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Legends to supplementary table 1 and figure 1

Clinicopathological characteristics, CTC and CEC counts, and primary tumor EpCAM and MCAM staining specified per patient.

Circulating tumor cells (CTC) are detected by the CellSearch System in 20% to 25% of patients with primary breast cancer (pBC). To improve CTC detection, we investigated melanoma cell adhesion molecule (MCAM) as enrichment marker next to epithelial cell adhesion molecule (EpCAM) and tested the clinical relevance of MCAM-positive CTCs in patients with HER2-negative stage II/III pBC starting neoadjuvant chemotherapy (NAC) in the NEOZOTAC trial. Using the CellSearch System, EpCAM-positive and MCAM-positive CTCs were separately enriched from 7.5 mL blood, at baseline and after the first NAC cycle. Circulating endothelial cells (CEC) were measured using flow cytometry. Primary objective was to improve the CTC detection rate to ≥40% combining EpCAM/MCAM. Correlations of CTC and CEC counts and pathologic complete response (pCR) were also explored. At baseline, we detected EpCAM-positive and MCAM-positive CTCs in 12 of 68 (18%) and 8 of 68 (12%) patients, respectively. After one cycle, this was 7 of 44 (16%) and 7 of 44 (16%) patients, respectively. The detection rate improved from 18% at baseline and 16% after one cycle with EpCAM to 25% (P = 0.08) and 30% (P = 0.02), respectively, with EpCAM/MCAM. No patients with MCAM-positive CTCs versus 23% of patients without MCAM-positive CTCs at baseline achieved pCR (P = 0.13). EpCAM-positive CTCs and CEC counts were not correlated to pCR. Combined EpCAM/MCAM CellSearch enrichment thus increased the CTC detection rate in stage II/III pBC. We found no associations of CTC and CEC counts with pCR to NAC. The clinical relevance of MCAM-positive CTCs deserves further study. Mol Cancer Ther; 14(3); 821–7. ©2014 AACR.

Examples of EpCAM and MCAM staining in primary tumor tissues from core needle biopsies taken before start of neoadjuvant chemotherapy

PDF file, 81K, Online Table 1. Clinical Characteristics of Patients in Both Datasets. Online Table 2. Distribution of Recurrences by Group (Karolinka Dataset).

Supplementary Figures S1-S2 from mRNA and microRNA Expression Profiles in Circulating Tumor Cells and Primary Tumors of Metastatic Breast Cancer Patients

Supplementary Data from Local Recurrence after Breast-Conserving Therapy in Relation to Gene Expression Patterns in a Large Series of Patients

Colon tumor sample overview, clinical characteristics, fusion genes and KRAS, NRAS, BRAF mutation status and sequencing statistics.

Purpose: Previously, we developed a radiosensitivity molecular signature [radiosensitivity index (RSI)] that was clinically validated in 3 independent datasets (rectal, esophageal, and head and neck) in 118 patients. Here, we test RSI in radiotherapy (RT)-treated breast cancer patients.Experimental Design: RSI was tested in 2 previously published breast cancer datasets. Patients were treated at the Karolinska University Hospital (n = 159) and Erasmus Medical Center (n = 344). RSI was applied as previously described.Results: We tested RSI in RT-treated patients (Karolinska). Patients predicted to be radiosensitive (RS) had an improved 5-year relapse-free survival when compared with radioresistant (RR) patients (95% vs. 75%, P = 0.0212), but there was no difference between RS/RR patients treated without RT (71% vs. 77%, P = 0.6744), consistent with RSI being RT-specific (interaction term RSI × RT, P = 0.05). Similarly, in the Erasmus dataset, RT-treated RS patients had an improved 5-year distant metastasis-free survival over RR patients (77% vs. 64%, P = 0.0409), but no difference was observed in patients treated without RT (RS vs. RR, 80% vs. 81%, P = 0.9425). Multivariable analysis showed RSI is the strongest variable in RT-treated patients (Karolinska, HR = 5.53, P = 0.0987, Erasmus, HR = 1.64, P = 0.0758) and in backward selection (removal α of 0.10), RSI was the only variable remaining in the final model. Finally, RSI is an independent predictor of outcome in RT-treated ER+ patients (Erasmus, multivariable analysis, HR = 2.64, P = 0.0085).Conclusions: RSI is validated in 2 independent breast cancer datasets totaling 503 patients. Including prior data, RSI is validated in 5 independent cohorts (621 patients) and represents, to our knowledge, the most extensively validated molecular signature in radiation oncology. Clin Cancer Res; 18(18); 5134–43. ©2012 AACR.

