Tumor Microenvironment in Male Breast Carcinoma with Emphasis on Tumor Infiltrating Lymphocytes and PD-L1 Expression.

Male breast cancer (MBC) is rare and usually presents as a locally advanced disease. Stromal tumor-infiltrating lymphocytes (sTILs) are associated with a better response to neoadjuvant chemotherapy and improved prognosis in all molecular subtypes of female breast cancer, but their role in MBC is less clear. We studied sTILs and the expression of programmed cell death ligand 1 (PD-L1) and pan-TRK in MBC. We retrospectively studied 113 cases of MBC surgically treated between 1988 and 2015. The tumors were evaluated for histological type and grade, stage, intrinsic subtype and sTILs. We performed immunohistochemistry for PD-L1 (clone SP142) and pan-TRK (clone EPR17341) on tissue microarrays. Pan-TRK positive cases were further analyzed by next-generation sequencing. The median age was 69 years (range 60−77). Invasive carcinoma of no special type was found in 94.7% of cases, of which 53.1% were grade 2. Estrogen receptor was positive in 92% of the tumors, progesterone receptor in 85.8%, androgen receptor in 70.8%; 4.4% were human epidermal growth factor receptor 2 (HER2)-positive, and 55.8% HER2-low. 40.7% of tumors were luminal A and 51.3% luminal B, 4.4% HER2-enriched and 3.5% triple negative carcinoma. sTILs density was <50% in 96.4% of the tumors, >50% in 3.6% of the tumors. PD-L1 immune cell score >1% was found in 7.1% of the tumors (all of luminal subtype). A weak focal cytoplasmic pan-TRK staining was present in 8.8% but without NTRK fusion. Neither sTILs nor PD-L1 had statistically significant outcomes. Our findings suggest that a subset of MBC patients harbors an immunological environment characterized by increased sTILs with PD-L1 expression. These patients may potentially benefit from immune checkpoint inhibitor therapy. Frequent HER2-low may offer novel anti-HER2 treatment options.

Transferring MINDACT to Daily Routine: Implementation of the 70-Gene Signature in Luminal Early Breast Cancer - Results from a Prospective Registry of the Austrian Group Medical Tumor Therapy (AGMT).

Background: For hormone receptor (HR)-positive/human epidermal growth factor receptor 2 (HER2)-negative early breast cancer (EBC), adjuvant chemotherapy (ACT) is recommended in the case of high-risk features only. The MINDACT trial showed that patients with high clinical risk (CR) but low genomic risk (GR) defined by the 70-gene signature (MammaPrint®; 70-GS) did not benefit from ACT. In this registry, we investigated the frequency and feasibility of 70-GS and concurrent 80-gene subtyping (BluePrint®) use in HR-positive, HER2-negative EBC. Furthermore, we recorded the frequency of ACT recommendation and the adherence to it when the "MINDACT strategy" was used. Methods: This prospective registry included patients from 2 Austrian cancer centers. Similar to MINDACT, a modified version of Adjuvant!Online was used to determine CR, and 70-GC was used to determine GR in high-CR patients. ACT was recommended to patients with high CR and high GR. Results: Of 224 enrolled patients, 76 (33.9%) had high CR and 67 (88.2%) received genomic testing. Of those, 43 (64.2%) were classified as low and 24 (35.8%) as high GR, respectively. All 24 patients with high CR and GR (10.7% of all patients) received the recommendation for ACT, but ACT was started in only 15 patients (62.5%). The median time from surgery to the start of ACT was 45 days (range 32-68), and the median time from test decision to the test result was 15 days (range 9-56). Conclusion: We showed that the results of the MINDACT trial are reproducible in an Austrian population. Incorporating 70-GS into the daily clinical routine is feasible and mostly accepted by physicians for the guidance of treatment recommendations.

Sensitive and robust liquid biopsy-based detection of PIK3CA mutations in hormone-receptor-positive metastatic breast cancer patients.

