Introduction Alterations in cell cycle regulators have been implicated in human malignancies including breast cancer. PD 0332991 is an orally active, highly selective inhibitor of the cyclin D kinases (CDK)4 and CDK6 with ability to block retinoblastoma (Rb) phosphorylation in the low nanomolar range. To identify predictors of response, we determined the in vitro sensitivity to PD 0332991 across a panel of molecularly characterized human breast cancer cell lines. Methods Forty-seven human breast cancer and immortalized cell lines representing the known molecular subgroups of breast cancer were treated with PD 0332991 to determine IC50 values. These data were analyzed against baseline gene expression data to identify genes associated with PD 0332991 response.

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PD 0332991, a selective cyclin D kinase 4/6 inhibitor, preferentially inhibits proliferation of luminal estrogen receptor-positive human breast cancer cell lines in vitro

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that is commonly upregulated in cancers such as in non-small-cell lung cancer, metastatic colorectal cancer, glioblastoma, head and neck cancer, pancreatic cancer, and breast cancer Various mechanisms mediate the upregulation of EGFR activity, including common mutations and truncations to its extracellular domain, such as in the EGFRvIII truncations, as well as to its kinase domain, such as the L858R and T790M mutations, or the exon 19 truncation These EGFR aberrations over-activate downstream pro-oncogenic signaling pathways, including the RAS-RAF-MEK-ERK MAPK and AKT-PI3K-mTOR pathways These pathways then activate many biological outputs that are beneficial to cancer cell proliferation, including their chronic initiation and progression through the cell cycle Here, we review the molecular mechanisms that regulate EGFR signal transduction, including the EGFR structure and its mutations, ligand binding and EGFR dimerization, as well as the signaling pathways that lead to G1 cell cycle progression We focus on the induction of CYCLIN D expression, CDK4/6 activation, and the repression of cyclin-dependent kinase inhibitor proteins (CDKi) by EGFR signaling pathways We also discuss the successes and challenges of EGFR-targeted therapies, and the potential for their use in combination with CDK4/6 inhibitors

Epidermal Growth Factor Receptor Cell Proliferation Signaling Pathways.

Breast cancer is the most common malignancy among women. Most of these cancers overexpress cyclin D1, a component of the core cell-cycle machinery. We previously generated mice lacking cyclin D1 using gene targeting. Here we report that these cyclin D1-deficient mice are resistant to breast cancers induced by the neu and ras oncogenes. However, animals lacking cyclin D1 remain fully sensitive to other oncogenic pathways of the mammary epithelium, such as those driven by c-myc or Wnt-1. Our analyses revealed that, in mammary epithelial cells, the Neu-Ras pathway is connected to the cell-cycle machinery by cyclin D1, explaining the absolute dependency on cyclin D1 for malignant transformation in this tissue. Our results suggest that an anti-cyclin D1 therapy might be highly specific in treating human breast cancers with activated Neu-Ras pathways.

Specific protection against breast cancers by cyclin D1 ablation

Uncontrolled cellular proliferation, mediated by dysregulation of the cell-cycle machinery and activation of cyclin-dependent kinases (CDKs) to promote cell-cycle progression, lies at the heart of cancer as a pathological process. Clinical implementation of first-generation, nonselective CDK inhibitors, designed to inhibit this proliferation, was originally hampered by the high risk of toxicity and lack of efficacy noted with these agents. The emergence of a new generation of selective CDK4/6 inhibitors, including ribociclib, abemaciclib and palbociclib, has enabled tumour types in which CDK4/6 has a pivotal role in the G1-to-S-phase cell-cycle transition to be targeted with improved effectiveness, and fewer adverse effects. Results of pivotal phase III trials investigating palbociclib in patients with advanced-stage oestrogen receptor (ER)-positive breast cancer have demonstrated a substantial improvement in progression-free survival, with a well-tolerated toxicity profile. Mechanisms of acquired resistance to CDK4/6 inhibitors are beginning to emerge that, although unwelcome, might enable rational post-CDK4/6 inhibitor therapeutic strategies to be identified. Extending the use of CDK4/6 inhibitors beyond ER-positive breast cancer is challenging, and will likely require biomarkers that are predictive of a response, and the use of combination therapies in order to optimize CDK4/6 targeting.

Treating cancer with selective CDK4/6 inhibitors

ERBB2 oncogene in human breast cancer and its clinical significance.