Purpose: In this study, we aimed to explore whether low levels of mitochondrial DNA (mtDNA) content in the primary tumor could predict better outcome for breast cancer patients receiving anthracycline-based therapies. We hypothesized that tumor cells with low mtDNA content are more susceptible to mitochondrial damage induced by anthracyclines, and thus are more susceptible to anthracycline treatment.Experimental Design: We measured mtDNA content by a qPCR approach in 295 primary breast tumor specimens originating from two well-defined cohorts: 174 lymph node–positive patients who received adjuvant chemotherapy and 121 patients with advanced disease who received chemotherapy as first-line palliative treatment. The chemotherapy regimens given were either anthracycline-based (FAC/FEC) or methotrexate-based (CMF).Results: In both the adjuvant and advanced settings, we observed increased benefit for patients with low mtDNA content in their primary tumor, but only when treated with FAC/FEC. In multivariable Cox regression analysis for respectively distant metastasis-free survival and progression-free survival, the HR for the FAC/FEC-treated mtDNA low group in the adjuvant setting was 0.46 [95% confidence interval (CI), 0.24–0.89; P = 0.020] and in the advanced setting 0.49 (95% CI, 0.27–0.90; P = 0.022) compared with the FAC/FEC-treated mtDNA high group. We did not observe these associations in the patients treated with CMF.Conclusions: In our two study cohorts, breast cancer patients with low mtDNA content in their primary tumor had better outcome from anthracycline-containing chemotherapy. The frequently observed decrease in mtDNA content in primary breast tumors may be exploited by guiding chemotherapeutic regimen decision making. Clin Cancer Res; 23(16); 4735–43. ©2017 AACR.

Supplementary Table 1: Clinicopathological variables of node-positive patients receiving adjuvant chemotherapy (FAC/FEC or CMF) (complete adjuvant cohort). Supplementary Table 2: Clinicopathological variables of the patients with recurrent disease receiving first-line chemotherapy (FAC/FEC or CMF) (complete advanced cohort). Supplementary Table 3: Patient response to first-line FAC/FEC or CMF chemotherapy after disease recurrence (advanced cohort). Supplementary Table 4: Performance of the calibration curves taken along in each plate for the used qPCR assays. Supplementary Table 5: Association between mtDNA content and chemotherapy given, either adjuvant in the adjuvant cohort, or as first-line for disease recurrence in the advanced cohort. Supplementary Table 6: Association between adjuvant chemotherapy given and clinicopathological variables of node-positive patients (adjuvant cohort). Supplementary Table 7: Association between first-line chemotherapy given and clinicopathological variables of patients with recurrent disease (advanced cohort). Supplementary Table 8: Cox regression analysis for DMFS of node-positive patients receiving FAC/FEC adjuvant chemotherapy (adjuvant cohort) limited to the 86 patients with no missing values. Supplementary Table 9: Cox regression analysis for PFS of patients receiving FAC/FEC first-line chemotherapy for their recurrent disease (advanced cohort) limited to the 70 patients with no missing values. Supplementary Table 10: Univariable Cox regression analysis of postulated markers for anthracycline sensitivity. Supplementary Table 11: Association between mtDNA content and postulated markers for anthracycline sensitivity.

Supplementary Data from DNA Methylation Markers Predict Outcome in Node-Positive, Estrogen Receptor-Positive Breast Cancer with Adjuvant Anthracycline-Based Chemotherapy

Supplementary Tables S1-S3 from Association of an Extracellular Matrix Gene Cluster with Breast Cancer Prognosis and Endocrine Therapy Response

XLS file - 1258K

Purpose: The majority of patients with early-stage breast cancer are treated with breast-conserving therapy (BCT). Several clinical risk factors are associated with local recurrence (LR) after BCT but are unable to explain all instances of LR after BCT. Here, gene expression microarrays are used to identify novel risk factors for LR after BCT.Experimental Design: Gene expression profiles of 56 primary invasive breast carcinomas from patients who developed a LR after BCT were compared with profiles of 109 tumors from patients who did not develop a LR after BCT. Both unsupervised and supervised methods of classification were used to separate patients into groups corresponding to disease outcome. In addition, for 15 patients, the gene expression profile in the recurrence was compared with that of the primary tumor.Results: The two main clusters found by hierarchical cluster analysis of all 165 primary invasive breast carcinomas revealed no association with LR. Predefined gene sets (molecular subtypes and “chromosomal instability” signature) are associated with LR (P = 0.0002 and 0.003, respectively). Significant analysis of microarrays revealed an association between LR and cell proliferation, not captured by histologic grading. Class prediction analysis constructed a gene classifier, which was successfully validated, cross-platform, on an independent data set of 161 patients (log-rank P = 0.041). In multivariate analysis, young age was the only independent predictor of LR.Conclusions: We have constructed and cross-platform validated a gene expression profile predictive for LR after BCT, which is characterized by genes involved in cell proliferation but not a surrogate for high histologic grade.

