Radium-223 in women with hormone receptor-positive bone-metastatic breast cancer receiving endocrine therapy: pooled analysis of two international, phase 2, randomized, double-blind, placebo-controlled trials.

BACKGROUND: Most women with advanced breast cancer have skeletal metastases. Radium-223 is an alpha-emitting radionuclide that selectively targets areas of bone metastases. METHODS: Two double-blind, placebo-controlled studies of radium-223 were conducted in women with hormone receptor-positive (HR+), bone-predominant metastatic breast cancer. All patients received endocrine therapy (ET), as a single agent of the investigator's choice (Study A) or exemestane + everolimus (Study B). Patients were randomized to receive radium-223 (55 kBq/kg) or placebo intravenously every 4 weeks for six doses. Accrual was halted following unblinded interim analyses per protocol amendments, and both studies were terminated. We report pooled analyses of symptomatic skeletal event-free survival (SSE-FS; primary endpoint), radiologic progression-free survival (rPFS) and overall survival (OS; secondary), and time to bone alkaline phosphatase (ALP) progression (exploratory). RESULTS: In total, 382 patients were enrolled, and 196 SSE-FS events (70% planned total) were recorded. Hazard ratios (95% confidence intervals) and nominal p values for radium-223 + ET versus placebo + ET were: SSE-FS 0.809 (0.610-1.072), p = 0.1389; rPFS 0.956 (0.759-1.205), p = 0.7039; OS 0.889 (0.660-1.199), p = 0.4410; and time to bone ALP progression 0.593 (0.379-0.926), p = 0.0195. Radium-223- or placebo-related treatment-emergent adverse events were reported in 50.3% versus 35.1% of patients (grade 3/4: 25.7% vs. 8.5%), with fractures/bone-associated events in 23.5% versus 23.9%. CONCLUSIONS: In patients with HR+ bone-metastatic breast cancer, numeric differences favoring radium-223 + ET over placebo + ET for the primary SSE-FS endpoint were suggestive of efficacy, in line with the primary outcome measure used in the underlying phase 2 studies. No similar evidence of efficacy was observed for secondary progression or survival endpoints. Adverse events were more frequent with radium-223 + ET versus placebo + ET, but the safety profile of the combination was consistent with the safety profiles of the component drugs. Clinical trial registration numbers Study A: NCT02258464, registered October 7, 2014. Study B: NCT02258451, registered October 7, 2014.

Late effects after breast cancer treatment. / Seneffekter etter behandling av brystkreft.

Breast cancer is the most common cancer among women in Norway. Nine out of ten will become long-term survivors. Being cancer-free does not necessarily mean feeling healthy, and many experience troublesome late effects, such as fatigue, pain and fear of recurrence. General practitioners represent the most important medical support for the majority of these women. This clinical review article summarises up-to-date knowledge about late effects after breast cancer treatment. Non-pharmacological interventions can have a positive effect on many of the most common late effects.

Compliance with recommended cancer patient pathway timeframes and choice of treatment differed by cancer type and place of residence among cancer patients in Norway in 2015-2016.

BACKGROUND: Cancer patient pathways (CPPs) were implemented in Norway to reduce unnecessary waiting times, regional variations, and to increase the predictability of cancer care for the patients. This study aimed to determine if 70% of cancer patients started treatment within the recommended time frames, and to identify potential delays. METHODS: Patients registered with a colorectal, lung, breast, or prostate cancer diagnosis at the Cancer Registry of Norway in 2015-2016 were linked with the Norwegian Patient Registry and Statistics Norway. Adjusting for sociodemographic variables, multivariable quantile (median) regressions were used to examine the association between place of residence and median time to start of examination, treatment decision, and start of treatment. RESULTS: The study included 20 668 patients. The proportions of patients who went through the CPP within the recommended time frames were highest among colon (84%) and breast (76%) cancer patients who underwent surgery and lung cancer patients who started systemic anticancer treatment (76%), and lowest for prostate cancer patients who underwent surgery (43%). The time from treatment decision to start of treatment was the main source of delay for all cancers. Travelling outside the resident health trust prolonged waiting time and was associated with a reduced odds of receiving surgery and radiotherapy for lung and rectal cancer patients, respectively. CONCLUSIONS: Achievement of national recommendations of the CCP times differed by cancer type and treatment. Identified bottlenecks in the pathway should be targeted to decrease waiting times. Further, CPP guidelines should be re-examined to determine their ongoing relevance.

