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AimsFGFR1 is located on 8p11.23 and regulates cell proliferation and survival. Increased copy number of FGFR1 is found in several cancers including cancer of the breast. ZNF703 is located close to FGFR1 at 8p11-12 and is frequently expressed in the luminal B subtype of breast cancer. Using tissue samples from a well-described cohort of patients with breast cancer with long-term follow-up, we studied associations between FGFR1 copy number in primary breast cancer tumours and axillary lymph node metastases, and proliferation status, molecular subtype and prognosis. Furthermore, we studied associations between copy number increase of FGFR1 and copy number of ZNF703.MethodsWe used fluorescence in situ hybridisation for FGFR1 and the chromosome 8 centromere applied to tissue microarray sections from a series of 534 breast cancer cases.ResultsWe found increased copy number (≥4) of FGFR1 in 74 (13.9%) of tumours. Only 6 of the 74 cases with increased copy number were non-luminal. Increased FGFR1 copy number was significantly associated with high Ki-67 status, high mitotic count and high histopathological grade, but not with prognosis. Forty-two (7.9%) cases had mean copy number ≥6. Thirty of these showed ZNF708 copy number ≥6.ConclusionsOur results show that FGFR1 copy number increase is largely found among luminal subtypes of breast cancer, particularly luminal B (HER2−). It is frequently accompanied by increased copy number of ZNF703. FGFR1 copy number increase is associated with high histopathological grade and high proliferation. However, we did not discover an association with prognosis.

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. 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. 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. In breast cancer, high ZNF703 copy number is associated with increased proliferation, Luminal B subtypes and poor prognosis.

Simple Summary Meningioma diagnostics and grading are currently based on subjective histopathological criteria given by the 2016 World Health Organization (WHO) classification. However, biomarkers may provide a more objective approach to diagnostics. This study was designed to elucidate the diagnostic and prognostic value of somatostatin receptors (SSTRs) as biomarkers in meningiomas, which could help to identify patients with a higher risk of recurrence and provide more personalized treatment. We have confirmed, in a population of 162 patients, that SSTRs have diagnostic value and may aid in the differentiation between WHO grade 1 and grade 2 tumors. Furthermore, SSTR1, SSTR2 and SSTR5 were associated with higher malignancy grades. SSTR2 expression was found to be characteristic in meningiomas. To maintain objectiveness, we scoped for a digital evaluation of immunoreactivity. We aim to impact and motivate researchers to further investigations towards more objective criteria in meningioma diagnostics, which in turn will improve patient care. Abstract Meningiomas have high recurrence rates despite frequently benign histopathological appearances. Somatostatin receptors (SSTRs) may be reliable biomarkers that could identify patients with increased risk of recurrence. Even though SSTRs are previously detected in meningiomas, their associations to clinicopathological features remain unclear. The aim of this study was to investigate the diagnostic and prognostic value of SSTRs in a large series of human meningiomas with long follow-up data. Immunohistochemistry was used to measure the expression of SSTR1-SSTR5 in tissue samples from 162 patients diagnosed with intracranial meningiomas of World Health Organization (WHO) grade 1 or 2. Digital scoring and a manual staining index were applied to assess immunoreactivity. All SSTRs, except SSTR4, were upregulated in our series of meningiomas. SSTR1 (p = 0.036), SSTR2 (p = 0.036) and SSTR5 (p = 0.029) were associated with a higher malignancy grade. SSTR2 presented as the most reliable marker. Only SSTR2 was associated with time to recurrence (TTR) in univariate Cox regression analyses. Manual staining index was strongly correlated with digital scoring for all SSTRs (r > 0.65, p < 0.001). SSTRs, and especially SSTR2, are useful in the diagnostics of meningiomas, even though their prognostic value appears limited. Digital scoring is valuable to ensure reproducibility.

