Prediction of recurrence from metabolites and expression of TOP2A and EZH2 in prostate cancer patients treated with radiotherapy.

BACKGROUND: The dual upregulation of TOP2A and EZH2 gene expression has been proposed as a biomarker for recurrence in prostate cancer patients to be treated with radical prostatectomy. A low tissue level of the metabolite citrate has additionally been connected to aggressive disease and recurrence in this patient group. However, for radiotherapy prostate cancer patients, few prognostic biomarkers have been suggested. The main aim of this study was to use an integrated tissue analysis to evaluate metabolites and expression of TOP2A and EZH2 as predictors for recurrence among radiotherapy patients. METHODS: From 90 prostate cancer patients (56 received neoadjuvant hormonal treatment), 172 transrectal ultrasound-guided (TRUS) biopsies were collected prior to radiotherapy. Metabolic profiles were acquired from fresh frozen TRUS biopsies using high resolution-magic angle spinning MRS. Histopathology and immunohistochemistry staining for TOP2A and EZH2 were performed on TRUS biopsies containing cancer cells (n = 65) from 46 patients, where 24 of these patients (n = 31 samples) received hormonal treatment. Eleven radical prostatectomy cohorts of a total of 2059 patients were used for validation in a meta-analysis. RESULTS: Among radiotherapy patients with up to 11 years of follow-up, a low level of citrate was found to predict recurrence, p = 0.001 (C-index = 0.74). Citrate had a higher predictive ability compared with individual clinical variables, highlighting its strength as a potential biomarker for recurrence. The dual upregulation of TOP2A and EZH2 was suggested as a biomarker for recurrence, particularly for patients not receiving neoadjuvant hormonal treatment, p = 0.001 (C-index = 0.84). While citrate was a statistically significant biomarker independent of hormonal treatment status, the current study indicated a potential of glutamine, glutamate and choline as biomarkers for recurrence among patients receiving neoadjuvant hormonal treatment, and glucose among patients not receiving neoadjuvant hormonal treatment. CONCLUSION: Using an integrated approach, our study shows the potential of citrate and the dual upregulation of TOP2A and EZH2 as biomarkers for recurrence among radiotherapy patients.

Long term trends of breast cancer incidence according to proliferation status.

BACKGROUND: Long-term breast cancer incidence trends according to proliferation status are poorly described. We studied time-trends in breast cancer incidence, using mitotic count and Ki-67 as markers of proliferation. METHODS: Among 83,298 Norwegian women followed for breast cancer occurrence 1961-2012, 2995 incident breast cancers were diagnosed. Ki-67 was assessed using immunohistochemistry on tissue microarrays and mitoses were counted on whole sections. We compared incidence rates according to proliferation status among women born 1886-1928 and 1929-1977, estimating age-specific incidence rate ratios. We performed multiple imputations to account for unknown proliferation status. Mean values of Ki-67 and mitotic counts were calculated, according to age and birth year. We performed separate incidence analyses for HER2+ and triple negative breast cancers. RESULTS: Among women aged 40-69 years, incidence rates of tumours with low-proliferative activity were higher among those born in 1929 or later, compared to before 1929, according to Ki-67 and mitotic count. Incidence rates of tumours with high-proliferative activity were also higher in women born in 1929 or later compared to before 1929 according to Ki-67, but not according to mitotic count. Mean values of Ki-67 and mitotic count varied according to age and birth year. In subtype-specific analyses we found an increase of high-proliferative HER2+ tumours according to Ki-67 in women born in 1929 or later, compared to before 1929. CONCLUSIONS: There has been a temporal increase in both low- and high-proliferative breast cancers.

Reproducible Lipid Alterations in Patient-Derived Breast Cancer Xenograft FFPE Tissue Identified with MALDI MSI for Pre-Clinical and Clinical Application.

