Dose-dense sequential adjuvant chemotherapy in the trastuzumab era: final long-term results of the Hellenic Cooperative Oncology Group Phase III HE10/05 Trial.

BACKGROUND: Dose-dense sequential chemotherapy with anthracyclines and taxanes achieved an 18% reduction of recurrence risk in early breast cancer (BC). The optimal chemotherapy schedule and interval between cycles remain under investigation. METHODS: Overall, 990 patients were randomised to receive either three cycles of epirubicin (E, 110 mg/m2) every 2 weeks followed by 3 cycles of paclitaxel (T, 200 mg/m2) every 2 weeks followed by three cycles of intensified CMF (Control Arm A, E-T-CMF) that was previously used in BC or three cycles of epirubicin followed by three cycles of CMF followed by nine consecutive weekly cycles of docetaxel (wD) 35 mg/m2 (Arm B, E-CMF-wD) or nine consecutive weekly cycles of paclitaxel (wT) 80 mg/m2 (Arm C, E-CMF-wT). Trastuzumab was administered for HER2-positive disease. RESULTS: At a median follow-up of 13.3 years, 330 disease-free survival (DFS) events (33.3%) were reported. DFS and overall survival (OS) did not differ between patients in the combined B and C arms versus arm A either in the entire cohort (HR = 0.90, P = 0.38 and HR = 0.85, P = 0.20) or among trastuzumab-treated patients (HR = 0.69, P = 0.13 and HR = 0.67, P = 0.13). Thirty-four patients (3.4%) developed secondary neoplasms. CONCLUSIONS: Overall, no significant differences in survival were found amongst the studied regimens after a long-term observational period. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry ACTRN12610000151033.

Phase 2 study of cabazitaxel as second-line treatment in patients with HER2-negative metastatic breast cancer previously treated with taxanes-a Hellenic Cooperative Oncology Group (HeCOG) Trial.

BACKGROUND: Cabazitaxel is a novel taxane that might be active in breast cancer resistant to first-generation taxanes. METHODS: The purpose of the current multicentre phase II trial was to evaluate the activity and safety of cabazitaxel, as second-line treatment, in patients with human epidermal growth factor receptor 2 (HER2)-negative metastatic breast cancer (MBC) previously treated with taxanes. The primary endpoint was objective response rate (ORR). RESULTS: Eighty-four patients were enrolled between October 2012 and November 2016. Taxane resistance to previous treatment was detected in 43 cases. The ORR was 22.6% in the intent-to-treat population, 23.3% in taxane-resistant and 20.5% in taxane-non-resistant cases. At a median follow-up of 39.6 months, the median progression-free survival and overall survival were 3.7 months (95% CI 2.2-4.4) and 15.2 months (95% CI 11.3-19.4), respectively. Regarding toxicity, grade 3-4 neutropenia was reported in 22.6% and febrile neutropenia in 6% of the patients, respectively. Two fatal events (one febrile neutropenia and one sepsis) were reported as being related to study treatment. CONCLUSIONS: This phase II trial suggests that cabazitaxel is active as second-line treatment in taxane-pretreated patients with HER2-negative MBC, with manageable toxicity.

Prognostic impact of stromal and intratumoral CD3, CD8 and FOXP3 in adjuvantly treated breast cancer: do they add information over stromal tumor-infiltrating lymphocyte density?

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/mm2) 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.

An MRI study of immune checkpoint inhibitor-induced musculoskeletal manifestations myofasciitis is the prominent imaging finding.