PurposeThe 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.MethodsUsing 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’sχ2test. We studied the effect of copy number increase on prognosis estimating cumulative incidence of breast cancer death and hazard ratios.ResultsMean 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.ConclusionsDTX3 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.

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. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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. This is a post-peer-review, pre-copyedit version of an article published in [Breast Cancer Research and Treatment]. Locked until 25.1.2019 due to copyright restrictions. The final authenticated version is available online at: https://link.springer.com/article/10.1007%2Fs10549-017-4125-8

Faciogenital dysplasia 5 (FGD5) amplification drives tumor cell proliferation, and is present in 9.5% of breast cancers. We describe FGD5 expression, assess associations between FGD5 amplification and FGD5 expression, and assess FGD5 expression in relation to proliferation and prognosis. FGD5 immunohistochemistry was done on primary tumors (n=829) and lymph node metastases (n=231) from a cohort of Norwegian patients. We explored associations between FGD5 amplification, FGD5 expression, and proliferation, and analyzed the prognostic value of FGD5 expression by estimating cumulative risks of death and hazard ratios (HRs). We identified nuclear and cytoplasmic expression in 64% and 73% of primary tumors, respectively, and found an association between gene amplification and nuclear expression (p=0.02). The proportion of cases with FGD5 expression was higher in lymph node metastases, compared with primary tumors (p=0.004 for nuclear and p=0.001 for cytoplasmic staining). Neither proliferation nor prognosis was associated with FGD5 expression (age-adjusted HR 1.12 [95% confidence interval = 0.89–1.41] for nuclear expression; and 0.88 [95% CI = 0.70–1.12] for cytoplasmic expression). FGD5 is expressed in a high proportion of breast cancers and lymph node metastases. There was a correlation between FGD5 amplification and nuclear expression, but no association between FGD5 expression and proliferation or prognosis. © 2018. This is the authors' accepted and refereed manuscript to the article. The final authenticated version is available online at: https://doi.org/10.1369/0022155418792032

Background Meningiomas are common intracranial tumors in humans that frequently recur despite having a predominantly benign nature. Even though these tumors have been shown to commonly express EGFR/c-erbB1 (epidermal growth factor receptor), results from previous studies are uncertain regarding the expression of either intracellular or extracellular domains, cellular localization, activation state, relations to malignancy grade, and prognosis. Aims This study was designed to investigate the expression of the intracellular and extracellular domains of EGFR and of the activated receptor as well as its ligands EGF and TGFα in a large series of meningiomas with long follow-up data, and investigate if there exists an association between antibody expression and clinical and histological data. Methods A series of 186 meningiomas consecutively operated within a 10-year period was included. Tissue microarrays were constructed and immunohistochemically analyzed with antibodies targeting intracellular and extracellular domains of EGFR, phosphorylated receptor, and EGF and TGFα. Expression levels were recorded as a staining index (SI). Results Positive immunoreactivity was observed for all antibodies in most cases. There was in general high SIs for the intracellular domain of EGFR, phosphorylated EGFR, EGF, and TGFα but lower for the extracellular domain. Normal meninges were negative for all antibodies. Higher SIs for the phosphorylated EGFR were observed in grade II tumors compared with grade I (p = 0.018). Survival or recurrence was significantly decreased in the time to recurrence analysis (TTR) with high SI-scores of the extracellular domain in a univariable survival analysis (HR 1.152, CI (1.036–1.280, p = 0.009)). This was not significant in a multivariable analysis. Expression of the other antigens did not affect survival. Conclusion EGFR is overexpressed and in an activated state in human meningiomas. High levels of ligands also support this growth factor receptor system to be involved in meningioma tumorigenesis. EGFR may be a potential candidate for targeted therapy.