Immune phenotype of tumor microenvironment predicts response to bevacizumab in neoadjuvant treatment of ER-positive breast cancer.

Antiangiogenic drugs are potentially a useful supplement to neoadjuvant chemotherapy for a subgroup of patients with human epidermal growth factor receptor 2 (HER2) negative breast cancer, but reliable biomarkers for improved response are lacking. Here, we report on a randomized phase II clinical trial to study the added effect of bevacizumab in neoadjuvant chemotherapy with FEC100 (5-fluorouracil, epirubicin and cyclophosphamide) and taxanes (n = 132 patients). Gene expression from the tumors was obtained before neoadjuvant treatment, and treatment response was evaluated by residual cancer burden (RCB) at time of surgery. Bevacizumab increased the proportion of complete responders (RCB class 0) from 5% to 20% among patients with estrogen receptor (ER) positive tumors (P = .02). Treatment with bevacizumab was associated with improved 8-year disease-free survival (P = .03) among the good responders (RCB class 0 or I). Patients treated with paclitaxel (n = 45) responded better than those treated with docetaxel (n = 21; P = .03). Improved treatment response was associated with higher proliferation rate and an immune phenotype characterized by high presence of classically activated M1 macrophages, activated NK cells and memory activated CD4 T cells. Treatment with bevacizumab increased the number of adverse events, including hemorrhage, hypertension, infection and febrile neutropenia, but despite this, the ECOG status was not affected.

Circulating Tumor Cells are an Independent Predictor of Shorter Survival in Patients Undergoing Resection for Pancreatic and Periampullary Adenocarcinoma.

OBJECTIVE: We evaluated the prognostic impact of circulating tumor cells (CTCs) for patients with presumed resectable pancreatic and periampullary cancers. SUMMARY OF BACKGROUND DATA: Initial treatment decisions for this group are currently taken without a reliable prognostic marker. The CellSearch system allows standardized CTC-testing and has shown excellent specificity and prognostic value in other applications. METHODS: Preoperative blood samples from 242 patients between September 2009 and December 2014 were analyzed. One hundred seventy-nine patients underwent tumor resection, of whom 30 with stage-I tumors and duodenal cancer were assigned to the low-risk group, and the others to the high-risk group. Further 33 had advanced disease, 30 benign histology. Observation ended in December 2016. Cancer-specific survival (CSS) and disease-free survival (DFS) were calculated by log-rank and Cox regression. RESULTS: CTCs (CTC-positive; ≥1 CTC/7.5 mL) were detected in 6.8% (10/147) of the high-risk patients and 6.2% (2/33) with advanced disease. No CTCs (CTC-negative) were detected in the low-risk patients or benign disease. In high-risk patients, median CSS for CTC-positive versus CTC-negative was 8.1 versus 20.0 months (P < 0.0001), and DFS 4.0 versus 10.5 months (P < 0.001). Median CSS in advanced disease was 7.7 months. Univariate hazard ratio (HR) of CTC-positivity was 3.4 (P < 0.001). In multivariable analysis, CTC-status remained independent (HR: 2.4, P = 0.009) when corrected for histological type (HR: 2.7, P = 0.030), nodal status (HR: 1.7, P = 0.016), and vascular infiltration (HR: 1.7, P = 0.001). CONCLUSION: Patients testing CTC-positive preoperatively showed a detrimental outcome despite successful tumor resections. Although the low CTC-rate seems a limiting factor, results indicate high specificity. Thus, preoperative analysis of CTCs by this test may guide treatment decisions and warrants further testing in clinical trials.

Patient and tumour characteristics associated with inclusion in Cancer patient pathways in Norway in 2015-2016.