BACKGROUND: The benefit of alpelisib in hormone-receptor-positive (HR+) metastatic breast cancer patients provided clinical evidence for the increasing importance of PIK3CA testing. We performed a comparison of liquid biopsy and tissue-based detection of PIK3CA mutations. MATERIALS AND METHODS: PIK3CA hotspot mutation analysis using a high-resolution SiMSen-Seq assay was performed in plasma from 93/99 eligible patients with HR+/HER2- breast cancer. Additionally, mFAST-SeqS was used to estimate the tumour fractions in plasma samples. In 72/93 patients, matched tissue was available and analysed using a customised Ion Torrent panel. RESULTS: PIK3CA mutations were detected in 48.6% of tissue samples and 47.3% of plasma samples, with identical PIK3CA mutation detected in 24/72 (33.3%) patients both in tissue and plasma. In 10 (13.9%) patients, mutations were only found in plasma, and in 6 (8.3%) patients, PIK3CA mutations found in tissue were not detectable in ctDNA. In 49/93 plasma samples without detectable PIK3CA mutations, 22 (44.9%) samples had elevated tumour fractions, implying true negative results. CONCLUSION: SiMSen-Seq-based detection of PIK3CA mutations in plasma shows advantageous concordance with the tissue analyses. A combination with an untargeted approach for detecting ctDNA fractions may confirm a negative PIK3CA result and enhance the performance of the SiMSen-Seq test.

Persistence of ctDNA in Patients with Breast Cancer During Neoadjuvant Treatment Is a Significant Predictor of Poor Tumor Response.

PURPOSE: Accurate response assessment during neoadjuvant systemic treatment (NST) poses a clinical challenge. Therefore, a minimally invasive assessment of tumor response based on cell-free circulating tumor DNA (ctDNA) may be beneficial to guide treatment decisions. EXPERIMENTAL DESIGN: We profiled 93 genes in tissue from 193 patients with early breast cancer. Patient-specific assays were designed for 145 patients to track ctDNA during NST in plasma. ctDNA presence and levels were correlated with complete pathological response (pCR) and residual cancer burden (RCB) as well as clinicopathologic characteristics of the tumor to identify potential proxies for ctDNA release. RESULTS: At baseline, ctDNA could be detected in 63/145 (43.4%) patients and persisted in 25/63 (39.7%) patients at mid-therapy (MT) and 15/63 (23.8%) patients at the end of treatment. ctDNA detection at MT was significantly associated with higher RCB (OR = 0.062; 95% CI, 0.01-0.48; P = 0.0077). Of 31 patients with detectable ctDNA at MT, 30 patients (96.8%) were nonresponders (RCB II, n = 8; RCB III, n = 22) and only one patient responded to the treatment (RCB I). Considering all 145 patients with baseline (BL) plasma, none of the patients with RCB 0 and only 6.7% of patients with RCB I had ctDNA detectable at MT, whereas 30.6% and 29.6% of patients with RCB II/III, respectively, had a positive ctDNA result. CONCLUSIONS: Overall, our results demonstrate that the detection and persistence of ctDNA at MT may have the potential to negatively predict response to neoadjuvant treatment and identify patients who will not achieve pCR or be classified with RCB II/III.

Circulating Tumor DNA as a Marker for Treatment Response in Metastatic Melanoma Patients Using Next-Generation Sequencing-A Prospective Feasibility Study.

We prospectively performed a longitudinal analysis of circulating tumor DNA (ctDNA) from 149 plasma samples and CT scans in Stage III and IV metastatic melanoma patients (n = 20) treated with targeted agents or immunotherapy using two custom next-generation sequencing (NGS) Ion AmpliSeq™ HD panels including 60 and 81 amplicons in 18 genes, respectively. Concordance of matching cancer-associated mutations in tissue and plasma was 73.3%. Mutant allele frequency (MAF) levels showed a range from 0.04% to 28.7%, well detectable with NGS technologies utilizing single molecule tagging like the AmpliSeq™ HD workflow. Median followup time of the tissue and/or plasma positive cohort (n = 15) was 24.6 months and median progression-free survival (PFS) was 7.8 months. Higher MAF ≥ 1% at baseline was not significantly associated with a risk of progression (Odds Ratio = 0.15; p = 0.155). Although a trend could be seen, MAF levels did not differ significantly over time between patients with and without a PFS event (p = 0.745). Depending on the cell-free DNA amount, NGS achieved a sensitivity down to 0.1% MAF and allowed for parallel analysis of multiple mutations and previously unknown mutations. Our study indicates that NGS gene panels could be useful for monitoring disease burden during therapy with ctDNA in melanoma patients.

MicroRNA expression profiling with a droplet digital PCR assay enables molecular diagnosis and prognosis of cancers of unknown primary.