Published article (CC BY-NC 3.0) Aims The clinical significance of TOP2A as a prognostic marker has not been clarified. The aims of this study were to investigate the frequency of TOP2A copy number change; to correlate TOP2A with HER2 status, hormone receptor (HR) status and molecular subtype, and further to explore differences in breast cancer-specific survival according to TOP2A and HER2. Methods In this study, TOP2A, HER2 and chromosome 17 copy number were assessed in 670 cases of breast cancer using in situ hybridisation techniques. Gene to chromosome ratios ≥2 were classified as amplification. TOP2A deletion (gene to chromosome ratio ≤0.8) or monosomy (only one signal for both gene and chromosome in more than 75% of nuclei) were classified as gene loss. Results A strong association between TOP2A change and HR and HER2 status was found. During the first 5 years after diagnosis, the risk of death from breast cancer was significantly higher for cases with HER2 amplification irrespective of TOP2A status. Conclusions TOP2A copy number change was strongly associated with HR and HER2 status and as a prognostic marker TOP2A is probably of limited value. This is an Open Access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 3.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial.

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.

BRCA1 associated tumours are found to express an oestrogen receptor negative "basal epithelial-like" phenotype. In contrast to ER negative tumours in general, such tumours rarely harbour amplification of the HER-2 gene. However, little is known about TOP2A gene amplification status in BRCA1-associated tumours. Such information may be of importance to therapy, as amplification of TOP2A has been associated with dose-dependent sensitivity to anthracycline therapy in breast cancer. We examined 40 breast carcinomas from BRCA1 mutation carriers and 40 sporadic breast carcinomas matched for age, tumour diameter and histological grade for HER-2 and TOP2A amplification status using fluorescence in situ hybridisation (FISH). Co-amplification of TOP2A and HER-2 was found in four of the mutation carriers and in three of the controls. While six tumours in the control group harboured HER-2 amplifications with normal TOP2A, this occurred in three of the BRCA1 associated tumours only. In contrast, three of the BRCA1-associated tumours but none of the controls harboured TOP2A amplification despite normal HER-2 status. Our findings have potential therapeutic implications. HER-2 assessment is routinely used to select breast cancer patients for trastuzumab but also dose-intensive anthracycline therapy. Our data suggest that BRCA1-associated breast cancers also need to be tested for TOP2A amplification.

We compared fluorescence in situ hybridization (FISH), immunohistochemical analysis, immunocytochemical analysis, and relative quantification assays using polymerase chain reaction (PCR) as methods for estimating HER-2 gene amplification and protein overexpression in fine-needle aspirate (FNA) specimens and paraffin-embedded tissue samples from 49 cases of breast cancer. FISH can be performed successfully on FNA smears. Immunohistochemical and immunocytochemical staining intensity of 3+ corresponds to a FISH ratio of more than 2.5. Immunohistochemical and immunocytochemical staining of 2+ and 1+ are not necessarily associated with gene amplification. Increased DNA PCR ratios might be seen without amplification, reflecting polysomy. HER-2 messenger RNA relative quantitation scores correlate well with HER-2 gene amplification. Owing to the ease with which it can be performed and interpreted, we conclude that FISH is the test of choice for HER-2 estimation and, when possible, should be performed on whole nuclei, which are readily available in FNA smears or imprint cytology. FISH may be used primarily or to confirm immunohistochemical, immunocytochemical, and PCR results.

The TOP2A gene is located on chromosome 17 close to the HER-2 gene. It encodes an enzyme involved in the regulation of cell proliferation. Using fluorescence in situ hybridization (FISH), we have examined fine needle aspiration smears from 42 cases of breast carcinoma with probes for TOP2A, HER-2 and chromosome 17. We found that amplification of TOP2A is a frequent finding in breast cancer and is often but not exclusively accompanied by HER-2 gene amplification. It is associated with high histological grade and oestrogen receptor (ER) negativity. TOP2A deletions may also be associated with high histological grade and loss of ER. TOP2A amplification in the absence of HER-2 amplification may be associated with lower histological grade and ER positivity. Testing for TOP2A aberrations may be useful in the search for individually tailored treatment regimes for breast cancer.