BACKGROUND: Cancer patient pathways (CPPs) were implemented in 2015 to reduce waiting time, regional variation in waiting time, and to increase the predictability of cancer care for the patients. The aims of this study were to see if the national target of 70% of all cancer patients being included in a CPP was met, and to identify factors associated with CPP inclusion. METHODS: All patients registered with a colorectal, lung, breast or prostate cancer diagnosis at the Cancer Registry of Norway in the period 2015-2016 were linked with the Norwegian Patient Registry for CPP information and with Statistics Norway for sociodemographic variables. Multivariable logistic regression examined if the odds of not being included in a CPP were associated with year of diagnosis, age, sex, tumour stage, marital status, education, income, region of residence and comorbidity. RESULTS: From 2015 to 2016, 30,747 patients were diagnosed with colorectal, lung, breast or prostate cancer, of whom 24,429 (79.5%) were included in a CPP. Significant increases in the probability of being included in a CPP were observed for colorectal (79.1 to 86.2%), lung (79.0 to 87.3%), breast (91.5 to 97.2%) and prostate cancer (62.2 to 76.2%) patients (p < 0.001). Increasing age was associated with an increased odds of not being included in a CPP for lung (p < 0.001) and prostate cancer (p < 0.001) patients. Colorectal cancer patients < 50 years of age had a two-fold increase (OR = 2.23, 95% CI: 1.70-2.91) in the odds of not being included in a CPP. The odds of no CPP inclusion were significantly increased for low income colorectal (OR = 1.24, 95%CI: 1.00-1.54) and lung (OR = 1.52, 95%CI: 1.16-1.99) cancer patients. Region of residence was significantly associated with CPP inclusion (p < 0.001) and the probability, adjusted for case-mix ranged from 62.4% in region West among prostate cancer patients to 97.6% in region North among breast cancer patients. CONCLUSIONS: The national target of 70% was met within 1 year of CPP implementation in Norway. Although all patients should have equal access to CPPs, a prostate cancer diagnosis, older age, high level of comorbidity or low income were significantly associated with an increased odds of not being included in a CPP.

Cytokeratin-positive cells in the bone marrow from patients with pancreatic, periampullary malignancy and benign pancreatic disease show no prognostic information.

BACKGROUND: Pancreatic and periampullary carcinoma are aggressive tumours where preoperative assessment is challenging. Disseminated tumour cells (DTC) in the bone marrow (BM) are associated with impaired prognosis in a variety of epithelial cancers. In a cohort of patients with presumed resectable pancreatic and periampullary carcinoma, we evaluated the frequency and the potential prognostic impact of the preoperative presence of DTC, defined as cytokeratin-positive cells detected by immunocytochemistry (ICC). METHODS: Preoperative BM samples from 242 patients selected for surgical resection of presumed resectable pancreatic and periampullary carcinoma from 09/2009 to 12/2014, were analysed for presence of CK-positive cells by ICC. The median observation time was 21.5 months. Overall survival (OS) and disease-free survival (DFS) were calculated by Kaplan-Meier and Cox regression analysis. RESULTS: Successful resections of malignant tumours were performed in 179 of the cases, 30 patients resected had benign pancreatic disease based on postoperative histology, and 33 were deemed inoperable intraoperatively due to advanced disease. Overall survival for patients with resected carcinoma was 21.1 months (95% CI: 18.0-24.1), for those with benign disease OS was 101 months (95% CI: 69.4-132) and for those with advanced disease OS was 8.8 months (95% CI: 4.3-13.3). The proportion of patients with detected CK-positive cells was 6/168 (3.6%) in resected malignant cases, 2/31 (6.5%) in advanced disease and 4/29 (13.8%) in benign disease. The presence of CK-positive cells was not correlated to OS or DFS, neither in the entire cohort nor in the subgroup negative for circulating tumour cells (CTC). CONCLUSIONS: The results indicate that CK-positive cells may be present in both patients with malignant and benign diseases of the pancreas. Detection of CK-positive cells was not associated with differences in prognosis for the entire cohort or any of the subgroups analysed. TRIAL REGISTRATION: clinicaltrials.gov ( NCT01919151 ).

NR2F1 stratifies dormant disseminated tumor cells in breast cancer patients.

BACKGROUND: The presence of disseminated tumor cells (DTCs) in bone marrow (BM) is an independent prognostic factor in early breast cancer but does not uniformly predict outcome. Tumor cells can persist in a quiescent state over time, but clinical studies of markers predicting the awakening potential of DTCs are lacking. Recently, experiments have shown that NR2F1 (COUP-TF1) plays a key role in dormancy signaling. METHODS: We analyzed the NR2F1 expression in DTCs by double immunofluorescence (DIF) staining of extra cytospins prepared from 114 BM samples from 86 selected DTC-positive breast cancer patients. Samples collected at two or more time points were available for 24 patients. Fifteen samples were also analyzed for the proliferation marker Ki67. RESULTS: Of the patients with detectable DTCs by DIF, 27% had ≥ 50% NR2F1high DTCs, chosen a priori as the cut-off for "dormant profile" classification. All patients with systemic relapse within 12 months after BM aspiration carried ≤ 1% NR2F1high DTCs, including patients who transitioned from having NR2F1high-expressing DTCs in previous BM samples. Of the patients with serial samples, half of those with no relapse at follow-up had ≥ 50% NR2F1high DTCs in the last BM aspiration analyzed. Among the 18 relapse-free patients at the time of the last DTC-positive BM aspiration with no subsequent BM analysis performed, distant disease-free intervals were favorable for patients carrying ≥ 50% NR2F1high DTCs compared with those with predominantly NR2F1low DTCs (p = 0.007, log-rank). No survival difference was observed by classification according to Ki67-expressing DTCs (p = 0.520). CONCLUSIONS: Our study translates findings from basic biological analysis of DTC dormancy to the clinical situation and supports further clinical studies of NR2F1 as a marker of dormancy.

