A Comprehensive Molecular Analysis of in Vivo Isolated EpCAM-Positive Circulating Tumor Cells in Breast Cancer.

BACKGROUND: Circulating tumor cell (CTC) analysis is highly promising for liquid biopsy-based molecular diagnostics. We undertook a comprehensive molecular analysis of in vivo isolated CTCs in breast cancer (BrCa). METHODS: In vivo isolated CTCs from 42 patients with early and 23 patients with metastatic breast cancer (MBC) were prospectively collected and analyzed for gene expression, DNA mutations, and DNA methylation before and after treatment. 19 healthy donor (HD) samples were analyzed as a control group. In identical blood draws, CTCs were enumerated using CellSearch® and characterized by direct IF staining. RESULTS: All 19 HD samples were negative for CK8, CK18, CK19, ERBB2, TWIST1, VEGF, ESR1, PR, and EGFR expression, while CD44, CD24, ALDH1, VIM, and CDH2 expression was normalized to B2M (reference gene). At least one gene was expressed in 23/42 (54.8%) and 8/13 (61.5%) CTCs in early BrCa before and after therapy, and in 20/23 (87.0%) and 5/7 (71.4%) MBC before and after the first cycle of therapy. PIK3CA mutations were detected in 11/42 (26.2%) and 3/13 (23.1%) in vivo isolated CTCs in early BrCa before and after therapy, and in 11/23 (47.8%) and 2/7 (28.6%) MBC, respectively. ESR1 methylation was detected in 5/32 (15.7%) and 1/10 (10.0%) CTCs in early BrCa before and after therapy, and in 3/15(20.0%) MBC before the first line of therapy. The comprehensive molecular analysis of CTC revealed a higher sensitivity in relation to CellSearch or IF staining when based on creatine kinase selection. CONCLUSIONS: In vivo-CTC isolation in combination with a comprehensive molecular analysis at the gene expression, DNA mutation, and DNA methylation level comprises a highly powerful approach for molecular diagnostic applications using CTCs.

Glycosylation Modulates Plasma Membrane Trafficking of CD24 in Breast Cancer Cells.

In breast cancer, expression of Cluster of Differentiation 24 (CD24), a small GPI-anchored glycoprotein at the cell periphery, is associated with metastasis and immune escape, while its absence is associated with tumor-initiating capacity. Since the mechanism of CD24 sorting is unknown, we investigated the role of glycosylation in the subcellular localization of CD24. Expression and localization of wild type N36- and/or N52-mutated CD24 were analyzed using immunofluorescence in luminal (MCF-7) and basal B (MDA-MB-231 and Hs578T) breast cancer cells lines, as well as HEK293T cells. Endogenous and exogenously expressed wild type and mutated CD24 were found localized at the plasma membrane and the cytoplasm, but not the nucleoplasm. The cell lines showed different kinetics for the sorting of CD24 through the secretory/endocytic pathway. N-glycosylation, especially at N52, and its processing in the Golgi were critical for the sorting and expression of CD24 at the plasma membrane of HEK293T and basal B type cells, but not of MCF-7 cells. In conclusion, our study highlights the contribution of N-glycosylation for the subcellular localization of CD24. Aberrant N-glycosylation at N52 of CD24 could account for the lack of CD24 expression at the cell surface of basal B breast cancer cells.

BRCA1 and BRCA2 germline testing in Cretan isolates reveals novel and strong founder effects.

Germline BRCA1 and BRCA2 loss-of-function variants have been linked to increased breast and ovarian cancer risk, with more than 5,000 distinct pathogenic variants being reported worldwide. Among individuals of Greek descent, the BRCA1/2 variant spectrum is heterogeneous, but characterized by strong founder effects. As patients from certain geographical regions of Greece (like Crete) were underrepresented in previous studies, we hypothesized that isolated Cretans, a southern Greece islanders' population with distinct demographic, cultural and genetic features, could harbor founder BRCA1/2 mutations. A total of 304 breast or/and ovarian cancer patients of Cretan descent, fulfilling NCCN criteria for genetic testing, were tested by NGS or Sanger sequencing, followed by MLPA. Haplotype analysis was subsequently performed to investigate potential founder effects of recurrent alleles. Overall, 16.5% (50/304) of the tested patients carried 22 different pathogenic variants; 48% in BRCA1, 52% in BRCA2. Three variants, namely two in BRCA2 (Δexons 12 and 13 and c.7806-2A>T) and one in BRCA1 (c.5492del), constituting approximately half (48%) of all detected pathogenic variants, were shown to have a founder effect, with all carriers sharing common haplotypes. Remarkably, these variants were confined to Cretans and have not been identified in other regions of Greece. The high prevalence of specific BRCA1/2 pathogenic variants among Cretans, provides the possibility of cost- and time-efficient screening of the Cretan population. Integrating this knowledge in local public health services may have a significant impact on cancer prevention, and may serve as a starting point for the implementation of testing on a population level.