OBJECTIVE: To assess: (i) the prevalence, and clinical and imaging characteristics of immune checkpoint inhibitor (ICI)-induced musculoskeletal immune-related adverse events (ir-AEs) in a prospective manner and (ii) whether serum levels of cytokines associated with the Th1/Th2/Th17 response are differentially expressed in patients with and without musculoskeletal Ir-AEs. METHODS: All patients treated with ICI who developed musculoskeletal manifestations were referred to the Rheumatology Department, and an MRI of the involved area(s) was performed. RESULTS: During the study period, a total of 130 patients were treated with ICIs. Of these, 10 (7.7%) developed ICI-induced Ir-AEs. The median time from ICI treatment since development of symptoms was 2.5 months. Three different patterns of musculoskeletal manifestations were found: (i) prominent joint involvement (n = 3); (ii) prominent 'periarticular' involvement (n = 4). These patients had diffuse swelling of the hands, feet or knees. MRI depicted mild synovitis with more prominent myositis and/or fasciitis in the surrounding tissues in all cases; (iii) myofasciitis (n = 3). Clinically, these patients presented with pain in the knee(s)/thigh(s), whereas MRI depicted myofasciitis of the surrounding muscles. Patients with musculoskeletal ir-AEs had significantly higher oncologic response rates compared with patients not exhibiting musculoskeletal ir-AEs (50% vs 12.5%, respectively, P = 0.0016). Cytokine levels associated with a Th1/Th2/Th17 response were similar between patients with and without musculoskeletal ir-AEs. Overall, symptoms were mild/moderate and responded well to treatment, with no need for ICI discontinuation. CONCLUSION: In our cohort, ICI-induced musculoskeletal manifestations developed in 7.7% of patients. Imaging evidence of myofasciitis was found in most patients, indicating that the muscle/fascia is more frequently involved than the synovium.

The combination of bevacizumab/temsirolimus after first-line anti-VEGF therapy in advanced renal-cell carcinoma: a clinical and biomarker study.

BACKGROUND: Vascular endothelial growth factor (VEGF) targeting represents the standard first-line therapy for metastatic renal-cell carcinoma (mRCC), while blocking the mammalian target of rapamycin (mTOR) is effective in relapsed disease. Since continuing blockade of VEGF may be of value, we studied the combination of bevacizumab with temsirolimus in mRCC patients relapsing after first-line treatment. METHODS: A prospective, phase II study of the combination of bevacizumab (10 mg/kg, every 2 weeks) with temsirolimus (25 mg weekly) in patients with mRCC who failed first-line anti-VEGF treatment. 6-month progression-free survival (PFS) rate was the primary end point. The association of VEGFa, VEGFR2, fibroblast growth factor (FGF) b, platelet-derived growth factor receptor (PDGFR) a and PDGFRb with prognostic factors and outcomes were also studied. RESULTS: 39 patients were enrolled. First-line therapy included: sunitinib (n = 16), bevacizumab/interferon (n = 12), pazopanib (n = 10), sorafenib (n = 1). After a median follow-up of 37 months, 6-month PFS rate was 50.9% [95% confidence interval (CI) 33.8-65.7], median time to progression 6.8 months (95% CI 5.5-9.2) and median overall survival (OS) 18.2 months (95% CI 12.9-27.2). Objective response rate was 27%. The most common AEs were metabolic (33%), renal (8%) and gastrointestinal (GI) (7%). The most common grade 3-5 AEs were GI (18%), infections (14%) and metabolic (25%). Toxicity was the most frequent cause of treatment discontinuation (40%). FGFb levels were associated with OS. CONCLUSIONS: In concert with recent data, our study confirms the efficacy of anti-VEGF/anti-mTOR combination in mRCC relapsing after anti-VEGF therapy. Toxicity was considerable leading to high rate of treatment discontinuations. TRIAL REGISTRATION: ClinicalTrials.gov: NCT01264341.

Prevalent somatic BRCA1 mutations shape clinically relevant genomic patterns of nasopharyngeal carcinoma in Southeast Europe.