Metastasis is responsible for the majority of cancer-related deaths. Particularly, challenging is the management of metastatic cancer of unknown primary site (CUP), whose tissue of origin (TOO) remains undetermined even after extensive investigations and whose therapy is rather unspecific and poorly effective. Molecular approaches to identify the most probable TOO of CUPs can overcome some of these issues. In this study, we applied a predetermined set of 89 microRNAs (miRNAs) to infer the TOO of 53 metastatic cancers of unknown or uncertain origin. The miRNA expression was assessed with droplet digital PCR in 159 samples, including primary tumors from 17 tumor classes (reference set) and metastases of known and unknown origin (test set). We combined two different statistical models for class prediction to obtain the most probable TOOs: the nearest shrunken centroids approach of Prediction Analysis of Microarrays (PAMR) and the least absolute shrinkage and selection operator (LASSO) models. The molecular test was successful for all formalin-fixed paraffin-embedded samples and provided a TOO identification within 1 week from the biopsy procedure. The most frequently predicted origins were gastrointestinal, pancreas, breast, lung, and bile duct. The assay was applied also to multiple metastases from the same CUP, collected from different metastatic sites: The predictions showed a strong agreement, intrinsically validating our assay. The final CUPs' TOO prediction was compared with the clinicopathological hypothesis of primary site. Moreover, a panel of 13 miRNAs proved to have prognostic value and be associated with overall survival in CUP patients. Our study demonstrated that miRNA expression profiling in CUP samples could be employed as diagnostic and prognostic test. Our molecular analysis can be performed on request, concomitantly with standard diagnostic workup and in association with genetic profiling, to offer valuable indications about the possible primary site, thereby supporting treatment decisions.

Patterns of Recurrence after Neoadjuvant Therapy in Early Breast Cancer, according to the Residual Cancer Burden Index and Reductions in Neoadjuvant Treatment Intensity.

BACKGROUND: The prognostic performance of the residual cancer burden (RCB) score is a promising tool for breast cancer patients undergoing neoadjuvant therapy. We independently evaluated the prognostic value of RCB scores in an extended validation cohort. Additionally, we analyzed the association between chemotherapy dose reduction and RCB scores. METHODS: In this extended validation study, 367 breast cancer patients with available RCB scores were followed up for recurrence-free survival (RFS), distant disease-free survival (DDFS), and overall survival (OS). We also computed standardized cumulative doses of anthracyclines and taxanes (A/Ts) to investigate a potential interaction between neoadjuvant chemotherapy dose reduction and RCB scores. RESULTS: Higher RCB scores were consistently associated with adverse clinical outcomes across different molecular subtypes (HR for RFS = 1.60, 95% CI 1.33-1.93, p < 0.0001; HR for DDFS = 1.70, 95% CI 1.39-2.05, p < 0.0001; HR for OS = 1.67, 95% CI 1.34-2.08, p < 0.0001). The adverse impact prevailed throughout 5 years of follow-up, with a peak for relapse risk between 1-2 years after surgery. Clinical outcomes of patients with RCB class 1 did not differ substantially at 5 years compared to RCB class 0. A total of 180 patients (49.1%) underwent dose reduction of neoadjuvant A/T chemotherapy. We observed a statistically significant interaction between dose reduction and higher RCB scores (interaction p-value = 0.042). CONCLUSION: Our results confirm RCB score as a prognostic marker for RFS, DDFS, and OS independent of the molecular subtype. Importantly, we show that lower doses of cumulative neoadjuvant A/T were associated with higher RCB scores in patients who required a dose reduction.

Safety and Clinical Evaluation of Dual Inhibition with Pertuzumab and Trastuzumab Biosimilar SB3 in HER2-Positive Breast Cancer Patients.