Serum concentrations of active tamoxifen metabolites predict long-term survival in adjuvantly treated breast cancer patients.

BACKGROUND: Controversies exist as to whether the genetic polymorphisms of the enzymes responsible for the metabolism of tamoxifen can predict breast cancer outcome in patients using adjuvant tamoxifen. Direct measurement of concentrations of active tamoxifen metabolites in serum may be a more biological plausible and robust approach. We have investigated the association between CYP2D6 genotypes, serum concentrations of active tamoxifen metabolites, and long-term outcome in tamoxifen treated breast cancer patients. METHODS: From an original observational study comprising 817 breast cancer patients, 99 women with operable breast cancer were retrospectively included in the present study. This cohort of patients were adjuvantly treated with tamoxifen, had provided serum samples suitable for measuring tamoxifen metabolites, and were relapse-free at 3 years after the primary treatment commenced. The median follow-up time from this entry point to breast cancer death was 13.9 years. Patients were CYP2D6 genotyped and grouped into four CYP2D6 phenotype groups (Ultra rapid, extensive, intermediate, and poor metabolizers). Tamoxifen and nine metabolites were quantified in serum (n = 86) and compared with CYP2D6 phenotype groups and outcome. RESULTS: Breast cancer patients with low concentrations of Z-4-hydroxy-tamoxifen (Z-4OHtam; ≤ 3.26 nM) had a breast cancer-specific survival (BCSS) of 60% compared to 84% in patients with Z-4OHtam concentrations > 3.26 nM (p = 0.020, log-rank hazard ratio (HR) = 3.56, 95% confidence interval (CI) = 1.14-11.07). For patients with Z-4-hydroxy-N-desmethyl-tamoxifen (Z-endoxifen) levels ≤ 9.00 nM BCSS was 57% compared to 84% for patients with concentrations > 9.00 nM (p = 0.029, HR = 3.73, 95% CI = 1.05-13.22). Low concentrations of Z-4OHtam and Z-endoxifen were associated with poorer survival also after adjusting for clinically relevant variables (HR = 4.27, 95% CI = 1.35-13.58, and HR = 3.70, 95% CI = 1.03-13.25, respectively). Overall survival analysis showed similar survival differences for both active metabolites. The Antiestrogen Activity Score showed comparable effects, but did not improve the prognostic information. CONCLUSIONS: Patients with Z-4OHtam and Z-endoxifen concentrations lower than 3.26 nM or 9.00 nM, respectively, showed an adverse outcome. Our results suggest that direct measurement of active tamoxifen metabolite concentrations could be of clinical value. Validation in larger study cohorts is warranted.

Prognostic value of PAM50 and risk of recurrence score in patients with early-stage breast cancer with long-term follow-up.