Disease-free survival as a surrogate for overall survival in patients with HER2-positive, early breast cancer in trials of adjuvant trastuzumab for up to 1 year: a systematic review and meta-analysis.

BACKGROUND: Although frequently used as a primary endpoint, disease-free survival has not been validated as a surrogate for overall survival in early breast cancer. We investigated this surrogacy in the adjuvant setting of treatment with anti-HER2 antibodies. METHODS: In a systematic review and meta-analysis, we identified published and non-published randomised controlled trials with completed accrual and available disease-free survival and overall survival results for the intention-to-treat population as of September 2016. Bibliographic databases (MEDLINE, Embase, and Cochrane Central Register of Controlled Trials), clinical trial registries (Clinicaltrials.gov, EU Clinical Trials Register, WHO International Clinical Trials Registry Platform, and PharmNet.Bund), and trial registries from relevant pharmaceutical companies were searched. Eligibility for treatment of HER2-positive early breast cancer required at least one group to have an anti-HER antibody treatment (ie, trastuzumab, pertuzumab, or trastuzumab emtansine) planned for 12 months, and at least one control arm with chemotherapy without the antibody, a lower total dose or duration of the antibody, or observation alone. Units of analysis were contrasts: two-group trials gave rise to one contrast, whereas trials with more than two groups gave rise to more than one contrast. We excluded trials enrolling patients with recurrent, metastatic, or non-invasive disease, and those testing neoadjuvant therapy exclusively. Our primary objective was to estimate patient-level and trial-level correlations between disease-free survival and overall survival. We measured the association between disease-free survival and overall survival using Spearman's correlation coefficient (rs), and the association between hazard ratios (HRs) for disease-free survival and overall survival using R2. We computed the surrogate threshold effect, the maximum HR for disease-free survival that statistically predicts an HR for overall survival less than 1·00 in a future trial. FINDINGS: Eight trials (n=21 480 patients) gave rise to a full set (12 contrasts). Patient-level associations between disease-free and overall survival were strong (rs=0·90 [95% CI 0·89-0·90]). Trial-level associations gave rise to values of R2 of 0·75 (95% CI 0·50-1·00) for the full set. Subgroups defined by nodal status and hormone receptor status yielded qualitatively similar results. Depending on the expected number of deaths in a future trial, the surrogate threshold effects ranged from 0·56 to 0·81, based on the full set. INTERPRETATION: These findings suggest that it is appropriate to continue to use disease-free survival as a surrogate for overall survival in trials in HER-2-positive, early breast cancer. The key limitation of this study is the dependence of its results on the trials included and on the existence of an outlying trial. FUNDING: Roche Pharma AG.

Expression of truncated human epidermal growth factor receptor 2 on circulating tumor cells of breast cancer patients.

INTRODUCTION: The truncated form of human epidermal growth factor receptor 2 (p95HER2) lacks the HER2 extracellular domain and has been associated with poor prognosis and resistance to trastuzumab. In the present study, the expression of p95HER2 was investigated on circulating tumor cells (CTCs) from breast cancer patients. METHODS: Triple-staining immunofluorescent experiments were performed on peripheral blood mononuclear cells' (PBMCs) cytospins obtained from patients with early (n = 24) and metastatic (n = 37) breast cancer. Cells were stained with the pancytokeratin (A45-B/B3) antibody coupled with antibodies against the extracellular (ECD) and the intracellular (ICD) domains of HER2. Slides were analyzed with either confocal laser scanning microscopy or with the Ariol system. RESULTS: HER2-positive CTCs were identified in 55.6 % of early and 65.2 % of metastatic CTC-positive breast cancer patients. p95HER2-positive CTCs were identified in 11.1 % of early and 39.1 % of metastatic breast cancer patients (p = 0.047). In 14 patients with metastatic HER2-positive breast cancer, CTCs were also analyzed before and after first-line trastuzumab therapy. Trastuzumab reduced the percentage of patients with full-length HER2-positive CTCs from 70 % at baseline to 50 % (p = 0.035) after treatment while increased the percentage of patients with p95HER2-positive CTCs from 40 % to 63 %. Moreover, the overall survival of metastatic patients with p95HER2-positive CTCs was significantly decreased (p = 0.03). CONCLUSIONS: p95HER2-positive CTCs can be detected in both early and metastatic breast cancer patients. Their incidence is increased in the metastatic setting and their presence is associated with poor survival. Longitudinal studies during anti-HER2 treatment are required to determine the clinical relevance of p95HER2-expressing CTCs.