XLS file - 1258K

Genomic rearrangements that give rise to oncogenic gene fusions can offer actionable targets for cancer therapy. Here we present a systematic analysis of oncogenic gene fusions among a clinically well-characterized, prospectively collected set of 278 primary colon cancers spanning diverse tumor stages and clinical outcomes. Gene fusions and somatic genetic variations were identified in fresh frozen clinical specimens by Illumina RNA-sequencing, the STAR fusion gene detection pipeline, and GATK RNA-seq variant calling. We considered gene fusions to be pathogenically relevant when recurrent, producing divergent gene expression (outlier analysis), or as functionally important (e.g., kinase fusions). Overall, 2.5% of all specimens were defined as harboring a relevant gene fusion (kinase fusions 1.8%). Novel configurations of BRAF, NTRK3, and RET gene fusions resulting from chromosomal translocations were identified. An R-spondin fusion was found in only one tumor (0.35%), much less than an earlier reported frequency of 10% in colorectal cancers. We also found a novel fusion involving USP9X-ERAS formed by chromothripsis and leading to high expression of ERAS, a constitutively active RAS protein normally expressed only in embryonic stem cells. This USP9X–ERAS fusion appeared highly oncogenic on the basis of its ability to activate AKT signaling. Oncogenic fusions were identified only in lymph node–negative tumors that lacked BRAF or KRAS mutations. In summary, we identified several novel oncogenic gene fusions in colorectal cancer that may drive malignant development and offer new targets for personalized therapy. Cancer Res; 77(14); 3814–22. ©2017 AACR.

Supplementary Tables S1-S2 from mRNA and microRNA Expression Profiles in Circulating Tumor Cells and Primary Tumors of Metastatic Breast Cancer Patients

Combined supplementary data file containing: -extended materials and methods, describing details of sample collection and isolation, sequencing data generation, cell culturing and protein analysis. -Supplementary Figure S1 showing patient inclusion in this study -Supplementary Figure S2 showing RT-PCR results of fusion gene verification -Supplementary Figure S3 showing Exonic and protein structure of three BRAF fusions -Supplementary Figure S4 showing the structure of an EML4-NTRK3 fusion gene and exonic expression of NTRK3 -Supplementary Figure S5 showing the structure and expression of a novel RRBP1-RET fusion -Supplementary Figure S6 showing the structure and expression of a novel USP9X-ERAS fusion gene. -Supplementary Figure S7 showing Rspondin fusion gene detection and expression -List of Three Supplementary Tables -Supplementary References

Purpose: Molecular characterization of circulating tumor cells (CTC) holds great promise. Unfortunately, routinely isolated CTC fractions currently still contain contaminating leukocytes, which makes CTC-specific molecular characterization extremely challenging. In this study, we determined mRNA and microRNA (miRNA) expression of potentially CTC-specific genes that are considered to be clinically relevant in breast cancer.Experimental Design: CTCs were isolated with the epithelial cell adhesion molecule–based CellSearch Profile Kit. Selected genes were measured by real-time reverse transcriptase PCR in CTCs of 50 metastatic breast cancer patients collected before starting first-line systemic therapy in blood from 53 healthy blood donors (HBD) and in primary tumors of 8 of the patients. The molecular profiles were associated with CTC counts and clinical parameters and compared with the profiles generated from the corresponding primary tumors.Results: We identified 55 mRNAs and 10 miRNAs more abundantly expressed in samples from 32 patients with at least 5 CTCs in 7.5 mL of blood compared with samples from 9 patients without detectable CTCs and HBDs. Clustering analysis resulted in 4 different patient clusters characterized by 5 distinct gene clusters. Twice the number of patients from cluster 2 to 4 had developed both visceral and nonvisceral metastases. Comparing transcript levels in CTCs with those measured in corresponding primary tumors showed clinically relevant discrepancies in estrogen receptor and HER2 levels.Conclusions: Our study shows that molecular profiling of low numbers of CTCs in a high background of leukocytes is feasible and shows promise for further studies on the clinical relevance of molecular characterization of CTCs. Clin Cancer Res; 17(11); 3600–18. ©2011 AACR.