BACKGROUND: The aim of this study was to investigate the prognostic value of the PAM50 intrinsic subtypes and risk of recurrence (ROR) score in patients with early breast cancer and long-term follow-up. A special focus was placed on hormone receptor-positive/human epidermal growth factor receptor 2-negative (HR+/HER2-) pN0 patients not treated with chemotherapy. METHODS: Patients with early breast cancer (n = 653) enrolled in the observational Oslo1 study (1995-1998) were followed for distant recurrence and breast cancer death. Clinicopathological parameters were collected from hospital records. The primary tumors were analyzed using the Prosigna® PAM50 assay to determine the prognostic value of the intrinsic subtypes and ROR score in comparison with pathological characteristics. The primary endpoints were distant disease-free survival (DDFS) and breast cancer-specific survival (BCSS). RESULTS: Of 653 tumors, 52.2% were classified as luminal A, 26.5% as luminal B, 10.6% as HER2-enriched, and 10.7% as basal-like. Among the HR+/HER2- patients (n = 476), 37.8% were categorized as low risk by ROR score, 22.7% as intermediate risk, and 39.5% as high risk. Median follow-up durations for BCSS and DDFS were 16.6 and 7.1 years, respectively. Multivariate analysis showed that intrinsic subtypes (all patients) and ROR risk classification (HR+/HER2- patients) yielded strong prognostic information. Among the HR+/HER2- pN0 patients with no adjuvant treatment (n = 231), 53.7% of patients had a low ROR, and their prognosis at 15 years was excellent (15-year BCSS 96.3%). Patients with intermediate risk had reduced survival compared with those with low risk (p = 0.005). In contrast, no difference in survival between the low- and intermediate-risk groups was seen for HR+/HER2- pN0 patients who received tamoxifen only. Ki-67 protein, grade, and ROR score were analyzed in the unselected, untreated pT1pN0 HR+/HER2- population (n = 171). In multivariate analysis, ROR score outperformed both Ki-67 and grade. Furthermore, 55% of patients who according to the PREDICT tool ( http://www.predict.nhs.uk/ ) would be considered chemotherapy candidates were ROR low risk (33%) or luminal A ROR intermediate risk (22%). CONCLUSIONS: The PAM50 intrinsic subtype classification and ROR score improve classification of patients with breast cancer into prognostic groups, allowing for a more precise identification of future recurrence risk and providing an improved basis for adjuvant treatment decisions. Node-negative patients with low ROR scores had an excellent outcome at 15 years even in the absence of adjuvant therapy.

Integrative clustering reveals a novel split in the luminal A subtype of breast cancer with impact on outcome.

BACKGROUND: Breast cancer is a heterogeneous disease at the clinical and molecular level. In this study we integrate classifications extracted from five different molecular levels in order to identify integrated subtypes. METHODS: Tumor tissue from 425 patients with primary breast cancer from the Oslo2 study was cut and blended, and divided into fractions for DNA, RNA and protein isolation and metabolomics, allowing the acquisition of representative and comparable molecular data. Patients were stratified into groups based on their tumor characteristics from five different molecular levels, using various clustering methods. Finally, all previously identified and newly determined subgroups were combined in a multilevel classification using a "cluster-of-clusters" approach with consensus clustering. RESULTS: Based on DNA copy number data, tumors were categorized into three groups according to the complex arm aberration index. mRNA expression profiles divided tumors into five molecular subgroups according to PAM50 subtyping, and clustering based on microRNA expression revealed four subgroups. Reverse-phase protein array data divided tumors into five subgroups. Hierarchical clustering of tumor metabolic profiles revealed three clusters. Combining DNA copy number and mRNA expression classified tumors into seven clusters based on pathway activity levels, and tumors were classified into ten subtypes using integrative clustering. The final consensus clustering that incorporated all aforementioned subtypes revealed six major groups. Five corresponded well with the mRNA subtypes, while a sixth group resulted from a split of the luminal A subtype; these tumors belonged to distinct microRNA clusters. Gain-of-function studies using MCF-7 cells showed that microRNAs differentially expressed between the luminal A clusters were important for cancer cell survival. These microRNAs were used to validate the split in luminal A tumors in four independent breast cancer cohorts. In two cohorts the microRNAs divided tumors into subgroups with significantly different outcomes, and in another a trend was observed. CONCLUSIONS: The six integrated subtypes identified confirm the heterogeneity of breast cancer and show that finer subdivisions of subtypes are evident. Increasing knowledge of the heterogeneity of the luminal A subtype may add pivotal information to guide therapeutic choices, evidently bringing us closer to improved treatment for this largest subgroup of breast cancer.

Prognostic significance of S100A4-expression and subcellular localization in early-stage breast cancer.