Variable expression levels of keratin and vimentin reveal differential EMT status of circulating tumor cells and correlation with clinical characteristics and outcome of patients with metastatic breast cancer.

BACKGROUND: CTCs expressing variable levels of epithelial and mesenchymal markers in breast cancer have previously been reported. However, no information exists for keratin expression levels of CTCs in association with disease status, whereas assays for the characterization of transitional EMT phenotypes of CTCs in breast cancer are rather lacking. We investigated the correlation between keratin expression of CTCs and patients' outcome and characterized the EMT status of CTCs via the establishment of a numerical "ratio" value of keratin and vimentin expression levels on a single cell basis. METHODS: Keratin expression was evaluated in 1262 CTCs from 61 CTC-positive patients with metastatic breast cancer, using analysis of images obtained through the CellSearch System. For the determination of vimentin/keratin (vim/K) ratios, expression levels of keratin and vimentin were measured in cytospin preparations of luminal (MCF-7 and T47D) and basal (MDA.MB231 and Hs578T) breast cancer cell lines and 110 CTCs from 5 CTC-positive patients using triple immunofluorescence laser scanning microscopy and image analysis. RESULTS: MCF-7 and T47D displayed lower vim/K ratios compared to MDA.MB231 and Hs578T cells, while MCF-7 cells that had experimentally undergone EMT were characterized by varying intermediate vim/K ratios. CTCs were consisted of an heterogeneous population presenting variable vim/K values with 46% of them being in the range of luminal breast cancer cell lines. Keratin expression levels of CTCs detected by the CellSearch System correlated with triple negative (p = 0.039) and ER-negative (p = 0.025) breast cancer, and overall survival (p = 0.038). CONCLUSIONS: Keratin expression levels of CTCs correlate with tumor characteristics and clinical outcome. Moreover, CTCs display significant heterogeneity in terms of the degree of EMT phenotype that probably reflects differential invasive potential. The assessment of the vim/K ratios as a surrogate marker for the EMT status of CTCs merits further investigation as a prognostic tool in breast cancer.

Evaluation of proliferation and apoptosis markers in circulating tumor cells of women with early breast cancer who are candidates for tumor dormancy.

INTRODUCTION: Clinical dormancy is frequently observed in breast cancer. In the present study, we aimed to characterize circulating tumor cells (CTCs) in dormancy candidates (DC) with early breast cancer in terms of proliferation and apoptosis. METHODS: Cytospins of peripheral blood mononuclear cells (PBMCs) were obtained from DC (n = 122) who were disease-free for at least 5 years and from metastatic patients (n = 40) who relapsed more than 5 years after surgery. Sequential samples from eight DC (n = 36) who maintained a prolonged disease-free status and from eight DC (n = 27) presenting late relapse during follow-up, were also analyzed. PBMCs were triple stained with a pancytokeratin, antibody along with anti-Ki67 and anti-M30 antibodies as proliferation and apoptosis markers, respectively. RESULTS: CTCs were identified in 40 (33%) of 122 DC and in 15 (37.5%) of 40 metastatic patients. In total, twenty-five (62.5%) DC had exclusively dormant (Ki67(-)/M30(-)), seven (17.5%) had proliferative Ki67(+)/M30(-), four (10%) had apoptotic Ki67(-)/M30(+) and four (10%) had both phenotypes of proliferative and apoptotic CTCs. In comparison, 53.4% of CTC-positive metastatic patients had exclusively dormant and 46.6% had proliferative CTCs; none had apoptotic CTCs (P = 0.039). Among all CTCs detected in DC patients, 82.4% were dormant, whereas in the nondormant population, 32.5% were proliferative and 67.5% apoptotic. The respective percentages in metastatic patients were 59.1%, 100% and 0% (P <0.0001). Moreover, apoptotic CTCs prevailed among nondormant CTCs detected in sequential samples from DC who remained in a prolonged disease-free status compared to those presenting late relapse during follow-up (70.6% versus 43.5% (P = 0.0002)). CONCLUSIONS: The apoptotic index of CTCs is increased during clinical dormancy, whereas the proliferation index is increased on relapse. In addition, apoptotic CTCs are more frequently encountered during follow-up in DC patients who remain disease-free compared to those with subsequent late relapse, suggesting that monitoring proliferation and apoptosis in CTCs during clinical dormancy merits further investigation as a tool for predicting late disease recurrence.