Genomic patterns of nasopharyngeal carcinomas (NPCs) have as yet been studied in Southeast Asian (SEA) patients. Here, we investigated genomic patterns of locally advanced NPC Southeast European (SEE) patients treated with chemoradiotherapy. We examined 126 tumors (89% EBV positive) from Greek and Romanian NPC patients with massively parallel sequencing. Paired tumor-cell-rich (TC) and infiltrating-lymphocyte-rich (TILs) samples were available in 19 and paired tumor-germline samples in 68 cases. Top mutated genes were BRCA1 (54% of all tumors); BRCA2 (29%); TP53 (22%); KRAS (18%). Based on the presence and number of mutations and mutated genes, NPC were classified as stable (no mutations, n = 27); unstable (>7 genes with multiple mutations, all BRCA1 positive, n = 21); and of intermediate stability (1-7 singly mutated genes, n = 78). BRCA1 p.Q563* was present in 59 tumors (48%), more frequently from Romanian patients (p < 0.001). No pathogenic germline mutations were identified. NPC exhibited APOBEC3A/B and nucleotide-excision-repair-related mutational signatures. As compared to TC, TILs demonstrated few shared and a higher number of low frequency private mutations (p < 0.001). In multivariate analysis models for progression-free survival, EBV positivity was a favorable prognosticator in stable tumors; BRCA1 mutations were unfavorable only in tumors of intermediate stability. In conclusion, other than described for SEA NPC, somatic BRCA1 mutations were common in SEE NPC; these were shared between TC and TILs, and appeared to affect patient outcome according to tumor genomic stability status. Along with the identified mutational signatures, these novel data may be helpful for designing new treatments for locally advanced NPC.

Association of osteopontin with specific prognostic factors and survival in adjuvant breast cancer trials of the Hellenic Cooperative Oncology Group.

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.

Effects of TP53 and PIK3CA mutations in early breast cancer: a matter of co-mutation and tumor-infiltrating lymphocytes.

The purpose of this study is to investigate whether the outcome of breast cancer (BC) patients treated with adjuvant chemotherapy is affected by co-mutated TP53 and PIK3CA according to stromal tumor-infiltrating lymphocytes (TILs). Paraffin tumors of all clinical subtypes from 1661 patients with operable breast cancer who were treated within 4 adjuvant trials with anthracycline-taxanes chemotherapy were informative for TP53 and PIK3CA mutation status (semiconductor sequencing genotyping) and for stromal TILs density. Disease-free survival (DFS) was examined. TP53 mutations were associated with higher (p < 0.001) and PIK3CA with lower (p = 0.004) TILs in an ER /PgR-specific manner (p < 0.001). Mutations did not affect the favorable DFS of patients with lymphocyte-predominant (LP) BC. Within non-LPBC, PIK3CA-only mutations conferred best, while TP53-PIK3CA co-mutations (6 % of all tumors) conferred worst DFS (HR 0.59; 95 % CI 0.44-0.79; p = 0.001 for PIK3CA-only). TP53-only mutations were unfavorable in patients with lower TILs, while patients with lower TILs performed worse if their tumors carried TP53-only mutations (interaction p = 0.046). Multivariate analysis revealed favorable PIK3CA-only mutations in non-LPBC (HR 0.64; 95 % CI 0.47-0.88; p = 0.007), and unfavorable TP53 mutations in ER/PgRpos/HER2neg (HR 1.55; 95 % CI 1.07-2.24; p = 0.021). Mutations did not interact with TILs in non-LP triple-negative and HER2-positive patients. TP53 and PIK3CA mutations appear to have diverse effects on the outcome of early BC patients, according to whether these genes are co-mutated or not, and for TP53 according to TILs density and ER/PgR-status. These findings need to be considered when evaluating the effect of these two most frequently mutated genes in the context of large clinical trials.

MYC copy gain, chromosomal instability and PI3K activation as potential markers of unfavourable outcome in trastuzumab-treated patients with metastatic breast cancer.