PURPOSE: Prognostic factors are useful in order to identify early-stage breast cancer patients who might benefit from adjuvant treatment. The metastasis-promoting protein S100A4 has previously been associated with poor prognosis in breast cancer patients. The protein is expressed in diverse subcellular compartments, including the cytoplasm, extracellular space, and nucleus. Nuclear expression is an independent predictor of poor outcome in several cancer types, but the significance of subcellular expression has not yet been assessed in breast cancer. METHODS: Nuclear and cytoplasmic expression of S100A4 was assessed by immunohistochemistry in prospectively collected tumor samples from early-stage breast cancer patients using tissue microarrays. RESULTS: In patients not receiving adjuvant systemic therapy, nuclear or cytoplasmic expression was found in 44/291 tumors (15%). Expression of either nuclear or cytoplasmic S100A4 was associated with histological grade III, triple-negative subtype, and Ki-67-expression. Patients with S100A4-positive tumors had inferior metastasis-free and overall survival compared to S100A4-negative. When expression was analyzed separately, nuclear S100A4 was a significant predictor of outcome, while cytoplasmic was not. In patients who received adjuvant treatment 23/300 tumors (8%) were S100A4-positive, but no tumors displayed nuclear staining alone. S100A4-expression was strongly associated with histological grade III and triple-negative subtype. Although not significant, metastasis-free and overall survival was numerically reduced in patients with S100A4-positive tumors. CONCLUSION: S100A4-expression was associated with poor outcome in early-stage breast cancer, but the low percentage of positive tumors and the modest survival differences imply that the clinical utility in selection of patients for adjuvant treatment is limited.

Survival Impact of Primary Tumor Lymph Node Status and Circulating Tumor Cells in Patients with Colorectal Liver Metastases.

OBJECTIVE: The aim of this study was to analyse the survival impact of primary tumor nodal status (N0/N+) in patients with resectable colorectal liver metastases (CLM), and to determine the value of circulating and disseminated tumor cells (CTCs/DTCs) in this setting. METHODS: In this prospective study of patients undergoing resection of CLM from 2008 to 2011, peripheral blood was analyzed for CTCs using the CellSearch System®, and bone marrow was sampled for DTC analyses just prior to hepatic resection. The presence of one or more tumor cells was scored as CTC/DTC-positive. Following resection of the primary tumor, the lymph nodes (LNs) were examined by routine histopathological examination. RESULTS: A total of 140 patients were included in this study; 38 patients (27.1%) were negative at the primary colorectal LN examination (N0). CTCs were detected in 12.1% of all patients; 5.3% of patients in the N0 group and 14.7% of patients in the LN-positive (N+) group (p = 0.156), with the LN-positive group (N+) consisting of both N1 and N2 patients. There was a significant difference in recurrence-free survival (RFS) when analysing the N0 group versus the N+ group (p = 0.007) and CTC-positive versus CTC-negative patients (p = 0.029). In multivariate analysis, CTC positivity was also significantly associated with impaired overall survival (OS) [p = 0.05], whereas DTC positivity was not associated with survival. CONCLUSION: In this cohort of resectable CLM patients, 27% had primary N0 colorectal cancer. Assessment of CTC in addition to nodal status may contribute to improved classification of patients into high- and low-risk groups, which has the potential to guide and improve treatment strategies.

Specific antibodies and sensitive immunoassays for the human epidermal growth factor receptors (HER2, HER3, and HER4).

The use of trastuzumab in patients with breast cancer that overexpresses human epidermal growth factor receptor 2 has significantly improved treatment outcomes. However, a substantial proportion of this patient group still experiences progression of the disease after receiving the drug. Evaluation of the changes in expression of the human epidermal growth factor receptors could be of interest. Monoclonal antibodies against the extracellular domain of the human growth factor receptors, 2, 3, and 4, have been raised, and specific and sensitive immunoassays have been established. Sera from healthy individuals (Nordic Reference Interval Project and Database) were analyzed in the human epidermal growth factor receptor 2 assay (N = 805) and the human epidermal growth factor receptor 3 and 4 assays (N = 114), and reference limits were calculated. In addition, sera from 208 individual patients with breast cancer were tested in all three assays. Finally, the human epidermal growth factor receptor 2 assay was compared with a chemiluminescent immunoassay for serum human epidermal growth factor receptor 2/neu. Reference values were as follows: human epidermal growth factor receptor 2, <2.5 µg/L; human epidermal growth factor receptor 3, <2.8 µg/L; and human epidermal growth factor receptor 4, <1.8 µg/L. There were significant differences in human epidermal growth factor receptor 2 and human epidermal growth factor receptor 3 serum levels between the patients with tissue human epidermal growth factor receptor 2-positive and tissue human epidermal growth factor receptor 2-negative ( p = 0.0026, p = 0.000011) tumors, but not in the serum levels of human epidermal growth factor receptor 4 ( p = 0.054). There was good agreement between the in-house human epidermal growth factor receptor 2 assay and the chemiluminescent immunoassay. Our new specific antibodies for all the three human epidermal growth factor receptors may prove valuable in the development of novel anti-human epidermal growth factor receptor targeted therapies with sensitive immunoassays for measuring serum levels of the respective targets and in monitoring established treatment.