International study on inter-reader variability for circulating tumor cells in breast cancer.

INTRODUCTION: Circulating tumor cells (CTCs) have been studied in breast cancer with the CellSearch® system. Given the low CTC counts in non-metastatic breast cancer, it is important to evaluate the inter-reader agreement. METHODS: CellSearch® images (N = 272) of either CTCs or white blood cells or artifacts from 109 non-metastatic (M0) and 22 metastatic (M1) breast cancer patients from reported studies were sent to 22 readers from 15 academic laboratories and 8 readers from two Veridex laboratories. Each image was scored as No CTC vs CTC HER2- vs CTC HER2+. The 8 Veridex readers were summarized to a Veridex Consensus (VC) to compare each academic reader using % agreement and kappa (κ) statistics. Agreement was compared according to disease stage and CTC counts using the Wilcoxon signed rank test. RESULTS: For CTC definition (No CTC vs CTC), the median agreement between academic readers and VC was 92% (range 69 to 97%) with a median κ of 0.83 (range 0.37 to 0.93). Lower agreement was observed in images from M0 (median 91%, range 70 to 96%) compared to M1 (median 98%, range 64 to 100%) patients (P < 0.001) and from M0 and <3CTCs (median 87%, range 66 to 95%) compared to M0 and ≥3CTCs samples (median 95%, range 77 to 99%), (P < 0.001). For CTC HER2 expression (HER2- vs HER2+), the median agreement was 87% (range 51 to 95%) with a median κ of 0.74 (range 0.25 to 0.90). CONCLUSIONS: The inter-reader agreement for CTC definition was high. Reduced agreement was observed in M0 patients with low CTC counts. Continuous training and independent image review are required.

Co-expression of putative stemness and epithelial-to-mesenchymal transition markers on single circulating tumour cells from patients with early and metastatic breast cancer.

BACKGROUND: The detection of circulating tumor cells (CTCs) in peripheral blood (PB) of patients with breast cancer predicts poor clinical outcome. Cancer cells with stemness and epithelial-to-mesenchymal transition (EMT) features display enhanced malignant and metastatic potential. A new methodology was developed in order to investigate the co-expression of a stemness and an EMT marker (ALDH1 and TWIST, respectively) on single CTCs of patients with early and metastatic breast cancer. METHODS: Triple immunofluorescence using anti-pancytokeratin (A45-B/B3), anti-ALDH1 and anti-TWIST antibodies was performed in cytospins prepared from hepatocellular carcinoma HepG2 cells and SKBR-3, MCF-7 and MDA.MB.231 breast cancer cell lines. Evaluation of ALDH1 expression levels (high, low or absent) and TWIST subcellular localization (nuclear, cytoplasmic or absent) was performed using the ARIOL system. Cytospins prepared from peripheral blood of patients with early (n = 80) and metastatic (n = 50) breast cancer were analyzed for CTC detection (based on pan-cytokeratin expression and cytomorphological criteria) and characterized according to ALDH1 and TWIST. RESULTS: CTCs were detected in 13 (16%) and 25 (50%) patients with early and metastatic disease, respectively. High ALDH1 expression (ALDH1high) and nuclear TWIST localization (TWISTnuc) on CTCs was confirmed in more patients with metastatic than early breast cancer (80% vs. 30.8%, respectively; p = 0.009). In early disease, ALDH1low/neg CTCs (p = 0.006) and TWISTcyt/neg CTCs (p = 0.040) were mainly observed. Regarding co-expression of these markers, ALDH1high/TWISTnuc CTCs were more frequently evident in the metastatic setting (76% vs. 15.4% of patients, p = 0.001; 61.5% vs. 12.9% of total CTCs), whereas in early disease ALDH1low/neg/TWISTcyt/neg CTCs were mainly detected (61.5% vs. 20% of patients, p = 0.078; 41.9% vs. 7.7% of total CTCs). CONCLUSIONS: A new assay is provided for the evaluation of ALDH1 and TWIST co-expression at the single CTC-level in patients with breast cancer. A differential expression pattern for these markers was observed both in early and metastatic disease. CTCs expressing high ALDH1, along with nuclear TWIST were more frequently detected in patients with metastatic breast cancer, suggesting that these cells may prevail during disease progression.

