RAD51 foci as a functional biomarker of homologous recombination repair and PARP inhibitor resistance in germline BRCA-mutated breast cancer.

Background: BRCA1 and BRCA2 (BRCA1/2)-deficient tumors display impaired homologous recombination repair (HRR) and enhanced sensitivity to DNA damaging agents or to poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi). Their efficacy in germline BRCA1/2 (gBRCA1/2)-mutated metastatic breast cancers has been recently confirmed in clinical trials. Numerous mechanisms of PARPi resistance have been described, whose clinical relevance in gBRCA-mutated breast cancer is unknown. This highlights the need to identify functional biomarkers to better predict PARPi sensitivity. Patients and methods: We investigated the in vivo mechanisms of PARPi resistance in gBRCA1 patient-derived tumor xenografts (PDXs) exhibiting differential response to PARPi. Analysis included exome sequencing and immunostaining of DNA damage response proteins to functionally evaluate HRR. Findings were validated in a retrospective sample set from gBRCA1/2-cancer patients treated with PARPi. Results: RAD51 nuclear foci, a surrogate marker of HRR functionality, were the only common feature in PDX and patient samples with primary or acquired PARPi resistance. Consistently, low RAD51 was associated with objective response to PARPi. Evaluation of the RAD51 biomarker in untreated tumors was feasible due to endogenous DNA damage. In PARPi-resistant gBRCA1 PDXs, genetic analysis found no in-frame secondary mutations, but BRCA1 hypomorphic proteins in 60% of the models, TP53BP1-loss in 20% and RAD51-amplification in one sample, none mutually exclusive. Conversely, one of three PARPi-resistant gBRCA2 tumors displayed BRCA2 restoration by exome sequencing. In PDXs, PARPi resistance could be reverted upon combination of a PARPi with an ataxia-telangiectasia mutated (ATM) inhibitor. Conclusion: Detection of RAD51 foci in gBRCA tumors correlates with PARPi resistance regardless of the underlying mechanism restoring HRR function. This is a promising biomarker to be used in the clinic to better select patients for PARPi therapy. Our study also supports the clinical development of PARPi combinations such as those with ATM inhibitors.

Senescence bypass screen identifies TBX2, which represses Cdkn2a (p19(ARF)) and is amplified in a subset of human breast cancers.

To identify new immortalizing genes with potential roles in tumorigenesis, we performed a genetic screen aimed to bypass the rapid and tight senescence arrest of primary fibroblasts deficient for the oncogene Bmi1. We identified the T-box member TBX2 as a potent immortalizing gene that acts by downregulating Cdkn2a (p19(ARF)). TBX2 represses the Cdkn2a (p19(ARF)) promoter and attenuates E2F1, Myc or HRAS-mediated induction of Cdkn2a (p19(ARF)). We found TBX2 to be amplified in a subset of primary human breast cancers, indicating that it might contribute to breast cancer development.

Cyclin D1 triggers autonomous growth of breast cancer cells by governing cell cycle exit.

Cyclin D1 controls G1-associated processes, including G0-to-G1 and G1-to-S transitions. This study demonstrates a novel aspect of cyclin D1 as a regulator of the transition between G1 and G0. Overexpression of cyclin D1 in MCF7 breast tumor cells resulted in a continued proliferation under low-serum conditions, whereas nonoverexpressing cells ceased to grow. This difference in growth was due to a reduced exit from G1 to G0 in cyclin D1-overexpressing cells. Our data therefore suggest a model in which cyclin D1 overexpression in tumor cells is responsible for hyperproliferation under growth factor-deprived conditions.

Overexpression of the HER-2 oncogene does not play a role in high-grade osteosarcomas.

The aim of our study was to determine whether or not the tyrosine kinase receptor, HER2 (also known as ErbB2/Her2/neu), is overexpressed in human osteosarcomas (OS). We studied 15 biopsy and 18 resection specimens at the mRNA and protein levels. HER2 status in the OS specimens was assessed by immunohistochemistry (IHC) and quantitative Real-Time Polymerase chain reaction (PCR). In moderately immunopositive cases fluorescent in situ hybridisation (FISH) analysis was used in order to identify any possible gene amplification. 27 samples were evaluable for IHC and only 1 case showed a moderately positive membrane staining. The remaining samples showed no staining or focal cytoplasmic staining (2 samples). In the moderately positive case, FISH analysis showed no HER-2 gene amplification. There was also no overexpression of HER2 mRNA suggesting this sample was a false-positive immunostain. HER2 mRNA expression was present in all samples at a similar level to that in the breast cancer cell line, MCF7, which does not overexpress HER2 and was used as a negative control. In conclusion, this study shows that HER2 mRNA or membranous HER2 protein overexpression is absent in human OS. We noted various inconsistencies in previous published studies, with regard to methodology and the interpretation of the results based on poor methodology. We therefore conclude that the positive data with regard to HER2 overexpression reported in these previous studies is not reliable. Our results suggest that the monoclonal antibody trastuzumab (Herceptin(R)), directed against the HER2-receptor, is not likely to be an effective therapeutic agent in OS.

Expression of a functional thyroglobulin fragment in a baculovirus system.

The synthesis is described of an N-terminal thyroglobulin (Tg) polypeptide of 27 kDa, which is capable of hormonogenesis, in a baculovirus system. This polypeptide was made using a 657 bp Tg cDNA cloned from human thyroid RNA by a polymerase chain reaction method. The cDNA contained the information for the Tg signal peptide, the N-terminally located site for thyroid hormone formation and, at the 3' end, a sequence coding for six histidine residues. The fragments produced were purified using a nickel-nitrilotriacetic acid column using these six histidine residues. The products were analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and showed two glycosylated fragments of 32 and 34 kDa, both of which started with asparagine. Iodination of the fragments with lactoperoxidase in vitro resulted in the formation of thyroxine (T4). The formation rate of T4 in the fragments was about five times lower than that of the mature Tg dimer of 660 kDa, but ten times more rapid than in bovine serum albumin under the same conditions.