BACKGROUND: There is an unmet need for more efficient patient stratification for receiving trastuzumab in the metastatic breast cancer (mBC) setting, since only part of such patients benefit from the addition of this agent to chemotherapy. The aim of this study was to investigate the prognostic value of biomarkers including MYC and MET in mBC patients treated with trastuzumab-based regimens. METHODS: mBC patients, locally tested as HER2-positive, treated with trastuzumab and chemotherapy between 1998 and 2010 were evaluated. Paraffin tumors (n = 229) were retrospectively centrally assessed by immunohistochemistry (IHC) for HER2, ER, PgR and Ki67; fluorescence in situ hybridization (FISH) for HER2, TOP2A and centromere (CEN) 17, MYC and CEN8, MET and CEN7; qPCR for MYC, MET copy number (CN); and, for PI3K activation (PIK3CA mutations; PTEN and phospho-mTOR protein expression). Increased CEN CN was assessed based on normal cut-offs. Time to progression (TTP) and survival were evaluated from the initiation of trastuzumab as first line treatment. RESULTS: Among all tumors, 90 were HER2-negative upon central testing (ambiguous HER2) and the rest were true HER2-positive. Further, 156 patients presented with mBC upon relapse of pre-treated disease (R-mBC) and 65 were diagnosed at stage IV (de novo mBC). Concordance between FISH and qPCR on gene CN status was fair for MYC (Kappa = 0.458) and absent for MET. The presence of MYC CN gain with qPCR and the absence of PI3K activation were infrequent events (7 and 8 % of evaluable tumors, respectively), while 41 % of tumors had increased CEN CN in one or more chromosomes, indicative of chromosomal instability. The most consistent finding in the entire cohort and in the above patient subgroups with respect to outcome was the unfavourable effect of MYC CN gain, which was retained upon multivariable analysis (e.g., survival in the entire cohort, HR 6.02; 95 % CI 2.67-13.6; p < 0.001). Further unfavourable prognosticators were increased CEN CN in one chromosome in R-mBC but not in de novo mBC (multivariable interaction p = 0.048), PI3K activation in R-mBC (multivariable p = 0.004) and increased Ki67 for patient TTP. CONCLUSIONS: MYC gene copies, centromere status and PI3K activation may adversely impact trastuzumab treated mBC patient outcome and seem worthy validating in larger series.

Evaluation of the prognostic significance of HER family mRNA expression in high-risk early breast cancer: a Hellenic Cooperative Oncology Group (HeCOG) validation study.

BACKGROUND: The aim of the study was to evaluate the prognostic ability of the transcriptional profiling of the HER family genes in early breast cancer, as a validation analysis of another previously published HeCOG study. METHODS: RNA was extracted from 663 formalin-fixed paraffin-embedded (FFPE) tumor tissue samples of high-risk early breast cancer patients enrolled in the randomized HE10/00 trial. Relative mRNA expression of all four HER family members was assessed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). RESULTS: In compliance with our previous study, the overall agreement between qRT-PCR and IHC/FISH for HER2 status determination was good (69%). Likewise, the overall concordance between qRT-PCR and IHC for EGFR status was high (81%). In line with our previously reported data, we demonstrated a positive association between HER2 and HER3 mRNA expression. Similarly, mRNA expression of HER3 and HER4 was positively associated with each other and negatively associated with EGFR. Regarding relationships with clinico-pathological parameters, our findings are also in agreement with our previous results. Generally, increased EGFR and HER2 mRNA expression was related to unfavorable, whereas high HER3 and HER4 mRNA expression was associated with favorable clinico-pathological parameters. In univariate analysis, no significant association between EGFR, HER2 and HER3 mRNA expression and overall survival (OS) or disease-free survival (DFS) was demonstrated. However, high EGFR protein expression was associated with significantly shorter OS (log-rank, p = 0.015). In compliance with our previously published data, increased HER4 mRNA expression had a significantly favorable prognostic value in terms of OS (p = 0.044) and DFS (p = 0.047). In multivariate analysis, among all HER receptors, only EGFR protein expression was found to affect OS (Wald's p = 0.028) and DFS (p = 0.015) independently. Concerning the combined expression of all four HER family receptors, the combination of high EGFR, high HER2, low HER3 and low HER4 mRNA expression was associated with a trend for shorter OS (log-rank, p = 0.065) and significantly worse DFS (p = 0.033), compared with all other co-expression profiles. CONCLUSIONS: These data indicate that qRT-PCR may represent a valid alternative method for evaluating the expression of HER family members in FFPE breast carcinoma tissue samples. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry ACTRN12609001036202.