Advances in circulating tumor cells (ACTC): from basic research to clinical practice.

The first 'Advances in Circulating Tumor Cells (ACTC): from Basic Research to Clinical Practice' meeting was held in Athens, Greece, September 26-29, 2012 (abstracts, presentations and a more detailed meeting report are freely available online: http://www.actc2012.org). We summarize in this report most major findings presented and the main conclusions derived during the expert panel sessions.

Pemetrexed versus erlotinib in pretreated patients with advanced non-small cell lung cancer: a Hellenic Oncology Research Group (HORG) randomized phase 3 study.

BACKGROUND: In this superiority study, pemetrexed was compared with erlotinib in pre-treated patients with metastatic non-small cell lung cancer (NSCLC). METHODS: Patients with stage IIIB/IV NSCLC who progressed after first-line or second-line treatment were randomized to receive either pemetrexed or erlotinib. In total, 21.7% of patients in the pemetrexed arm and 23.5% of patients in the erlotinib arm had squamous cell histology, and treatment was third line in 39.2% and 46.4% of patients, respectively. The primary study endpoint was time to tumor progression (TTP). Epidermal growth factor receptor/v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (EGFR/KRAS) mutation status also was investigated. RESULTS: There was no difference in terms of the TTP (P = .195), the objective response rate (P = .469), or overall survival (P = .986) between the 2 treatment arms. In patients who had squamous cell histology, erlotinib resulted in a superior TTP compared with pemetrexed (4.1 months vs 2.5 months, respectively; P = .006). The incidence of grade 3 and 4 neutropenia, thrombocytopenia, and asthenia was significantly higher in the pemetrexed arm, whereas the incidence of grade 3 and 4 skin rash was higher in the erlotinib arm. CONCLUSIONS: Both pemetrexed and erlotinib had comparable efficacy in pre-treated patients with metastatic NSCLC, and the current results indicated that genotyping of tumor cells may have an important effect on treatment efficacy.

Combined analysis of KRAS and PIK3CA mutations, MET and PTEN expression in primary tumors and corresponding metastases in colorectal cancer.

Metastasis is the main cause of mortality in patients with colorectal cancer. However, most of the targeted therapies and predictive molecular biomarkers were developed based mainly on primary tumors. The current study was conducted to determine the degree of discordance between potential predictive and/or prognostic molecular markers in primary colorectal tumors and corresponding metastases, as this could have an impact on the efficacy of targeted therapies in the advanced colorectal cancer. KRAS, PIK3CA and BRAF mutations were determined by Sanger sequencing and mutant-enriched polymerase chain reaction (PCR) assays in 83 paired samples, MET gene copy number by quantitative PCR in 59, MET expression by immunohistochemistry in 73 and nuclear and cytoplasmic expression of PTEN by immunohistochemistry in 78 and 71 pairs, respectively. A certain degree of discordance between primary tumors and corresponding metastases was demonstrated for all examined biomarkers except BRAF mutations. PIK3CA exon 9 mutations in primary tumors and loss of PTEN nuclear expression in metastases correlated with KRAS mutations. KRAS wild-type status in primary tumors was associated with loss of PTEN cytoplasmic expression and high gene copy number of MET. Survival and clinical data were available for 68 patients. The multiple regression analysis revealed that the right-sided tumor localization and overexpression of MET were associated with shorter overall survival.

Effect of body tissue composition on the outcome of patients with metastatic non-small cell lung cancer treated with PD-1/PD-L1 inhibitors.