BACKGROUND: The addition of trastuzumab to standard chemotherapy has improved survival in patients with HER2-positive breast cancer in neoadjuvant, adjuvant, and metastatic settings. In higher tumor stages, the addition of pertuzumab is now a standard of care and associated with a favorable toxicity profile. We evaluated the safety and efficacy of the trastuzumab biosimilar SB3 in combination with pertuzumab in HER2-positive breast cancer patients. METHODS: Seventy-eight patients with HER2-positive breast cancer treated at the Division of Oncology at the Medical University of Graz were included. Summary measures are reported as medians (25th to 75th percentile) for continuous variables and as absolute frequencies (%) for count data. RESULTS: Thirty-five patients received a median of 4 (3-7) cycles of trastuzumab biosimilar SB3 plus pertuzumab. All patients had a normal baseline left ventricular ejection fraction (LVEF; >50%) prior to the initiation of SB3 plus pertuzumab treatment with a median LVEF of 60% (60-65). Twenty-one patients had a median absolute LVEF decline of 1% (-5 to 0). Two patients (5.7%) had a LVEF reduction ≤50%, but none ≥10%. There were no unexpected adverse events. Twenty-two of 35 patients (63%) were treated with trastuzumab biosimilar SB3 and pertuzumab in the neoadjuvant setting and 11 patients (50%) achieved a pathological complete response. The safety and the efficacy in this setting was comparable to the trastuzumab plus pertuzumab combination in neoadjuvantly treated matched samples. CONCLUSION: In this series of HER2-positive breast cancer patients, the combination of SB3 plus pertuzumab was consistent with the known safety and efficacy profile of trastuzumab and pertuzumab combination.

Longitudinal tumor fraction trajectories predict risk of progression in metastatic HR+ breast cancer patients undergoing CDK4/6 treatment.

Despite improved clinical outcomes, intrinsic or acquired resistance to CDK4/6 inhibitor treatment has limited the success of this treatment in HR+ HER2- metastatic breast cancer patients. Biomarkers are urgently needed, and longitudinal biomarker measurements may harbor more dynamic predictive and prognostic information compared to single time point measurements. The aim of this study was to explore the longitudinal evolution of circulating tumor fractions within cell-free DNA assessed by an untargeted sequencing approach during CDK4/6 therapy and to quantify the potential association between longitudinal z-score measurements and clinical outcome by using joint models. Forty-nine HR+ HER2- metastatic breast cancer patients were enrolled, and z-score levels were measured at baseline and during 132 follow-up visits (median number of measurements per patient = 3, 25th -75th percentile: 3-5, range: 1-8). We observed higher baseline z-score levels (estimated difference 0.57, 95% CI: 0.147-0.983, P-value = 0.008) and a constant increase of z-score levels over follow-up time (overall P-value for difference in log z-score over time = 0.024) in patients who developed progressive disease. Importantly, the joint model revealed that elevated z-score trajectories were significantly associated with higher progression risk (HR of log z-score at any time of follow-up = 3.3, 95% CI, 1.44-7.55, P = 0.005). In contrast, single z-score measurement at CDK4/6 inhibitor treatment start did not predict risk of progression. In this prospective study, we demonstrate proof-of-concept that longitudinal z-score trajectories rather than single time point measurements may harbor important dynamic information on the development of disease progression in HR+ HER2- breast cancer patients undergoing CDK4/6 inhibitor treatment.

Evaluation of Blood-based Biomarkers for Prediction of Response in Carboplatin-treated Metastatic Castration-resistant Prostate Cancer Patients.

BACKGROUND/AIM: Carboplatin-containing treatment regimens demonstrate moderate efficacy in metastatic castration-resistant prostate cancer (mCRPC). In this study, we retrospectively analyzed the efficacy of carboplatin in relation to blood-based parameters. PATIENTS AND METHODS: A retrospective chart review was performed for 20 patients with mCRPC who received carboplatin in a single center. RESULTS: Median overall survival was 3.8 months (95%CI=1.5-7.1), median progression-free survival was 1.7 months. We observed two partial remissions (PR, 10%), four stable diseases (SD, 20%) and 14 disease progressions (PD, 70%), resulting in a clinical benefit rate of 30%. A doubling of NSE (neurone specific enolase) values was associated with a 19% absolute higher response rate (95%CI=14-23, p=0.027). All other laboratory parameters failed as predictive markers of response to carboplatin. In univariate Cox regression analysis, only NSE was significantly associated with impaired PFS (HR=0.7, 95%CI=0.56-0.96, p=0.030). CONCLUSION: Carboplatin showed moderate efficacy against mCRPC in this unselected population of patients and NSE levels may help to predict the success of this treatment.

Comparison of three commercial decision support platforms for matching of next-generation sequencing results with therapies in patients with cancer.