The association between lipid metabolism and long-term outcomes is relevant for tumor diagnosis and therapy. Archival material such as formalin-fixed and paraffin embedded (FFPE) tissues is a highly valuable resource for this aim as it is linked to long-term clinical follow-up. Therefore, there is a need to develop robust methodologies able to detect lipids in FFPE material and correlate them with clinical outcomes. In this work, lipidic alterations were investigated in patient-derived xenograft of breast cancer by using a matrix-assisted laser desorption ionization mass spectrometry (MALDI MSI) based workflow that included antigen retrieval as a sample preparation step. We evaluated technical reproducibility, spatial metabolic differentiation within tissue compartments, and treatment response induced by a glutaminase inhibitor (CB-839). This protocol shows a good inter-day robustness (CV = 26 ± 12%). Several lipids could reliably distinguish necrotic and tumor regions across the technical replicates. Moreover, this protocol identified distinct alterations in the tissue lipidome of xenograft treated with glutaminase inhibitors. In conclusion, lipidic alterations in FFPE tissue of breast cancer xenograft observed in this study are a step-forward to a robust and reproducible MALDI-MSI based workflow for pre-clinical and clinical applications.

DTX3 copy number increase in breast cancer: a study of associations to molecular subtype, proliferation and prognosis.

PURPOSE: The degree of cell proliferation is important for subclassification of breast cancers into prognostic and therapeutic groups. DTX3 has been identified as a driver of proliferation in luminal breast cancer. In this study, we describe DTX3 copy number in breast cancer primary tumours and corresponding axillary lymph node metastases, and studied associations with molecular subtype, proliferation and prognosis. METHODS: Using fluorescence in situ hybridization, we assessed DTX3 and chromosome 12 centromere (CEP12) copy number in 542 primary breast cancers and 117 lymph node metastases, from a well-described cohort of Norwegian breast cancer patients. Proliferation was expressed as mitotic counts and Ki67 score. Associations between DTX3 copy number and molecular subtype and proliferation were assessed using Pearson's χ2 test. We studied the effect of copy number increase on prognosis estimating cumulative incidence of breast cancer death and hazard ratios. RESULTS: Mean DTX3 copy number ≥ 4 was found in 23 tumours (4%), and mean ≥ 5 in 9 tumours (1.7%). Copy number increase was found within all molecular subtypes except the 5 negative phenotype and the Luminal B (HER2 +) subtype. DTX3 copy number increase was not accompanied by an increase in CEP12. Point estimates showed that there were associations between DTX3 copy number increase and high proliferation and poor prognosis; however, precision depended on copy number cut-off. CONCLUSIONS: DTX3 copy number increase was present in a small proportion of breast cancer cases. There was an association between copy number increase and high tumour cell proliferation and poor prognosis.

ZNF703 gene copy number and protein expression in breast cancer; associations with proliferation, prognosis and luminal subtypes.

PURPOSE: Amplification of 8p12 is frequent in breast cancer and associated with poor prognosis in luminal subtypes. ZNF703 has been identified as the driver gene of proliferation in the A1 amplicon situated in 8p12. In this study, the aims were to investigate associations between ZNF703 copy number alterations and molecular subtypes, proliferation and prognosis, and using immunohistochemistry, examine associations between ZNF703 copy number and ZNF703 protein expression. METHODS: Copy number alterations in 702 primary breast tumours and corresponding lymph node metastases were examined using fluorescence in situ hybridization with probes for ZNF703 and centromere 8. In addition, protein expression was studied in 869 tumours from the same cohort. Associations between copy number alterations and protein expression and tumour characteristics were assessed using Pearson chi square test. The prognostic impact of ZNF703 copy number increase and protein expression was assessed estimating cumulative incidence of breast cancer death and hazard ratios. RESULTS: We found mean ZNF703 copy number ≥ 6 in 7% of tumours, most frequently in Luminal B subtypes. We found a positive association between increased copy number, and high proliferation, high histological grade, and poor prognosis. Luminal A tumours with high copy number had high histological grade and poor prognosis (borderline significant). We found positive nuclear staining in 76% of primary tumours. There was an association between copy number status and protein expression, but no association between protein expression and prognosis. CONCLUSIONS: In breast cancer, high ZNF703 copy number is associated with increased proliferation, Luminal B subtypes and poor prognosis.