Comparison of filgrastim and pegfilgrastim to prevent neutropenia and maintain dose intensity of adjuvant chemotherapy in patients with breast cancer.

PURPOSE: The aim of this study was to compare the effectiveness of prophylactic single fixed dose of pegfilgrastim and daily administration of filgrastim on febrile neutropenia (FN), severe neutropenia, treatment delay, and dose reduction in patients with breast cancer receiving dose-dense adjuvant chemotherapy. METHODS: A retrospective cohort study with 1058 breast cancer patients matched by age and chemotherapy was conducted. The primary endpoints were FN, severe (grade 3, 4) neutropenia, dose reduction (>10 % reduction of the dose planned), and treatment delay (dose given more than 2 days later). RESULTS: Eighteen episodes of FN (3.4%) in the filgrastim group and 23 (4.3%) in the pegfilgrastim group (p = 0.500) were recorded. More than half of the total episodes (27/41) occurred during the first 4 cycles of treatment. Patients who received filgrastim were almost three times more likely to experience a severe neutropenia episode and were significantly more likely to experience a dose reduction (18.5%) compared to those who received pegfilgrastim (10.8%) (p < 0.001). The percentage of patients, who received their planned dose on time, was significantly lower in patients receiving filgrastim (58%) compared to those receiving pegfilgrastim (72.4%, p < 0.001). CONCLUSIONS: No significant difference was detected on FN rate between daily administration of filgrastim and single administration of pegfilgrastim. However, patients receiving pegfilgrastim had a significantly lower rate of severe neutropenia, as well as dose reduction and treatment delay, thus, achieving a higher dose density.

Dose-dense sequential adjuvant chemotherapy followed, as indicated, by trastuzumab for one year in patients with early breast cancer: first report at 5-year median follow-up of a Hellenic Cooperative Oncology Group randomized phase III trial.

BACKGROUND: Dose-dense sequential chemotherapy including anthracyclines and taxanes has been established in the adjuvant setting of high-risk operable breast cancer. However, the preferable taxane and optimal schedule of administration in a dose-dense regimen have not been defined yet. METHODS: From July 2005 to November 2008, 1001 patients (990 eligible) were randomized to receive, every 2 weeks, 3 cycles of epirubicin 110 mg/m2 followed by 3 cycles of paclitaxel 200 mg/m2 followed by 3 cycles of intensified CMF (Arm A; 333 patients), or 3 cycles of epirubicin followed by 3 cycles of CMF, as in Arm A, followed 3 weeks later by 9 weekly cycles of docetaxel 35 mg/m2 (Arm B; 331), or 9 weekly cycles of paclitaxel 80 mg/m2 (Arm C; 326). Trastuzumab was administered for one year to HER2-positive patients post-radiation. RESULTS: At a median follow-up of 60.5 months, the 3-year disease-free survival (DFS) rate was 86%, 90% and 88%, for Arms A, B and C, respectively, while the 3-year overall survival (OS) rate was 96% in all arms. No differences were found in DFS or OS between the combined B and C Arms versus Arm A (DFS: HR = 0.81, 95% CI: 0.59-1.11, P = 0.20; OS: HR = 0.84, 95% CI: 0.55-1.30, P = 0.43). Among the 255 patients who received trastuzumab, 189 patients (74%) completed 1 year of treatment uneventfully. In all arms, the most frequently reported severe adverse events were neutropenia (30% vs. 27% vs. 26%) and leucopenia (12% vs. 13% vs. 12%), while febrile neutropenia occurred in fifty-one patients (6% vs. 4% vs. 5%). Patients in Arm A experienced more often severe pain (P = 0.002), neurological complications (P = 0.004) and allergic reactions (P = 0.004), while patients in Arm B suffered more often from severe skin reactions (P = 0.020). CONCLUSIONS: No significant differences in survival between the regimens were found in the present phase III trial. Taxane scheduling influenced the type of severe toxicities. HER2-positive patients demonstrated comparable 3-year DFS and OS rates with those reported in other similar studies. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry ACTRN12610000151033.