Obesity and sarcopenia have been reported to affect outcomes in patients with non-small cell lung cancer (NSCLC) treated with immune checkpoint inhibitors (ICIs). We analyzed prospective data from 52 patients with non-oncogene driven metastatic NSCLC treated with ICIs. Body tissue composition was calculated by measuring the fat and muscle densities at the level of 3rd lumbar vertebra in each patient computed tomography scan before ICI initiation using sliceOmatic tomovision. We converted the densities to indices [Intramuscular Fat Index (IMFI), Visceral Fat Index (VFI), Subcutaneous Fat Index (SFI), Lumbar Skeletal Muscle Index (LSMI)] by dividing them by height in meters squared. Patients were dichotomized based on their baseline IMFI, VFI and SFI according to their gender-specific median value. The cut-offs that were set for LMSI values were 55 cm2/m2 for males and 39 cm2/m2 for females. SFI distribution was significantly higher (p = 0.040) in responders compared to non-responders. None of the other variables affected response rates. Low LSMI HR: 2.90 (95% CI: 1.261-6.667, p = 0.012) and low SFI: 2.20 (95% CI: 1.114-4.333, p = 0.023) values predicted for inferior OS. VFI and IMFI values did not affect survival. Subcutaneous adipose and skeletal muscle tissue composition significantly affected immunotherapy outcomes in our cohort.

Immunological responses in cancer patients after vaccination with the therapeutic telomerase-specific vaccine Vx-001.

Vx-001, an HLA-A*0201 restricted telomerase (TERT)-specific anti-tumor vaccine, is composed of the 9-mer cryptic TERT(572) peptide and its optimized variant TERT(572Y). We have previously shown that Vx-001 is non-toxic, highly immunogenic and in vaccinated NSCLC patients early specific immune response is associated with prolonged survival. The aim of the present study was to investigate the specific T-cell immune response against Vx-001. Fifty-five patients with chemo-resistant advanced solid tumors were vaccinated with TERT(572Y) (2 subcutaneous injections) followed by TERT(572) peptide (4 subcutaneous injections) every 3 weeks. Specific immune response was evaluated by IFN-γ and perforin ELISpot and intracellular cytokine staining assays. TERT-reactive T cells were detected in 27 (51%) out of 53 evaluable patients after the 2nd vaccination and in 22 (69%) out of 32 evaluable patients after the completion of 6 vaccinations. Immune responses developed irrespective of the stage of disease and disease status before vaccination. Patients with disease progression at study entry who developed a post-vaccination-induced immunological response had a significant overall survival benefit compared to the post-vaccination non-responders. The Vx-001 vaccine is a promising candidate for cancer immunotherapy since it can induce a TERT-specific T-cell immune response that is associated with prolonged survival.

Baseline plasma levels of interleukin-8 in stage IV non-small-cell lung cancer patients: relationship with nutritional status and prognosis.

Interleukin (IL)-8 promotes cellular proliferation and angiogenesis in patients with non-small-cell lung cancer (NSCLC) and may be related to cachexia. Our aim was to investigate the relationship of IL-8 levels with nutritional status, and clinical outcome of patients with NSCLC. Patients with metastatic NSCLC referred for first-line therapy were eligible. Baseline IL-8 levels were measured in plasma. The Mini Nutritional Assessment (MNA) was used for the evaluation of the nutritional status, and patients were classified into 3 groups: A (score 24-30) "well nourished," B (score 17-23.5) "risk of malnutrition," and C (0-16.5) "malnourishment." Response to first-line chemotherapy, time-to-tumor progression (TTP), and overall survival (OS) were also recorded. In total, 114 patients (101 males, 88.5%; mean age = 67.5 yr) were evaluated. Performance status was 0-1 in 62% of the patients. According to the MNA, the majority of patients (71%) was either at nutritional risk or malnourished. IL-8 levels were significantly different between MNA groups (P = 0.023) and correlated with TTP (P = 0.013) and OS (P = 0.001) in univariate analysis. Baseline IL-8 levels correlate with the nutritional status of patients with metastatic NSCLC, suggesting that this cytokine may be related with cachexia.

Nutritional status, acute phase response and depression in metastatic lung cancer patients: correlations and association prognosis.