The expression of the long NEAT1_2 isoform is associated with human epidermal growth factor receptor 2-positive breast cancers.

The long non-coding RNA NEAT1 locus is transcribed into two overlapping isoforms, NEAT1_1 and NEAT1_2, of which the latter is essential for the assembly of nuclear paraspeckles. NEAT1 is abnormally expressed in a wide variety of human cancers. Emerging evidence suggests that the two isoforms have distinct functions in gene expression regulation, and recently it was shown that NEAT1_2, but not NEAT1_1, expression predicts poor clinical outcome in cancer. Here, we report that NEAT1_2 expression correlates with HER2-positive breast cancers and high-grade disease. We provide evidence that NEAT1_1 and NEAT1_2 have distinct expression pattern among different intrinsic breast cancer subtypes. Finally, we show that NEAT1_2 expression and paraspeckle formation increase upon lactation in humans, confirming what has previously been demonstrated in mice.

MRPS23 amplification and gene expression in breast cancer; association with proliferation and the non-basal subtypes.

PURPOSE: MRPS23 is recognized as a driver of proliferation in luminal breast cancer. The aims of the present study were to describe MRPS23 copy number change in breast cancer, and to assess associations between MRPS23 copy number change and molecular subtype, proliferation and prognosis, and between MRPS23 gene expression and molecular subtype and prognosis. METHODS: Using fluorescence in situ hybridization (FISH), we examined MRPS23 and centromere 17 copy number in 590 formalin-fixed, paraffin-embedded primary tumours and 144 corresponding lymph node metastases from a cohort of Norwegian breast cancer patients. Furthermore, we analysed MRPS23 gene expression data in 1971 primary breast cancer tumours from the METABRIC dataset. We used Pearson's χ2 test to assess associations between MRPS23 copy number and molecular subtype and proliferation, and between MRPS23 expression and molecular subtype. We studied prognosis by estimating hazard ratios and cumulative incidence of death from breast cancer according to MRPS23 copy number and MRPS23 expression status. RESULTS: We found MRPS23 amplification (mean MRPS23 copy number ≥ 6 and/or MRPS23/chromosome 17 ratio ≥ 2) in 8% of primary tumours. Copy number increase associated with non-basal subtypes and higher tumour cell proliferation (Ki67). Higher MRPS23 expression associated with the Luminal B subtype. We found no significant association between MRPS23 amplification or MRSP23 gene expression, and prognosis. CONCLUSION: Amplification of MRPS23 is associated with higher proliferation and non-basal subtypes in breast cancer. High MRPS23 expression is associated with the Luminal B subtype.

The prognostic value of androgen receptors in breast cancer subtypes.

PURPOSE: Androgen receptor (AR) expression is frequent in breast cancer and has been associated with good prognosis in several studies. The present study investigates AR-expression in relation to molecular subtypes, clinicopathological features and prognosis in 1297 primary tumours and 336 paired axillary lymph node metastases (LNM) from two cohorts of Norwegian patients. METHODS: Immunohistochemistry for AR was performed on tumours previously reclassified into molecular subtypes using immunohistochemistry and in situ hybridisation. Associations between AR-expression and clinical features were studied using Chi-square tests. Cumulative incidence of breast cancer death and Cox regression analyses were used to assess prognosis. RESULTS: AR-positivity was found in 78.0% of all cases, 84.9% of luminal and 45.1% of non-luminal tumours. The highest proportion of AR-positivity was found in Luminal B tumours, and the lowest in the Basal phenotype. Discordance in AR-status between primary tumours and lymph node metastases was observed in 21.4% of cases. A switch from AR- primary tumour to AR+ lymph node metastasis was seen in 60/72 discrepant cases. AR-expression in primary tumours was an independent and favourable prognostic marker (HR 0.70, 95% CI 0.55-0.90), particularly in the Luminal A subtype, and in grade 3 tumours. CONCLUSIONS: AR is an independent predictor of good prognosis in BC, particularly in grade 3 and Luminal A tumours. Discordant AR-expression between primary tumour and LNM was observed in 21.4% of cases and most often there was a switch from AR- primary tumour to AR+ axillary LNM.