Quantitative multigene FISH on breast carcinomas identifies der(1;16)(q10;p10) as an early event in luminal A tumors.

In situ detection of genomic alterations in cancer provides information at the single cell level, making it possible to investigate genomic changes in cells in a tissue context. Such topological information is important when studying intratumor heterogeneity as well as alterations related to different steps in tumor progression. We developed a quantitative multigene fluorescence in situ hybridization (QM FISH) method to detect multiple genomic regions in single cells in complex tissues. As a "proof of principle" we applied the method to breast cancer samples to identify partners in whole arm (WA) translocations. WA gain of chromosome arm 1q and loss of chromosome arm 16q are among the most frequent genomic events in breast cancer. By designing five specific FISH probes based on breakpoint information from comparative genomic hybridization array (aCGH) profiles, we visualized chromosomal translocations in clinical samples at the single cell level. By analyzing aCGH data from 295 patients with breast carcinoma with known molecular subtype, we found concurrent WA gain of 1q and loss of 16q to be more frequent in luminal A tumors compared to other molecular subtypes. QM FISH applied to a subset of samples (n = 26) identified a derivative chromosome der(1;16)(q10;p10), a result of a centromere-close translocation between chromosome arms 1q and 16p. In addition, we observed that the distribution of cells with the translocation varied from sample to sample, some had a homogenous cell population while others displayed intratumor heterogeneity with cell-to-cell variation. Finally, for one tumor with both preinvasive and invasive components, the fraction of cells with translocation was lower and more heterogeneous in the preinvasive tumor cells compared to the cells in the invasive component.

Circulating tumor cells in patients with colorectal liver metastasis predict impaired survival.

OBJECTIVE: The aim of the study is to assess the prognostic and predictive value of circulating tumor cells (CTCs) and disseminated tumor cells (DTCs) in bone marrow (BM) in patients with colorectal liver metastasis referred to surgery. BACKGROUND: A total of 194 patients were included. Treatment of the patients was decided in a multidisciplinary team. METHODS: BM aspirates and blood samples were collected at surgery, or in local anesthesia in nonresectable patients. CTCs were disclosed with CellSearch System, DTC with immunocytology. RESULTS: Liver resection was completed in 153 patients. Forty-one patients were nonresectable, 22 preoperatively and 19 intraoperatively. The median follow-up was 22 (range 1-61) months. Relapse was diagnosed in 103 of the resected patients. Totally, 67 patients died of cancer. CTCs were detected in 19.6% of the patients. CTC positivity was significantly higher in nonresectable (46%) than in resectable patients (11.7%), P < 0.001. 13.8% of the patients had 2 or more CTCs, 31% of the nonresectable and 9.1% of the resectable patients (P = 0.001). Patients with 2 or more CTCs experienced reduced time to relapse/progression, both analyzing all patients (P = 0.002) and analyzing resectable patients (P < 0.001). Two or more CTCs was a strong predictor of progression and mortality in all subgroups of patients, together with more than 3 liver metastases, R1 resection, and extrahepatic disease. DTCs were detected in 9.9% of the patients, but not associated with clinical outcome in resectable patients. CONCLUSIONS: CTCs predict nonresectability and impaired survival. CTC analysis should be considered as a tool for decision-making before liver resection in these patients.

Detection of Circulating Tumor Cells at Surgery and at Follow-Up Assessment to Predict Survival After Two-Stage Liver Resection of Colorectal Liver Metastases.

BACKGROUND: The presence of circulating tumor cells (CTCs) is negatively associated with survival after resection of colorectal liver metastases (CLM). The current study aimed to determine the prognostic value of CTCs and disseminated tumor cells (DTCs) at the time of surgery and the prognostic value of CTCs at follow-up assessment, for patients scheduled to undergo two-stage hepatectomy with portal vein embolization (PVE) for CLM. METHODS: Samples were collected at surgery (blood and bone marrow) and at follow-up assessment (blood) for the period 2008 through 2011. In this study, CTCs were detected with the CellSearch system, and DTCs were detected using standard immunocytochemical analysis. RESULTS: Of 24 patients, 18 completed both stages, and no patients were lost to follow-up. The median overall survival (OS) was 37 months, and the median recurrence-free survival (RFS) was 7 months. At surgery, CTCs were found in nine patients (38 %), and their presence was associated with reduced OS (p < 0.001) and RFS (p = 0.006). Follow-up CTC status was available for 11 patients. All eight patients with positive CTC status experienced recurrence. Two of three patients with negative CTC status remained recurrence free. In seven patients (32 %), DTCs were detected but were not associated with OS or RFS. CONCLUSIONS: The presence of CTCs at surgery is associated with worse OS and RFS for patients undergoing two-stage hepatectomy with PVE for CLM. Analysis of CTCs should be explored further for their potential to assist in treatment decisions and monitoring for CLM patients.