BACKGROUND AND AIM: Cancer cachexia is a metabolic syndrome related with poor outcome. Cytokines play a key role in the pathophysiology of that syndrome. The aim of this study was to investigate the potential correlations between nutritional status, systemic inflammation, and psychological distress in cancer patients. The prognostic significance of the recorded parameters was also assessed. PATIENTS AND METHODS: Patients with metastatic lung cancer were eligible. Mini Nutritional Assessment (MNA) was used for the evaluation of nutritional status, Glasgow Prognostic Score (GPS) for the estimation of systemic inflammation, and Hospital Anxiety and Depression Scale (HADS) for psychological assessment. RESULTS: Totally, 122 patients were enrolled (71.3% with NSCLC and 28.7% with SCLC). The following correlations were observed: MNA and GPS (r = 0.289, p = 0.001), MNA and HADS (depression scale) (r = 0.275, p = 0.002), GPS and HADS (depression scale) (r = 0.256, p = 0.004), and GPS and HADS (anxiety scale) (r =0.194, p =0.033). In univariate analysis, GPS (p = 0.002) and MNA (p = 0.010) emerged as significant predictors of survival. In multivariate analysis, both MNA (p = 0.032) and GPS (p = 0.020) retained their importance. CONCLUSIONS: This study highlights the associations between nutritional status, systemic inflammation, and psychological distress, supporting their common underlying pathophysiological mechanisms and further suggesting the necessity of a holistic anti-cachectic approach.

Cancer Cachexia and Antitumor Immunity: Common Mediators and Potential Targets for New Therapies.

Cancer cachexia syndrome (CCS) is a multifactorial metabolic syndrome affecting a significant proportion of patients. CCS is characterized by progressive weight loss, alterations of body composition and a systemic inflammatory status, which exerts a major impact on the host's innate and adaptive immunity. Over the last few years, the development of immune checkpoint inhibitors (ICIs) transformed the treatment landscape for a wide spectrum of malignancies, creating an unprecedented opportunity for long term remissions in a significant subset of patients. Early clinical data indicate that CCS adversely impairs treatment outcomes of patients receiving ICIs. We herein reviewed existing evidence on the potential links between the mechanisms that promote the catabolic state in CCS and those that impair the antitumor immune response. We show that the biological mediators and processes leading to the development of CCS may also participate in the modulation and the sustainment of an immune suppressive tumor microenvironment and impaired anti-tumor immunity. Moreover, we demonstrate that the deregulation of the host's metabolic homeostasis in cancer cachexia is associated with resistance to ICIs. Further research on the interrelation between cancer cachexia and anti-tumor immunity is required for the effective management of resistance to immunotherapy in this specific but large subgroup of ICI treated individuals.

Epithelial to mesenchymal transition markers expressed in circulating tumour cells of early and metastatic breast cancer patients.

INTRODUCTION: Epithelial to mesenchymal transition (EMT) is considered an essential process in the metastatic cascade. EMT is characterised by upregulation of vimentin, Twist, Snail, Slug and Sip1 among others. Metastasis is also associated with the presence of circulating tumour cells (CTCs) and disseminated tumour cells in the blood and bone marrow, respectively, of breast cancer patients, but the expression of EMT markers in these cells has not been reported so far. METHODS: The expression of Twist and vimentin in CTCs of 25 metastatic and 25 early breast cancer patients was investigated by using double-immunofluorescence experiments in isolated peripheral blood mononuclear cell cytospins using anti-cytokeratin (anti-CK) anti-mouse (A45-B/B3) and anti-Twist or anti-vimentin anti-rabbit antibodies. RESULTS: Among early breast cancer patients, vimentin-and Twist-expressing CK(+) CTCs were identified in 77% and 73% of the patients, respectively, and in 100% of the patients with metastatic breast cancer for both markers (P = 0.004 and P = 0.037, respectively). Among patients with early disease, 56% and 53% of the CK(+) CTCs were double-stained with vimentin and Twist, and the corresponding values for metastatic patients were 74% and 97%, respectively (P = 0.005 and P = 0.0001, respectively). The median expression of CK(+)vimentin(+) and CK(+)Twist(+) cells per patient in metastatic patients was 98% and 100%, and in an adjuvant chemotherapy setting the corresponding numbers were 56% and 40.6%, respectively. Triple-staining experiments revealed that all CK(+)Twist(+) or CK(+)vimentin(+) cells were also CD45(-), confirming their epithelial origin. Immunomagnetic separation of CTCs and triple-immunofluorescence with anti-CK/anti-Twist/anti-vimentin antibodies demonstrated that both mesenchymal markers could be coexpressed in the same CK(+) cell, since 64% of the total identified CTCs were triple-stained. There was a significant correlation (P = 0.005) between the number of CTCs expressing Twist and vimentin within the same setting. CONCLUSIONS: CTCs expressing Twist and vimentin, suggestive of EMT, are identified in patients with breast cancer. The high incidence of these cells in patients with metastatic disease compared to early stage breast cancer strongly supports the notion that EMT is involved in the metastatic potential of CTCs.