FGD5 amplification in breast cancer patients is associated with tumour proliferation and a poorer prognosis.

PURPOSE: Proliferation is a hallmark of cancer. Using a combined genomic approach, FGD5 amplification has been identified as a driver of proliferation in Luminal breast cancer. We aimed to describe FGD5 copy number change in breast cancer, and to assess a possible association with tumour proliferation and prognosis. METHODS: We used fluorescence in situ hybridization targeting FGD5 and chromosome 3 centromere (CEP3) on formalin-fixed, paraffin-embedded tissue from 430 primary breast cancers and 108 lymph node metastases, from a cohort of Norwegian breast cancer patients. We tested the association between FGD5 copy number status and proliferation (assessed by Ki67 levels and mitotic count) using Pearson's Chi square test, and assessed the prognostic impact of FGD5 copy number change by estimating cumulative risks of death and hazard ratios. RESULTS: We identified FGD5 amplification (defined as FGD5/CEP3 ratio ≥2 or mean FGD5/tumour cell ≥4) in 9.5% of tumours. Mitotic count and Ki67 levels were higher in tumours with FGD5 copy number increase, compared to tumours with no copy number change. After 10 years of follow-up, cumulative risk of death from breast cancer was higher among cases with FGD5 amplification [48.1% (95% CI 33.8-64.7)], compared to non-amplified cases [27.7% (95% CI 23.4-32.6)]. CONCLUSIONS: FGD5 is a new prognostic marker in breast cancer, and increased copy number is associated with higher tumour proliferation and poorer long-term prognosis.

Molecular Subtypes of Breast Cancer: Long-term Incidence Trends and Prognostic Differences.

BACKGROUND: Secular trends in incidence and prognosis of molecular breast cancer subtypes are poorly described. We studied long-term trends in a population of Norwegian women born 1886-1977. METHODS: A total of 52,949 women were followed for breast cancer incidence, and 1,423 tumors were reclassified into molecular subtypes using IHC and in situ hybridization. We compared incidence rates among women born 1886-1928 and 1929-1977, estimated age-specific incidence rate ratios (IRR), and performed multiple imputations to account for unknown subtype. Prognosis was compared for women diagnosed before 1995 and in 1995 or later, estimating cumulative risk of death and HRs. RESULTS: Between 50 and 69 years of age, incidence rates of Luminal A and Luminal B (HER2-) were higher among women born in 1929 or later, compared with before 1929 [IRRs 50-54 years; after imputations: 3.5; 95% confidence interval (CI), 1.8-6.9 and 2.5; 95% CI, 1.2-5.2, respectively], with no clear differences for other subtypes. Rates of death were lower in women diagnosed in 1995 or later, compared to before 1995, for Luminal A (HR 0.4; 95% CI, 0.3-0.5), Luminal B (HER2-; HR 0.5; 95% CI, 0.3-0.7), and Basal phenotype (HR 0.4; 95% CI, 0.2-0.9). CONCLUSIONS: We found a strong secular incidence increase restricted to Luminal A and Luminal B (HER2-) subtypes, combined with a markedly improved prognosis for these subtypes and for the Basal phenotype. IMPACT: This study documents a clear secular increase in incidence and a concomitant improved prognosis for specific molecular breast cancer subtypes. Cancer Epidemiol Biomarkers Prev; 25(12); 1625-34. ©2016 AACR.