Overexpression of cyclin D1 indicates a poor prognosis in squamous cell carcinoma of the head and neck.

OBJECTIVES: To evaluate the overexpression of cyclin D1 and p53 as a prognostic marker of squamous cell carcinoma of the head and neck and to investigate whether deregulation of these genes is associated with an unfavorable course of disease. DESIGN: Retrospective study. MATERIALS AND METHODS: Formalin-fixed, paraffin-embedded tumor materials that were obtained from a well-characterized series of 115 patients with resectable head and neck cancer at The Netherlands Cancer Institute, Amsterdam, were analyzed by immunohistochemical methods using antiserum samples that were directed against 2 proteins (ie, cyclin D1 and p53), which are crucial in the regulation of the G1 phase of the cell cycle. RESULTS: Overexpression of cyclin D1 protein was found in 49% of the patients with squamous cell carcinoma of the head and neck. This overexpression was not associated with known prognostic factors (eg, the T and N stages). Tumors recurred more frequently and in a shorter period in patients whose primary tumors showed an overexpression of cyclin D1 protein. This difference (P = .05) was statistically significant in a stepwise proportional hazard regression analysis. However, since a discrepancy in staining results was observed between the biopsy and resection materials that were taken from the same patient, this result may not have been applicable in the evaluation of biopsy specimens only. This discrepancy is most likely owing to tissue heterogeneity. The overexpression of p53 that was found in 42% of the patients was of no prognostic significance. CONCLUSIONS: These data provide evidence that overexpression of cyclin D1 protein in resection material of squamous cell carcinomas of the head and neck is indicative of a poor prognosis, independently of other known prognostic factors. Whether overexpression of cyclin D1 may therefore be used to select patients for more intensive treatment should be examined in the context of a clinical trial.

Molecular subtypes of breast cancer and amplification of topoisomerase II alpha: predictive role in dose intensive adjuvant chemotherapy.

Benefit from chemotherapy treatment in breast cancer patients is determined by the molecular make-up of the tumour. In a retrospective analysis, we determined the molecular subtypes of breast cancer originally defined by expression microarrays by immunohistochemistry in tumours of patients who took part in a randomised study of adjuvant high-dose chemotherapy in breast cancer. In addition, the topoisomerase II alpha (TOP2A) amplification status was determined by fluorescence in situ hybridisation and chromogenic in situ hybridisation. 411 of the 753 tumours (55%) were classified as luminal-like, 137 (18%) as basal-like and 205 (27%) as human epithelial receptor type 2 (HER2) amplified. The basal-like tumours were defined as having no expression of ER and HER2; 98 of them did express epidermal growth factor receptor and/or cytokeratin 5/6. The luminal-like tumours had a significantly better recurrence free and overall survival than the other two groups. From the 194 HER2-positive tumours, 47 (24%) were shown to harbour an amplification of TOP2A. Patients with an HER2-amplified tumour randomised to the high-dose therapy arm did worse than those in the conventional treatment arm, possibly caused by the lower cumulative anthracycline dose in the high-dose arm. The tumours with a TOP2A amplification contributed hardly to this difference, suggesting that TOP2A amplification is not the cause of the steep dose-response curve for anthracyclines in breast cancer. Possibly, the difference of the cumulative dose of only 25% between the treatment arms was insufficient to yield a survival difference.

Determining MDR1/P-glycoprotein expression in breast cancer.

The mechanism of chemotherapy resistance in breast cancer is unresolved. MDR1/P-glycoprotein (P-Gp) over-expression confers multidrug resistance in vitro and might play a role in clinical breast cancer. Studies using clinical samples have yielded conflicting results. MDR1/P-Gp mRNA expression was determined relative to the expression in normal human liver using TaqMan real-time RT-PCR (corrected for expression of the housekeeping gene PBGD). Immunohistochemistry (IHC) was performed with monoclonal antibodies against P-Gp (JSB1, C219). The positive control was SW1573/2R160, the intermediate control SW1573 and the negative control GLC4/ADR. We assayed 9 breast-cancer cell lines by RT-PCR and IHC, 52 carcinoma samples by RT-PCR and 168 samples by IHC. SW1573/2R160 contained high levels of MDR1/P-Gp mRNA (1.0, equal to liver) and showed strong membranous staining. Expression of MDR1/P-Gp mRNA in SW1573 (0.05) and GLC4/ADR (3.2 x 10(-5)) was not detectable by IHC. Very low levels of MDR1/P-Gp mRNA were measured in breast-cancer cell lines (mean 3.1 x 10(-4), range 1 to 12 x 10(-4)), but P-Gp was not detected by IHC. In 25 specimens from chemotherapy-naive patients, MDR1/P-Gp mRNA levels varied from 1 to 11 x 10(-2) (mean 3.9 x 10(-2)). In sections of 80 chemotherapy-naive tumors, no membrane-bound staining was observed in the tumor cells. Tumors of 27 anthracycline-treated patients had comparable MDR1/P-Gp mRNA expression levels (mean 5.4 x 10(-2)). P-Gp was undetectable in 88 tumor samples of patients who had received anthracycline-based chemotherapy. In breast cancer, MDR1/P-Gp mRNA is low or absent and P-Gp levels in cancer cells are too low to detect by IHC. Chemotherapy exposure does not result in detectable MDR1/P-Gp over-expression.