ß-Blocker Use and Risk of Recurrence in Patients with Early Breast Cancer.

AIM: We evaluated whether the concurrent ß-blocker use in early breast cancer patients influenced the outcome in terms of preventing tumor recurrence after adjuvant chemotherapy. METHODS: We retrospectively reviewed the medical records of 610 patients with breast cancer. Thereafter, we compared overall disease-free survival (DFS) between ß-blocker users and nonusers. RESULTS: Those not receiving ß-blockers had a relatively longer mean DFS (10.8 vs. 9.7 years), although the difference did not reach statistical significance (p = 0.651). When the survival analysis was adjusted for age, tumor stage, hormone receptor status and HER2 status, the results remained unaltered, suggesting that ß-blocker use did not significantly improve overall DFS (HR, 0.849; 95% CI, 0.537-1.343; p = 0.485). CONCLUSION: Our findings failed to confirm previous results indicating a potential antitumor effect of ß-blockers.

alphaB-crystallin is a marker of aggressive breast cancer behavior but does not independently predict for patient outcome: a combined analysis of two randomized studies.

BACKGROUND: alphaB-crystallin is a small heat shock protein that has recently been characterized as an oncoprotein correlating with the basal core phenotype and with negative prognostic factors in breast carcinomas. The purpose of this study was to evaluate alphaB-crystallin with respect to clinicopathological parameters and the outcome of patients with operable high-risk breast cancer. METHODS: A total of 940 tumors were examined, derived from an equal number of patients who had participated in two randomized clinical trials (paclitaxel-containing regimen in 793 cases). Immunohistochemistry for ER, PgR, HER2, Ki67, CK5, CK14, CK17, EGFR, alphaB-crystallin, BRCA1 and p53 was performed. BRCA1 mutation data were available in 89 cases. RESULTS: alphaß-crystallin was expressed in 170 cases (18.1%) and more frequently in triple-negative breast carcinomas (TNBC) (45% vs. 14.5% non-TNBC, p < 0.001). alphaB-crystallin protein expression was significantly associated with high Ki67 (Pearson chi-square test, p < 0.001), p53 (p = 0.002) and basal cytokeratin protein expression (p < 0.001), BRCA1 mutations (p = 0.045) and negative ER (p < 0.001) and PgR (p < 0.001). Its overexpression, defined as >30% positive neoplastic cells, was associated with adverse overall survival (Wald's p = 0.046). However, alphaB-crystallin was not an independent prognostic factor upon multivariate analysis. No interaction between taxane-based therapy and aß-crystallin expression was observed. CONCLUSIONS: In operable high-risk breast cancer, alphaB-crystallin protein expression is associated with poor prognostic features indicating aggressive tumor behavior, but it does not seem to have an independent impact on patient survival or to interfere with taxane-based therapy. TRIAL REGISTRATIONS: ACTRN12611000506998 (HE10/97 trial) and ACTRN12609001036202 (HE10/00 trial).

Current Status and Future Perspectives of Antibody-Drug Conjugates in Hormone Receptor-Positive Breast Cancer.

Breast cancer is the most common cancer type in women. The vast majority of breast cancer patients have hormone receptor-positive (HR+) tumors. In advanced HR+ breast cancer, the combination of endocrine therapy with cyclin-dependent kinase 4/6 (CDK4/6) inhibitors is considered the standard of care in the front-line setting. Nevertheless, resistance to hormonal therapy and CDK4/6 inhibitors eventually occurs, leading to progression of the disease. Antibody-drug conjugates (ADCs) comprise a promising therapeutic choice with significant efficacy in patients with HR+ breast cancer, which is resistant to endocrine treatment. ADCs typically consist of a cytotoxic payload attached by a linker to a monoclonal antibody that targets a specific tumor-associated antigen, offering the advantage of a more selective delivery of chemotherapy to cancer cells. In this review, we focus on the ADC mechanisms of action, their toxicity profile and therapeutic uses as well as on related biomarkers and future perspectives in advanced HR+ breast cancer.