XLS file - 208K

Purpose: A fibrotic focus, the scar-like area found in the center of an invasive breast tumor, is a prognostic parameter associated with an expansive growth pattern, hypoxia, and (lymph)angiogenesis. Little is known about the molecular pathways involved.Experimental Design: Sixty-five patients were selected of whom microarray data of the tumor and H&E slides for histologic analysis were available. The growth pattern and the presence and size of a fibrotic focus were assessed. Differences in biological pathways were identified with global testing. The correlations of growth pattern and fibrotic focus with common breast cancer signatures and with clinicopathologic variables and survival were investigated.Results: Tumors with a large fibrotic focus showed activation of Ras signaling and of the hypoxia-inducible factor-1α pathway. Furthermore, unsupervised hierarchical cluster analysis with hypoxia- and (lymph)angiogenesis-related genes showed that hypoxia-inducible factor-1α, vascular endothelial growth factor A, and carbonic anhydrase 9 were overexpressed. The presence of a fibrotic focus, especially a large fibrotic focus, was associated with the basal-like subtype (P = 0.009), an activated wound-healing signature (P = 0.06), and a poor-prognosis 76-gene signature (P = 0.004). The presence of a fibrotic focus (P = 0.02) and especially of a large fibrotic focus (P = 0.004) was also associated with early development of distant metastasis.Conclusions: Our results sustain the hypothesis that hypoxia-driven angiogenesis is essential in the biology of a fibrotic focus. Ras and Akt might play a role as downstream modulators. Our data furthermore suggest that vascular endothelial growth factor A does not only drive angiogenesis but also lymphangiogenesis in tumors with a fibrotic focus. Our data also show an association between the presence of a fibrotic focus and infaust molecular signatures.

Supplementary Methods from Copy Number Alterations that Predict Metastatic Capability of Human Breast Cancer

Supplementary Table S2 from Association of DNA Methylation of Phosphoserine Aminotransferase with Response to Endocrine Therapy in Patients with Recurrent Breast Cancer

Supplementary Table 3 from Copy Number Alterations that Predict Metastatic Capability of Human Breast Cancer

PDF file - 56K, Supplementary Table S3 Read count, filtering and number of peaks.

Supplementary Figure 3 from Subtypes of Breast Cancer Show Preferential Site of Relapse

PDF file - 101K, Supplementary Figure S5 Venn diagram, depicting the level of overlap of genes between 'proximity' and 'functionality' genes.

PDF file - 46K, Supplementary Table S2 Clinicopathological characteristics of the aromatase inhibitor cohort.

Supplementary Table 2 from Copy Number Alterations that Predict Metastatic Capability of Human Breast Cancer

PDF file - 100K, Supplementary Figure S2 Overlap of peaks from the same ChIP-seq condition between tumors.

Supplementary Figure 3 from Inhibition of Multiple Vascular Endothelial Growth Factor Receptors (VEGFR) Blocks Lymph Node Metastases but Inhibition of VEGFR-2 Is Sufficient to Sensitize Tumor Cells to Platinum-Based Chemotherapeutics

Supplementary Figure 1 from Copy Number Alterations that Predict Metastatic Capability of Human Breast Cancer

PDF file - 129K, Supplementary Table S4 Differential bound regions for ERalpha, H3K4me3 or H3K27me3 in 'poor' and 'good' outcome patients.

Supplementary Table 1 from Copy Number Alterations that Predict Metastatic Capability of Human Breast Cancer

Supplementary Figure 3 from Copy Number Alterations that Predict Metastatic Capability of Human Breast Cancer

We explored whether the five previously reported molecular subtypes in breast cancer show a preference for organ-specific relapse and searched for molecular pathways involved. The “intrinsic” gene list describing the subtypes was used to classify 344 primary breast tumors of lymph node–negative patients. Fisher exact tests were used to determine the association between a tumor subtype and a particular site of distant relapse in these patients who only received local treatment. Modulated genes and pathways were identified in the various groups using Significance Analysis of Microarrays and Global Testing. Bone relapse patients were most abundant in the luminal subtypes but were found less than expected in the basal subtype. The reverse was true for lung and brain relapse patients with the remark that absence of lung relapse was luminal A specific. Finally, a pleura relapse, although rare, was found almost exclusively in both luminal subtypes. Many differentially expressed genes were identified, of which several were in common in a subtype and the site to which the subtype preferentially relapsed. WNT signaling was up-regulated in the basal subtype and in brain-specific relapse, and down-modulated in the luminal B subtype and in bone-specific relapse. Focal adhesion was found up-regulated in the luminal A subtype but down-regulated in lung relapse. The five major molecular subtypes in breast cancer are evidently different with regard to their ability to metastasize to distant organ(s), and share biological features and pathways with their preferred distant metastatic site. [Cancer Res 2008;68(9):3108–14]

Supplementary Figure 2 from Subtypes of Breast Cancer Show Preferential Site of Relapse

PDF file - 198K, Supplementary Figure S6 Differential response to aromatase inhibitors, using ChiA-PET associated genes and genes overlapping between proximity and ChIA-PET data.

Supplementary Figure 1 from Inhibition of Multiple Vascular Endothelial Growth Factor Receptors (VEGFR) Blocks Lymph Node Metastases but Inhibition of VEGFR-2 Is Sufficient to Sensitize Tumor Cells to Platinum-Based Chemotherapeutics

PDF file - 192K, Supplementary Figure S4 Clustering heatmap analysis for ERalpha, H3K4me3 and H3K27me3.