Recent years have seen development and implementation of anticancer therapies targeted to particular gene mutations, but methods to assay clinical cancer specimens in a comprehensive way for the critical mutations remain underdeveloped. We have developed UW-OncoPlex, a clinical molecular diagnostic assay to provide simultaneous deep-sequencing information, based on >500× average coverage, for all classes of mutations in 194 clinically relevant genes. To validate UW-OncoPlex, we tested 98 previously characterized clinical tumor specimens from 10 different cancer types, including 41 formalin-fixed paraffin-embedded tissue samples. Mixing studies indicated reliable mutation detection in samples with ≥ 10% tumor cells. In clinical samples with ≥ 10% tumor cells, UW-OncoPlex correctly identified 129 of 130 known mutations [sensitivity 99.2%, (95% CI, 95.8%-99.9%)], including single nucleotide variants, small insertions and deletions, internal tandem duplications, gene copy number gains and amplifications, gene copy losses, chromosomal gains and losses, and actionable genomic rearrangements, including ALK-EML4, ROS1, PML-RARA, and BCR-ABL. In the same samples, the assay also identified actionable point mutations in genes not previously analyzed and novel gene rearrangements of MLL and GRIK4 in melanoma, and of ASXL1, PIK3R1, and SGCZ in acute myeloid leukemia. To best guide existing and emerging treatment regimens and facilitate integration of genomic testing with patient care, we developed a framework for data analysis, decision support, and reporting clinically actionable results.

There is a need to image excised tissues during tumor-resection procedures in order to identify residual tumors at the margins and to guide their complete removal. The imaging of dysregulated cell-surface receptors is a potential means of identifying the presence of diseases with high sensitivity and specificity. However, due to heterogeneities in the expression of protein biomarkers in tumors, molecular-imaging technologies should ideally be capable of visualizing a multiplexed panel of cancer biomarkers. Here, we demonstrate that the topical application and quantification of a multiplexed cocktail of receptor-targeted surface-enhanced Raman scattering (SERS) nanoparticles (NPs) enables rapid quantitative molecular phenotyping (QMP) of the surface of freshly excised tissues to determine the presence of disease. In order to mitigate the ambiguity due to nonspecific sources of contrast such as off-target binding or uneven delivery, a ratiometric method is employed to quantify the specific vs. nonspecific binding of the multiplexed NPs. Validation experiments with human tumor cell lines, fresh human tumor xenografts in mice, and fresh human breast specimens demonstrate that QMP imaging of excised tissues agrees with flow cytometry and immunohistochemistry, and that this technique may be achieved in less than 15 minutes for potential intraoperative use in guiding breast-conserving surgeries.

Oncotype DX(TM) is an RT-PCR-based assay used to predict chemotherapy benefit in patients with estrogen receptor (ER) positive breast cancers. We were interested if routinely available pathologic parameters could predict Oncotype DX Recurrence Scores (RS) in subsets of patients. We identified 173 breast cancers with available RSs and used 104 of these as a test set and 69 cases as a validation set. Pathologic characteristics including size, histologic type, Nottingham grade, and lymphatic invasion were recorded. Test set cases were stained for ER, progesterone receptor (PR), HER2, Ki67, CyclinD1, BCL2, D2-40, and P53. Statistical correlations with RS and regression tree analysis were performed. The validation set was subjected to analysis on the basis of grade, PR, and Ki67. In the test set, grade, PR levels and Ki67 had the strongest correlation with RS (P = 0.0002-0.0007). Regression tree analysis showed grade and PR as factors that could segregate cases into RS categories, with Ki67 adding value in certain subsets. A subset of cancers with a high likelihood of having a low RS (0-18) was identified with the following characteristics: grade 1, strong PR expression (Allred score ≥ 5) and Ki67 ≤ 10%. No cases with these characteristics had a high RS (≥ 31) and 73% had a low RS. Cancers highly likely to have a high RS were grade 3, low to absent PR expression (Allred score <5) and Ki67 > 10%. 80% of cases with these characteristics had a high RS and no cases had a low RS. Our validation set had similar findings in these two subsets. In conclusion, When cost and time are a consideration and the added value of Oncotype DX(TM) testing is in question, it may be reasonable to assume the results of this test in two specific subsets of breast cancers: (1) grade 1, high PR, low Ki67 cancers (low RS), and (2) grade 3, low PR, high Ki67 cancers (high RS).