Protein biomarkers for subtyping breast cancer and implications for future research

Abstract

Breast cancer subtypes are currently defined by a combination of morphologic, genomic, and proteomic characteristics. These subtypes provide a molecular portrait of the tumor that aids diagnosis, prognosis, and treatment escalation/de-escalation options. Gene expression signatures describing intrinsic breast cancer subtypes for predicting risk of recurrence have been rapidly adopted in the clinic. Despite the use of subtype classifications, many patients develop drug resistance, breast cancer recurrence, or therapy failure. Areas covered: This review provides a summary of immunohistochemistry, reverse phase protein array, mass spectrometry, and integrative studies that are revealing differences in biological functions within and between breast cancer subtypes. We conclude with a discussion of rigor and reproducibility for proteomic-based biomarker discovery. Expert commentary: Innovations in proteomics, including implementation of assay guidelines and standards, are facilitating refinement of breast cancer subtypes. Proteomic and phosphoproteomic information distinguish biologically functional subtypes, are predictive of recurrence, and indicate likelihood of drug resistance. Actionable, activated signal transduction pathways can now be quantified and characterized. Proteomic biomarker validation in large, well-designed studies should become a public health priority to capitalize on the wealth of information gleaned from the proteome.

Keywords: Basal-like; HER2; biomarker; breast cancer; estrogen receptor; mass spectrometry; progesterone receptor; reverse phase protein array; signal transduction; triple negative breast cancer.

Figure 1.. Current scheme for assigning breast cancer subtypes.

Histopathology is integrated with proteomic and genomic biomarkers to characterize breast tumors into clinically relevant subtypes. The first step is pathologist review of the tumor histology which is described as in situ or invasive, with the corresponding architectural pattern [–4,11,211,212]. If the tumor is infiltrating ductal carcinoma, the stage of cellular differentiation is also described. Next selected protein biomarkers are semi-quantitatively scored based on the immunohistochemical staining pattern [,,,,–215]. For ER+/PR+/HER2neg, lymph node negative tumors the Predictive Analysis of Microarray 50 gene signature (Prosigna, PAM50 assay) provides a predictive risk of recurrence score [40,41,44]. Integration of proteomic biomarker scores with gene expression signatures and clinical information aids therapy escalation and de-escalation decisions [33,37].

Figure 2.. Intra-tumor heterogeneity in breast cancer

Breast tumors may contain cellular sub-clones harboring a variety of genomic and proteomic alterations. Clonal cooperation and emergence of sub-clones during treatment have a profound influence on the biological phenotype of the tumor and treatment resistance. Combination treatment to simultaneously, or sequentially, eliminate every clonal population could potentially provide greater ther efficacy.

Figure 3.. IHC provides protein biomarker cellular context and subcellular location

Example IHC staining patterns for (a) Ki-67 and (b) HER2 using Ki-67 antibody clone MIB-1 and Dako HercepTest™, respectively. Positive staining, which appears brown, occurs via deposition of diaminobenzidine at the site of the antigen-antibody interaction. The subcellular location of the staining is used for quality control; Ki-67 should be localized in the nucleus, whereas HER2 should be localized to the plasma membrane. IHC scoring can be qualitative (0, 1+, 2+, 3+) or semi-quantitative, based on the intensity of the stain and the proportion of positively stained cells. All IHC biomarker scoring is interpreted within the context of appropriate positive and negative controls. HER2 scoring using the HercepTest™ (Agilent/Dako) is considered negative if the membrane staining score is 0 or 1+. A score of 2+ is considered weakly positive. A score of 3+ is strongly positive. Note: HerceptTest™ staining is recommended for invasive cancers and any cytoplasmic staining should not be scored. The DCIS image depicted in panel B was for research use only and to illustrate IHC staining that is localized to the plasma membrane [216] (Ki-67 magnification 100x, HER2 magnification 200x).

Figure 4.. Protein carry-overin liquid chromatography mass spectrometry potentially confounds results.

(a) Experimental procedure to assess protein carry-over in LC-MS/MS based on insertion of blank samples or adjustment of mobile phase flow rate. One human serum sample, used as a control, was analyzed by LC-MS/MS to ensure no E. coli contamination. A subsequent E. coli sample, consisting of recombinant E. coli DXP reductoisomerase (DXR) prepared in E. coli BL21 (DE3) RIL Codon Plus cells and purified to >90% purity, was analyzed for high and low abundance proteins. Carry-over of E. coli proteinswere examined in 5 subsequently analyzed human serum samples. All human serum samples were from the same serum aliquot. (b) Carry-over of E. coli proteins in subsequent human serum samples. No significant carryover of E. coli proteins were observed in any subsequent human serum samples using any of the five defined experimental protocols. (c) Carry-over of human serum proteins in E. coli samples. Carry-over was observed in all samples, with the greatest carry-over seen at the lowest volume in the blanking protocol and with the highest flow rates. (d) Human serum proteins carried over in E. coli samples. Albumin was found in all five E. coli samples, while apoliopprotein A-II preprotein, hemopexin, and trypsin-1 Isoform X1 were also found in the majority of the E. coli samples.