Cyclin D1 plays a critical role in the timing of the initiation of DNA synthesis in the normal cell cycle of mammalian cells Deregulated expression of this protein has been seen in a variety of tumours either as a result of gene amplification or chromosomal translocation, in breast cancer and B cell malignancies respectively In order to determine the role this putative oncoprotein plays in breast cancer, we have applied a new monoclonal antibody, recently produced in our laboratory, in an immunohistochemical study of 93 primary breast carcinomas We show that approximately 28% of the cases displayed enhanced expression of the cyclin D1 protein Furthermore, either cyclin D1, cyclin D3, or both, were expressed in 69% of cases, suggesting that overexpression of any one member of this family may relieve cancer cells of their mitogenic stimulatory requirement In addition, we show that those patients whose breast cancers co-express cyclin D1 with either epidermal growth factor receptor (EGFR) or the retinoblastoma protein (pRB) have a significantly poorer prognosis in comparison to those expressing cyclin D1 alone Our observations indicate that, in a subset of breast cancers, aberrant cyclin D1 expression is a contributory factor to tumorigenesis and in association with EGFR or pRB expression, identify those tumours which may require more aggressive therapy

Determination of the prognostic value of cyclin D1 overexpression in breast cancer

We examined the reactivity of four p53-specific monoclonal antibodies--PAb 1801, p53-BP-12, D07 and CM1--on sections of formalin-fixed tissue collected from 245 breast carcinomas. Immunodetection of p53 varied between 37.6% and 46.6%. The greatest variation was observed among lobular carcinomas and low-grade tumors in which immunodetection varied between 8.3% and 27.3%. In contrast, immunodetection of p53 in invasive ductal carcinomas was subject to a lower degree of variability with between 40.6% and 49.7% of these tumours proving to be positive. In general, we found antibodies PAb 1801 and DO7 to be the most effective in immunolocalising p53. Immunodetection of p53 with each of the four antibodies was found to correlate strongly with tumour grade. In survival analysis, the results gained using antibody PAb 1801 proved to be of greatest statistical significance and to provide the strongest index of prognosis. A significant relationship was observed between immunodetection of p53 with each of the four antibodies and poor responsiveness to endocrine therapy. In addition, relationships were also observed between p53 immunostaining and tumour oestrogen receptor (ER) status as well as c-jun expression. We observed no correlation between abnormalities of the p53 and the Rb gene products or between elevated c-erbB-2 or epidermal growth factor receptor (EGFR) expression and immunodetection of p53.

p53 protein as a prognostic indicator in breast carcinoma: a comparison of four antibodies for immunohistochemistry

Aims

Assessment of the expression of antigens CD5, CD10 and CD23 can be of value in the differential diagnosis of small B-cell lymphoma. Correct subclassification is important since optimal treatment regimes differ between the subtypes. The aim of this study was to generate monoclonal antibodies recognizing these antigens in paraffin-embedded tissue and to assess their efficacy using a panel of cases of small B-cell lymphoma of various subtypes.



Methods and results

For each antibody synthetic recombinant protein and conventional murine hybridoma technology was employed. Monoclonal antibodies effective in formalin-fixed, paraffin-embedded tissue were successfully generated, designated NCL-CD5-4C7, NCL-CD10-270 and NCL-CD23-1B12, respectively. A series of 58 cases of small B-cell lymphoma including examples of each subtype (lymphocytic, follicle centre cell, mantle cell, marginal zone and lymphoplasmacytoid) was assembled and immunostaining for the respective antigens carried out using the monoclonal antibodies produced. Our results indicate that the antibodies are specific for their respective antigens and give the predicted phenotypic profile in the small B-cell lymphoma subtypes.



Conclusions

These novel monoclonal antibodies may be of value in routine diagnostic practice.

Monoclonal antibodies recognizing CD5, CD10 and CD23 in formalin-fixed, paraffin-embedded tissue: production and assessment of their value in the diagnosis of small B-cell lymphoma.

G. G. Mcintosh

Papers

Determination of the prognostic value of cyclin D1 overexpression in breast cancer

p53 protein as a prognostic indicator in breast carcinoma: a comparison of four antibodies for immunohistochemistry

Monoclonal antibodies recognizing CD5, CD10 and CD23 in formalin-fixed, paraffin-embedded tissue: production and assessment of their value in the diagnosis of small B-cell lymphoma.