Numerical change in chromosome 8 is an acquired abnormality associated with high clinical stage and may be involved in the conversion of carcinoma in situ in the breast to invasive carcinoma. Fine needle aspiration smears from 53 cases of breast carcinoma were hybridized with centromeric probes for chromosome 8 and the X chromosome. Thirty-eight cases revealed chromosome 8 copy gain. Of the 45 grade II and III tumours, 28 showed polysomy (>3 signals) and six showed trisomy. Of the eight grade I tumours, four were trisomic, none were polysomic. There were only two cases of chromosome 8 copy loss (one each of grade I and III). X chromosome polysomy was also a frequent finding although the signal counts were similar to those for chromosome 8 in only a few cases. Chromosome 8 polysomy occurs frequently in breast carcinoma and high copy number (>3) is associated with high malignancy grade.

Formalin-fixed and paraffin-embedded autopsy material from 10 fetuses and infants with unknown karyotype and anomalies suggestive of trisomy 18 were subjected to fluorescence in situ hybridization (FISH). Nuclei were extracted from the tissues and hybridized with a chromosome 18–specific centromere probe. The hybridization was successful in 9 of 10 cases. Two cases showed three hybridization signals in most of the nuclei (74% and 85%). These had anomalies frequently occurring with trisomy 18 (congenital heart defect, omphalocele, and horseshoe kidney). Two cases showed a mixture of two and three signals (47%/49% and 59%/36%), suggesting the possibility of mosaicism. One of these cases had anomalies consistent with a trisomy 18 phenotype. In the other case intrauterine growth retardation and syndactylies suggested triploidy. Hybridization with a chromosome 8–specific probe gave a distribution of two and three signals (34% and 62%, respectively). This result strengthened the suspicion of a possible triploid mosaicism. In five of the cases most of the nuclei showed two signals (85% to 88%). However, as only one type of tissue was examined for enumeration of chromosome 18, the possibility of organ mosaicism or other chromosome aberrations cannot be excluded. The FISH technique is applicable on macerated and autolysed formalin-fixed tissue, making it possible to retrospectively analyze autopsy material from aborted and stillborn fetuses and infants. This analysis contributes to a better quality of perinatal autopsies and is helpful in parental counseling.

The epidermal growth factor receptor (EGFR) family, consisting of four tyrosine kinase receptors, c-erbB1-4, seems to be influential in gliomagenesis. The aim of this study was to investigate EGFR gene amplification and expression of c-erbB1-4 receptor proteins in human anaplastic astrocytomas. Formalin-fixed and paraffin-embedded sections from 31 cases were investigated by standard immunohistochemical procedures for expression of c-erbB1-4 receptor proteins using commercial antibodies. EGFR gene amplification was studied by fluorescence in situ hybridization using paraffin-embedded tissues. Two monoclonal antibodies, NCL-EGFR-384 and NCL-EGFR, were used for EGFR detection and they displayed positive immunoreactivity in 97% and 71%, respectively. For c-erbB2 detection three monoclonal antibodies, CB11, 3B5, and 5A2, were applied and they displayed positive immunoreactivity in 45%, 100%, and 52%, respectively. Positive immunostaining for c-erbB3 and c-erbB4 was encountered in 97% and 74%, respectively. The EGFR gene was amplified in 9 out of 31 tumors (29%). After adjusting for age, Karnofsky performance status, and extent of surgical resection, Cox multiple regression analysis with overall survival as the dependent variable revealed that c-erbB2 overexpression detected by the monoclonal antibody clone CB11 was a statistically significant poor prognostic factor (P = 0.004). This study shows the convenience and feasibility of immunohistochemistry when determining the expression of receptor proteins in tissue sections of human astrocytomas. The synchronous overexpression of c-erbB1-4 proteins in anaplastic astrocytomas supports their role in the pathogenesis of these tumors. Further, c-erbB2 overexpression seems to predict aggressive behaviour.