally assessed in tissue microarrays by IHC for 6 biological markers, that is, estrogen receptor (ER), progesterone receptor (PgR), HER2, Ki67, cytokeratin 5 (CK5) and EGFR. All cases were further evaluated for HER2 amplification by FISH. Patients were classified as: luminal A (ER/PgR-positive, HER2-negative, Ki67low, N=382, 49.5%); luminal B (ER/PgR-positive, HER2-negative, Ki67high, N=136, 17.6%); luminal-HER2 (ER/PgR-positive, HER2-positive, N=125, 16.2%); HER2-enriched (ER-negative, PgR-negative, HER2-positive, N=63, 8.2%); triple-negative (TNBC) (ER-negative, PgR-negative, HER2-negative, N=65, 8.4%); and basal core phenotype (BCP) (TNBC, CK5-positive and/or EGFR-positive, N=53, 6.9%). Results: At a median follow-up of 60.5 months, the 3-year disease-free survival (DFS) and overall survival (OS) rates for the total patient population were 88.3% and 96.0%, respectively. The 3-year DFS rates for luminal A, luminal B, luminal-HER2, HER2-enriched, TNBC and BCP patients were 91.1%, 88.2%, 86.4%, 93.7%, 87.7%, and 89.4%, respectively, while the corresponding 3-year OS rates were 95.8%, 95.6%, 97.6%, 95.2%, 95.4%, and 95.0%, respectively. No significant differences were detected for either 3-year DFS or OS in the immunohistochemically defined subtypes, except a trend for significantly worse DFS in patients with luminal-HER2 tumors compared to patients with HER2-enriched tumors (log-rank, p=0.069). Conclusions: In the post-trastuzumab era, at a relatively short follow-up, the luminal-HER2 patients show a trend for worse DFS compared to patients with HER2-enriched tumors treated with dose-dense sequential adjuvant chemotherapy followed by trastuzumab. No other significant differences were detected, with follow-up however being continued. Citation Format: George Fountzilas, Eleni Timotheadou, Georgia Gourgioti, Petroula Arapantoni-Dadioti, Sotiris Lakis, Anna Batistatou, Triantafyllia Koletsa, Olympia Tzaida, Mattheos Bobos, Alexandra Papoudou-Bai, Eleftheria Tsolaki, Sofia Chrisafi, Elena Fountzilas, Ioannis Efstratiou, Helen Gogas, Flora Zagouri, Dimitrios Pectasides. Prognostic value of immunophenotypically defined subtypes in patients treated with dose-dense sequential adjuvant chemotherapy in the trastuzumab era. A Hellenic Cooperative Oncology Group study [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-09-07.

Newly diagnosed invasive breast cancers should be evaluated for Human Epidermal Growth Factor Receptor 2 (HER2) status by immunohistochemistry (IHC) and/or in situ hybridisation (ISH) to determine eligibility for trastuzumab or other HER2-targeted therapies. Previous reports of high discordance rates between IHC and ISH have raised concerns over the accuracy of HER2 testing, especially when IHC is conducted locally. This study aimed to determine the rate of false-negative IHC results at three pathology centres (one central, two local) in Greece by central retesting of 240 prospectively collected invasive breast cancers scored as IHC 0/1+ at initial testing. All samples were from female patients (median age 58.0 years). Initial IHC tests utilised either the CB11 (159/239; 66.5%) or 4B5 (80/239; 33.5%) antibodies and were scored as 0 in 105/240 cases (43.8%) and 1+ in 135/240 cases (56.3%). All samples were centrally retested by automated silver in situ hybridisation (SISH). Of 237 samples with SISH staining suitable for assessment, 223 (94.1%; 95% confidence interval 90.3–96.5%) were classed as SISH-negative (HER2:chromosome enumeration probe 17 (CEP17) <1.8). Eight tested SISH-positive (HER2:CEP17 >2.2), providing a false-negative rate of 3.4%. A further four samples (1.7%) exhibited equivocal amplification status (HER2:CEP17 1.8–2.2) and two (0.8%) showed polysomy of chromosome 17. The proportion of SISH-negative results did not significantly differ between the IHC 0 and 1+ subgroups (95.2% vs. 93.2%; p=0.505). In conclusion, the low observed rate of false-negative IHC results in this study supports the use of IHC for initial HER2 status assessment in local or central laboratories in Greece.

BACKGROUNDDespite improvement in therapeutic techniques, patients with early-stage laryngeal cancer still recur after treatment. Gene expression prognostic models could suggest which of these patients would be more appropriate for testing adjuvant strategies.MATERIALS AND METHODSExpression profiling using whole-genome DASL arrays was carried out on 56 formalin-fixed paraffin-embedded tumor samples of patients with early-stage laryngeal cancer. We split the samples into a training and a validation set. Using the supervised principal components survival analysis in the first cohort, we identified gene expression profiles that predict the risk of recurrence. These profiles were then validated in an independent cohort.RESULTSGene models comprising different number of genes identified a subgroup of patients who were at high risk of recurrence. Of these, the best prognostic model distinguished between a high- and a low-risk group (log-rank P<0.005). The prognostic value of this model was reproduced in the validation cohort (median disease-free survival: 38 versus 161 months, log-rank P=0.018), hazard ratio=5.19 (95% confidence interval 1.14-23.57, P<0.05).CONCLUSIONSWe have identified gene expression prognostic models that can refine the estimation of a patient's risk of recurrence. These findings, if further validated, should aid in patient stratification for testing adjuvant treatment strategies.