OBJECTIVE: Precision oncology depends on translating molecular data into therapy recommendations. However, with the growing complexity of next-generation sequencing-based tests, clinical interpretation of somatic genomic mutations has evolved into a formidable task. Here, we compared the performance of three commercial clinical decision support tools, that is, NAVIFY Mutation Profiler (NAVIFY; Roche), QIAGEN Clinical Insight (QCI) Interpret (QIAGEN) and CureMatch Bionov (CureMatch). METHODS: In order to obtain the current status of the respective tumour genome, we analysed cell-free DNA from patients with metastatic breast, colorectal or non-small cell lung cancer. We evaluated somatic copy number alterations and in parallel applied a 77-gene panel (AVENIO ctDNA Expanded Panel). We then assessed the concordance of tier classification approaches between NAVIFY and QCI and compared the strategies to determine actionability among all three platforms. Finally, we quantified the alignment of treatment suggestions across all decision tools. RESULTS: Each platform varied in its mode of variant classification and strategy for identifying druggable targets and clinical trials, which resulted in major discrepancies. Even the frequency of concordant actionable events for tier I-A or tier I-B classifications was only 4.3%, 9.5% and 28.4% when comparing NAVIFY with QCI, NAVIFY with CureMatch and CureMatch with QCI, respectively, and the obtained treatment recommendations differed drastically. CONCLUSIONS: Treatment decisions based on molecular markers appear at present to be arbitrary and dependent on the chosen strategy. As a consequence, tumours with identical molecular profiles would be differently treated, which challenges the promising concepts of genome-informed medicine.

Hazard Curves for Tumor Recurrence and Tumor-Related Death Following Esophagectomy for Esophageal Cancer.

BACKGROUND: The knowledge of both patterns and risk of relapse following resection for esophageal cancer is crucial for establishing appropriate surveillance schedules. The aim of this study was to evaluate the pattern of hazards for tumor recurrence and tumor-related death in the postoperative long-term follow-up after esophagectomy. METHODS: Retrospective single-center analysis of 362 patients, with resected esophageal cancer. Multivariate Cox proportional hazard model was used. RESULTS: A total of 192 (53%) had postoperative tumor recurrence. The relapse patterns of adenocarcinoma and squamous-cell carcinoma showed that each had a single peak, 12 months after surgery. After induction there was one peak at 5 months, the non-induced patients peaked 11 months, postoperatively. At 18 months, the recurrence hazard declined sharply in all cases. The hazard curves for tumor-related death were bimodal for adenocarcinoma, with two peaks at 6 and 22 months and one single peak for squamous-cell carcinoma at 18 months after surgery, showing pronounced decline later on. CONCLUSION: In curatively resected esophageal cancer, both tumor recurrence hazard and hazard for tumor-related death showed distinct, partly bimodal patterns. It could be justified to intensify the surveillance during the first two postoperative years by initiating a close-meshed follow-up to detect and treat tumor recurrence, as early as possible.

Chronic Oxidative Stress Promotes Molecular Changes Associated with Epithelial Mesenchymal Transition, NRF2, and Breast Cancer Stem Cell Phenotype.

Oxidative stress plays a role in carcinogenesis, but it also contributes to the modulation of tumor cells and microenvironment caused by chemotherapeutics. One of the consequences of oxidative stress is lipid peroxidation, which can, through reactive aldehydes such as 4-hydroxy-2-nonenal (HNE), affect cell signaling pathways. On the other hand, cancer stem cells (CSC) are now recognized as a major factor of malignancy by causing metastasis, relapse, and therapy resistance. Here, we evaluated whether oxidative stress and HNE modulation of the microenvironment can influence CSC growth, modifications of the epithelial to mesenchymal transition (EMT) markers, the antioxidant system, and the frequency of breast cancer stem cells (BCSC). Our results showed that oxidative changes in the microenvironment of BCSC and particularly chronic oxidative stress caused changes in the proliferation and growth of breast cancer cells. In addition, changes associated with EMT, increase in glutathione (GSH) and Nuclear factor erythroid 2-related factor 2 (NRF2) were observed in breast cancer cells grown on HNE pretreated collagen and under chronic oxidative stress. Our results suggest that chronic oxidative stress can be a bidirectional modulator of BCSC fate. Low levels of HNE can increase differentiation markers in BCSC, while higher levels increased GSH and NRF2 as well as certain EMT markers, thereby increasing therapy resistance.

Validation of Residual Cancer Burden as Prognostic Factor for Breast Cancer Patients After Neoadjuvant Therapy.