Ten-year clinical outcome, toxicity and compliance of dose-dense sequential adjuvant administration of cyclophosphamide & epirubicin followed by docetaxel in patients with early breast cancer: A hellenic cooperative oncology group observational study (HE 10/10) with concurrent investigation of significance of tumor infiltrating lymphocytes.

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.

Evaluation of the prognostic role of centromere 17 gain and HER2/topoisomerase II alpha gene status and protein expression in patients with breast cancer treated with anthracycline-containing adjuvant chemotherapy: pooled analysis of two Hellenic Cooperative Oncology Group (HeCOG) phase III trials.

BACKGROUND: The HER2 gene has been established as a valid biological marker for the treatment of breast cancer patients with trastuzumab and probably other agents, such as paclitaxel and anthracyclines. The TOP2A gene has been associated with response to anthracyclines. Limited information exists on the relationship of HER2/TOP2A gene status in the presence of centromere 17 (CEP17) gain with outcome of patients treated with anthracycline-containing adjuvant chemotherapy. METHODS: Formalin-fixed paraffin-embedded tumor tissue samples from 1031 patients with high-risk operable breast cancer, enrolled in two consecutive phase III trials, were assessed in a central laboratory by fluorescence in situ hybridization for HER2/TOP2A gene amplification and CEP17 gain (CEP17 probe). Amplification of HER2 and TOP2A were defined as a gene/CEP17 ratio of >2.2 and ≥2.0, respectively, or gene copy number higher than 6. Additionally, HER2, TopoIIa, ER/PgR and Ki67 protein expression was assessed by immunohistochemistry (IHC) and patients were classified according to their IHC phenotype. Treatment consisted of epirubicin-based adjuvant chemotherapy followed by hormonal therapy and radiation, as indicated. RESULTS: HER2 amplification was found in 23.7% of the patients and TOP2A amplification in 10.1%. In total, 41.8% of HER2-amplified tumors demonstrated TOP2A co-amplification. The median (range) of HER2, TOP2A and CEP17 gain was 2.55 (0.70-45.15), 2.20 (0.70-26.15) and 2.00 (0.70-26.55), respectively. Forty percent of the tumors had CEP17 gain (51% of those with HER2 amplification). Adjusting for treatment groups in the Cox model, HER2 amplification, TOP2A amplification, CEP17 gain and HER2/TOP2A co-amplification were not associated with time to relapse or time to death. CONCLUSION: HER2 amplification, TOP2A amplification, CEP17 gain and HER2/TOP2A co-amplification were not associated with outcome in high-risk breast cancer patients treated with anthracycline-based adjuvant chemotherapy. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry (ANZCTR) ACTRN12611000506998 and ACTRN12609001036202.

The combination of antitumor drugs, exemestane and erlotinib, induced resistance mechanism in H358 and A549 non-small cell lung cancer (NSCLC) cell lines.

Abstract Context: Estrogens in non-small-cell lung cancer (NSCLC) are important, and their interaction with epidermal growth factor receptor (EGFR) might be crucial. Objective: This study investigates the effect of exemestane, an aromatase inhibitor, and erlotinib, an EGFR inhibitor, on human NSCLC cell lines; H23, H358 and A549. Materials and methods: A cell proliferation assay was used for measuring cell number, apoptosis assay for detecting apoptosis and necrosis and immunoblotting for beclin-1 and Bcl-2 proteins detection. An immunofluorescence assay was used for EGFR localization. A migration assay and zymography were used for cell motility and metalloproteinases (MMPs) expression, respectively. Results: Exemestane, erlotinib or their combination decreased cell proliferation and increased apoptosis. Exemestane's half maximal inhibitory concentration (IC50) was 50 µM for H23 and H358 cells and 20 µM for A549. The IC50 of erlotinib was 25 µM for all cell lines. Apoptosis increase induced by exemestane was 58.0 (H23), 186.3 (H358) and 34.7% (A549) and by erlotinib was 16.7 (H23), 65.3 (H358) and 66.3% (A549). A synergy effect was observed only in H23 cells. Noteworthy, the combination of exemestane and erlotinib decreased beclin-1 protein levels (32.3 ± 19.2%), an indicator of autophagy, in H23 cells. The combination of exemestane and erlotinib partially reversed the EGFR translocation to mitochondria and decreased MMP levels and migration. Discussion and conclusions: The benefit from a dual targeting of aromatase and EGFR seems to be regulated by NSCLC cell content. The diverse responses of cells to agents might be influenced by the dominance of certain molecular pathways.