Cytokeratin-19 mRNA-positive circulating tumor cells during follow-up of patients with operable breast cancer: prognostic relevance for late relapse.

BACKGROUND: The detection of cytokeratin-19 (CK-19) mRNA-positive circulating tumor cells (CTC) before and/or after adjuvant chemotherapy in patients with operable breast cancer is associated with poor clinical outcome. Reliable prognostic markers for late disease relapse are not available. In this study we investigated the value of CTC detection during the first five years of follow-up in predicting late disease relapse. METHODS: Blood was analyzed from 312 women with operable breast cancer who had not experienced disease relapse during the first two years of follow-up. A real-time reverse transcriptase polymerase chain reaction (RT-PCR) for CK-19 mRNA was used to detect CTC three months after the completion of adjuvant chemotherapy and every six months thereafter for a follow-up period of five years. RESULTS: Eighty patients (25.6% of the study population) remained CTC free throughout the five-year period. A change in CTC status was observed in 133 patients (42.6%); 64 patients (20.5%) with initially CK-19 mRNA-positive CTC during the first 24 months turned CTC-negative afterwards while 69 (22.1%) who were initially CTC-negative became CTC-positive. Ninety-nine patients (31.7%) remained persistently CK-19 mRNA-positive. After a median follow-up period of 107 months (range: 38 to 161 months), the persistently CTC-positive patients with either hormonal receptor positive or negative tumors, had a higher risk of late-disease relapse compared to the persistently CTC-negative patients (36.4% versus 11.2%, P <0.001). Multivariate analysis revealed that persistently CTC-positive patients also had a shorter disease-free (P = 0.001) and overall survival (P = 0.001). CONCLUSIONS: Persistent detection of CK-19 mRNA-positive CTC during the first five years of follow-up is associated with an increased risk of late relapse and death in patients with operable breast cancer and indicates the presence of chemo-and hormonotherapy-resistant residual disease. This prognostic evaluation may be useful when deciding on subsequent adjuvant systemic therapy.

Gene expression profile of circulating tumor cells in breast cancer by RT-qPCR.

BACKGROUND: Circulating tumor cells (CTCs) have been associated with prognosis especially in breast cancer and have been proposed as a liquid biopsy for repeated follow up examinations. Molecular characterization of CTCs is difficult to address since they are very rare and the amount of available sample is very limited. METHODS: We quantified by RT-qPCR CK-19, MAGE-A3, HER-2, TWIST1, hTERT α+ß+, and mammaglobin gene transcripts in immunomagnetically positively selected CTCs from 92 breast cancer patients, and 28 healthy individuals. We also compared our results with the CellSearch system in 33 of these patients with early breast cancer. RESULTS: RT-qPCR is highly sensitive and specific and can detect the expression of each individual gene at the one cell level. None of the genes tested was detected in the group of healthy donors. In 66 operable breast cancer patients, CK-19 was detected in 42.4%, HER-2 in 13.6%, MAGE-A3 in 21.2%, hMAM in 13.6%, TWIST-1 in 42.4%, and hTERT α+ß+ in 10.2%. In 26 patients with verified metastasis, CK-19 was detected in 53.8%, HER-2 in 19.2%, MAGE-A3 in 15.4%, hMAM in 30.8%, TWIST-1 in 38.5% and hTERT α+ß+in 19.2%. Our preliminary data on the comparison between RT-qPCR and CellSearch in 33 early breast cancer patients showed that RT-qPCR gives more positive results in respect to CellSearch. CONCLUSIONS: Molecular characterization of CTCs has revealed a remarkable heterogeneity of gene expression between breast cancer patients. In a small percentage of patients, CTCs were positive for all six genes tested, while in some patients only one of these genes was expressed. The clinical significance of these findings in early breast cancer remains to be elucidated when the clinical outcome for these patients is known.