BACKGROUND: Assessing the residual cancer burden (RCB) predictive performance, the potential subgroup effects, and time-dependent impact on breast cancer patients who underwent neoadjuvant therapy in a developer's independent cohort is essential for its usage in clinical routine. METHODS: Between 2011 and 2016, the RCB scores of 184 female breast cancer patients were prospectively collected, and subsequent clinicopathological and follow-up data were obtained retrospectively. Recurrence-free survival (RFS), overall survival (OS), as well as subgroup analysis, and time-dependent variables were calculated with multivariate, complex, or linear statistical models. RESULTS: A total of 184 patients (HER2 33%, TNBC 27%), with a mean follow-up time of 4 years, treated with neoadjuvant systemic therapy (92% anthracycline-taxane based) were analyzed revealing 43 events (38 recurrences, 28 deaths). High RCB scores were associated with recurrence (median index: 2.34 vs. 1.39 points, rank-sum p < 0.0001), decreased RFS (hazard ratio [HR] = 1.80, 95% confidence interval [CI] 1.44-2.24, p < 0.0001) and reduced OS (HR 1.96, 95% CI 1.49-2.59, p < 0.0001). The RCB score showed proportionality of hazards (interaction HR with linear follow-up time = 1.00, p = 0.896) and good discriminating power (Harrell's c index 0.7). CONCLUSIONS: Our results confirm the RCB score as externally valid prognostic marker and being independent of molecular subtype for RFS and OS in a clinical setting.

Untargeted Assessment of Tumor Fractions in Plasma for Monitoring and Prognostication from Metastatic Breast Cancer Patients Undergoing Systemic Treatment.

The aim of this study was to assess the prognostic and predictive value of an untargeted assessment of tumor fractions in the plasma of metastatic breast cancer patients and to compare circulating tumor DNA (ctDNA) with circulating tumor cells (CTC) and conventional tumor markers. In metastatic breast cancer patients (n = 29), tumor fractions in plasma were assessed using the untargeted mFAST-SeqS method from 127 serial blood samples. Resulting z-scores for the ctDNA were compared to tumor fractions established with the recently published ichorCNA algorithm and associated with the clinical outcome. We observed a close correlation between mFAST-SeqS z-scores and ichorCNA ctDNA quantifications. Patients with mFAST-SeqS z-scores above three (34.5%) showed significantly worse overall survival (p = 0.014) and progression-free survival (p = 0.018) compared to patients with lower values. Elevated z-score values were clearly associated with radiologically proven progression. The baseline CTC count, carcinoembryonic antigen (CEA), and cancer antigen (CA)15-5 had no prognostic impact on the outcome of patients in the analyzed cohort. This proof of principle study demonstrates the prognostic impact of ctDNA levels detected with mFAST-SeqS as a very fast and cost-effective means to assess the ctDNA fraction without prior knowledge of the genetic landscape of the tumor. Furthermore, mFAST-SeqS-based ctDNA levels provided an early means of measuring treatment response.

Multigene methylation analysis of enriched circulating tumor cells associates with poor progression-free survival in metastatic breast cancer patients.

Blood-based biomarkers such as circulating tumor cells (CTCs) provide dynamic real-time assessment of molecular tumor characteristics beyond the primary tumor. The aim of this study was to evaluate the feasibility of a size-based microfilter to assess multigene methylation analysis of enriched CTCs in a prospective proof-of principle study. We examined the quantitative methylation status of nine genes (AKR1B1, BMP6, CST6, HOXB4, HIST1H3C, ITIH5, NEUROD1, RASSF1, SOX17) in enriched CTCs from metastatic breast cancer patients. Feasibility and clinical performance testing were assessed in a test set consisting of 37 patients and 25 healthy controls. With established cut-off values from the healthy control group, methylation of enriched CTCs was detected in at least one gene in 18/37 patients (48.6%), while 97.8% of all control samples were unmethylated. Patients with CTCs unmethylated for CST6, ITIH5, or RASSF1 showed significantly longer PFS compared to patients with corresponding enriched methylated CTCs. This proof-of-principle study shows the feasibility of a size-based microfilter to enrich and analyze multigene methylation profile of CTCs from metastatic breast cancer patients. For the first time, we report that multigene methylation analysis of enriched CTCs provides prognostic information in metastatic breast cancer patients.

Frequency and clinical impact of preoperative circulating tumor cells in resectable non-metastatic lung adenocarcinomas.