Health-related quality of life in patients with solid tumors receiving implantable venous access ports for chemotherapy: A prospective randomized controlled trial.

PURPOSE: Implantable venous access ports are widely used in patients receiving chemotherapy, but there is still scarce evidence about any patient-reported outcome measures. This prospective randomized controlled trial examined the impact on patients' quality-of-life following the placement of an implantable port device for long-term chemotherapy treatment. METHOD: A total of 120 chemotherapy naïve adult outpatients scheduled to receive chemotherapy (duration ≥12 weeks) for solid tissue tumors in a single academic oncology unit were randomly allocated (n = 60 in each arm) between radiologically guided insertion of an implantable venous access port (PORT arm) or standard repeated peripheral venous access (Control arm). Health-related quality-of-life scores (HRQoL) were assessed with the EQ-5D-5L and the oncology-specific EORTC QLQ-C30 (version 3.0) questionnaires at baseline, 3- and 6-months post randomization. Non-parametric tests were applied and differences between medians (Δ) are reported because of skewed-left HRQoL data. RESULTS: Baseline clinical and demographic characteristics were well balanced between the two groups. There were no complications during insertion and no infection or device failure in the PORT subjects through the 6-month follow-up. The functional and symptom scales of the EORTC QLQ-C30 questionnaire were similar between both study arms at all time intervals. The EORTC QLQ-C30 global health status was significantly improved in the PORT subjects both at 3 months (Δ: 8.3 out of 100; P = 0.04) and 6 months follow-up (Δ: 16.7 out of 100; P = 0.003). Changes in EQ-5D-5L scores were significantly improved at 6 months in the PORT arm compared to control (Δ: 0.074 out of 1; P = 0.01). CONCLUSIONS: Implantable venous access ports may confer significantly improved patient-reported quality-of-life benefits in patients receiving chemotherapy for solid tissue tumors.

Small Extracellular Vesicles (sEVs) Biogenesis Molecular Players Are Associated with Clinical Outcome of Colorectal Cancer Patients.

A growing number of studies have shed light on the role of small extracellular vesicles (sEVs), including exosomes, in colorectal cancer (CRC). Available data regarding the clinical significance of molecular players in CRC, implicated in sEVs biogenesis, is limited. In this study, we assessed the expression of the most important genes which are implicated in sEVs biogenesis and their association with sEVs plasma levels, investigated with a double sandwich ELISA assay, as well as with the clinical outcome of patients with CRC. Our study shows that RAB27A, RAB27B, RAB2B, and RAB3B mRNA levels were lower in tumor tissues compared to tumor adjacent, non-malignant tissues (p < 0.001, p = 0.009, p = 0.011, and p < 0.001, respectively). In addition, high tumor expression of RAB27A, RAB27B, RAB9A, RAB11B, and STX1A was favorable of a 5-year survival (p = 0.038, p = 0.015, p = 0.008, p = 0.002, and p = 0.028, respectively). Furthermore, patients with adenomas had lower overall plasma sEVs concentrations, compared to healthy volunteers (p = 0.026), while no statistically significant differences were observed in the overall or tumor-derived plasma sEVs concentration (p = 0.885 and p = 0.330, respectively) of CRC patients. In conclusion, sEVs biogenesis has a potentially significant role in CRC, with RAB27A, RAB27B, RAB9A, RAB11B, and STX1A having a promising role in survival outcomes.