Expression of an estrogen-regulated variant transcript of the peroxisomal branched chain fatty acid oxidase ACOX2 in breast carcinomas.

BACKGROUND: Alternate transcripts from a single gene locus greatly enhance the combinatorial flexibility of the human transcriptome. Different patterns of exon usage have been observed when comparing normal tissue to cancers, suggesting that variant transcripts may play a role in the tumor phenotype. METHODS: Ribonucleic acid-sequencing (RNA-seq) data from breast cancer samples was used to identify an intronic start variant transcript of Acyl-CoA oxidase 2, ACOX2 (ACOX2-i9). Difference in expression between Estrogen Receptor (ER) positive and ER negative patients was assessed by the Wilcoxon rank sum test, and the findings validated in The Cancer Genome Atlas (TCGA) breast cancer dataset (BRCA). ACOX2-i9 expression was also assessed in cell lines using both quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and Western blot analysis. Knock down by short hairpin RNA (shRNA) and colony formation assays were used to determine whether ACOX2-i9 expression would influence cellular fitness. The effect of ACOX2-i9 expression on patient survival was assessed by the Kaplan-Meier survival function, and association to clinical parameters was analyzed using a Fisher exact test. RESULTS: The expression and translation of ACOX2-i9 into a 25 kDa protein was demonstrated in HepG2 cells as well as in several breast cancer cell lines. shRNA knock down of the ACOX2-i9 variant resulted in decreased cell viability of T47D and MDA-MB 436 cells. Moreover, expression of ACOX2-i9 was shown to be estrogen regulated, being induced by propyl pyrazoletriol and inhibited by tamoxifen and fulvestrant in ER+ T47D and Mcf-7 cells, but not in the ER- MDA-MB 436 cell line. This variant transcript showed expression predominantly in ER-positive breast tumors as assessed in our initial set of 53 breast cancers and further validated in 87 tumor/normal pairs from the TCGA breast cancer dataset, and expression was associated with better outcome in ER positive patients. CONCLUSIONS: ACOX2-i9 is specifically enriched in ER+ breast cancers where expression of the variant is associated with improved outcome. These data identify variant ACOX2 as a potential novel therapeutic biomarker in ER+ breast tumors.

Integrated molecular profiles of invasive breast tumors and ductal carcinoma in situ (DCIS) reveal differential vascular and interleukin signaling.

We use an integrated approach to understand breast cancer heterogeneity by modeling mRNA, copy number alterations, microRNAs, and methylation in a pathway context utilizing the pathway recognition algorithm using data integration on genomic models (PARADIGM). We demonstrate that combining mRNA expression and DNA copy number classified the patients in groups that provide the best predictive value with respect to prognosis and identified key molecular and stromal signatures. A chronic inflammatory signature, which promotes the development and/or progression of various epithelial tumors, is uniformly present in all breast cancers. We further demonstrate that within the adaptive immune lineage, the strongest predictor of good outcome is the acquisition of a gene signature that favors a high T-helper 1 (Th1)/cytotoxic T-lymphocyte response at the expense of Th2-driven humoral immunity. Patients who have breast cancer with a basal HER2-negative molecular profile (PDGM2) are characterized by high expression of protumorigenic Th2/humoral-related genes (24-38%) and a low Th1/Th2 ratio. The luminal molecular subtypes are again differentiated by low or high FOXM1 and ERBB4 signaling. We show that the interleukin signaling profiles observed in invasive cancers are absent or weakly expressed in healthy tissue but already prominent in ductal carcinoma in situ, together with ECM and cell-cell adhesion regulating pathways. The most prominent difference between low and high mammographic density in healthy breast tissue by PARADIGM was that of STAT4 signaling. In conclusion, by means of a pathway-based modeling methodology (PARADIGM) integrating different layers of molecular data from whole-tumor samples, we demonstrate that we can stratify immune signatures that predict patient survival.

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.