OBJECTIVES: Despite successful surgery, 30-50% of patients with resectable non-small cell lung cancer develop tumor recurrence within 5 years of surgery. MATERIALS AND METHODS: In this prospective study, we performed CTC enumerations in 40 patients with non-metastatic lung adenocarcinoma (NMLA) using a size-based microfilter. Additionally, cfDNA isolated from plasma was analyzed in 35 out of 40 patients. RESULTS: CTCs were identified in 15 out of 40 patients (37.5%) with a range of 1-44 cells, whereas mutated cfDNA was only detected in 3 out of 35 patients (8.6%). Disease-free survival (DFS) was significantly associated with CTC positivity (log-rank p=0.025), grading (log-rank p=0.019), tumor stage (log-rank p=0.025) and lymph node status (log-rank p=0.029). Multivariate analysis, including tumor stage and grading, showed that CTC positivity (p=0.006), grading (0.039) and tumor stage (p=0.022) were independently associated with DFS. CONCLUSION: Our study found that microfilter-based CTC enumeration in NMLA patients is an independent predictor of worse DFS. The used NGS-based cfDNA characterization had limited sensitivity to be clinically informative in our study cohort. CTC assessment before surgery can thus identify NMLA patients at high risk of disease recurrence.

Primary patient-derived lung adenocarcinoma cell culture challenges the association of cancer stem cells with epithelial-to-mesenchymal transition.

The cancer stem cell (CSC) and epithelial-to-mesenchymal transition (EMT) models have been closely associated and used to describe both the formation of metastasis and therapy resistance. We established a primary lung cell culture from a patient in a clinically rare and unique situation of primary resistant disease. This culture consisted of two biologically profoundly distinct adenocarcinoma cell subpopulations, which differed phenotypically and genotypically. One subpopulation initiated and sustained in spheroid cell culture (LT22s) whereas the other subpopulation was only capable of growth and proliferation under adherent conditions (LT22a). In contrast to our expectations, LT22s were strongly associated with the epithelial phenotype, and expressed additionally CSC markers ALDH1 and CD133, whereas the LT22a was characterized as mesenchymal with lack of CSC markers. The LT22s cells also demonstrated an invasive behavior and mimicked gland formation. Finally, LT22s were more resistant to Cisplatin than LT22a cells. We demonstrate a primary lung adenocarcinoma cell culture derived from a patient with resistant disease, with epithelial aggressive subpopulation of cells associated with stem cell features and therapy resistance. Our findings challenge the current model associating CSC and disease resistance mainly to mesenchymal cells and may have important clinical implications.

Genetic profiling of putative breast cancer stem cells from malignant pleural effusions.

A common symptom during late stage breast cancer disease is pleural effusion, which is related to poor prognosis. Malignant cells can be detected in pleural effusions indicating metastatic spread from the primary tumor site. Pleural effusions have been shown to be a useful source for studying metastasis and for isolating cells with putative cancer stem cell (CSC) properties. For the present study, pleural effusion aspirates from 17 metastatic breast cancer patients were processed to propagate CSCs in vitro. Patient-derived aspirates were cultured under sphere forming conditions and isolated primary cultures were further sorted for cancer stem cell subpopulations ALDH1+ and CD44+CD24-/low. Additionally, sphere forming efficiency of CSC and non-CSC subpopulations was determined. In order to genetically characterize the different tumor subpopulations, DNA was isolated from pleural effusions before and after cell sorting, and compared with corresponding DNA copy number profiles from primary tumors or bone metastasis using low-coverage whole genome sequencing (SCNA-seq). In general, unsorted cells had a higher potential to form spheres when compared to CSC subpopulations. In most cases, cell sorting did not yield sufficient cells for copy number analysis. A total of five from nine analyzed unsorted pleura samples (55%) showed aberrant copy number profiles similar to the respective primary tumor. However, most sorted subpopulations showed a balanced profile indicating an insufficient amount of tumor cells and low sensitivity of the sequencing method. Finally, we were able to establish a long term cell culture from one pleural effusion sample, which was characterized in detail. In conclusion, we confirm that pleural effusions are a suitable source for enrichment of putative CSC. However, sequencing based molecular characterization is impeded due to insufficient sensitivity along with a high number of normal contaminating cells, which are masking genetic alterations of rare cancer (stem) cells.