Suzanne M. Dintzis

Validation and implementation of targeted capture and sequencing for the detection of actionable mutation, copy number variation, and gene rearrangement in clinical cancer specimens

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.

Quantitative molecular phenotyping with topically applied SERS nanoparticles for intraoperative guidance of breast cancer lumpectomy

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.

Upper Gastrointestinal Tract

Publisher Summary The upper gastrointestinal tract is composed of the oral cavity and salivary glands, esophagus, stomach, and small intestine (duodenum, jejunum, and ileum). In both species, the basic mural structure of the tract is maintained. The tract is essentially a smooth muscle–enveloped tube with innermost mucosa (barrier epithelium, lamina propria, and muscularis mucosae), submucosa, muscularis propria, and variable serosa or adventitia. The functions of the upper gastrointestinal tract include transport of the swallowed food bolus, enzymatic digestion, and absorption of nutrients, in addition to protective barrier function against the external environment. The morphologic appearance of the different sections of the upper digestive tract reflects the primary function of each segment and is variable between the species.

Raman-Encoded Molecular Imaging with Topically Applied SERS Nanoparticles for Intraoperative Guidance of Lumpectomy

Intraoperative identification of carcinoma at lumpectomy margins would enable reduced re-excision rates, which are currently as high as 20% to 50%. Although imaging of disease-associated biomarkers can identify malignancies with high specificity, multiplexed imaging of such biomarkers is necessary to detect molecularly heterogeneous carcinomas with high sensitivity. We have developed a Raman-encoded molecular imaging (REMI) technique in which targeted nanoparticles are topically applied on excised tissues to enable rapid visualization of a multiplexed panel of cell surface biomarkers at surgical margin surfaces. A first-ever clinical study was performed in which 57 fresh specimens were imaged with REMI to simultaneously quantify the expression of four biomarkers HER2, ER, EGFR, and CD44. Combined detection of these biomarkers enabled REMI to achieve 89.3% sensitivity and 92.1% specificity for the detection of breast carcinoma. These results highlight the sensitivity and specificity of REMI to detect biomarkers in freshly resected tissue, which has the potential to reduce the rate of re-excision procedures in cancer patients. Cancer Res; 77(16); 4506-16. ©2017 AACR.

Disclosing Harmful Pathology Errors to Patients

Imagine this scenario: You receive multiple stomach biopsy fragments showing diffuse surface ulceration, marked inflammation, and reactive atypia. In a small focus of 1 gastric mucosal fragment, there is a subtle, signet-ring adenocarcinoma. You notice on review of the patient’s history that she has had multiple previous stomach biopsies for dyspepsia and weight loss. With a sinking feeling, you pull the preceding biopsy specimens and find that signet-ring adenocarcinoma was present in a biopsy you signed out as benign 8 months earlier. After confirming your diagnoses with other pathologists in your department, you call the gastroenterologist to inform him that the patient currently has gastric adenocarcinoma and that the diagnosis was missed on a previous biopsy. You tell the gastroenterologist that you will issue an amended report correcting the missed adenocarcinoma. The gastroenterologist emphatically tells you not to amend the previous report and insists that the missed diagnosis will not alter current care options or patient survival, and that disclosing this information will only serve to upset “his” patient. As a clinician, and the person responsible for a diagnostic error, how would you handle this situation? Should you issue a corrected report regardless of the opinion of the treating physician? If you decide to issue a corrected report, because the treating physician most likely serves as the gatekeeper for patient information, the patient may never be informed of the delay in diagnosis. Should you contact the patient directly to share this information? Many would argue that because the pathologist has no established relationship with the patient, …

Biome representational in silico karyotyping

Metagenomic characterization of complex biomes remains challenging. Here we describe a modification of digital karyotyping-biome representational in silico karyotyping (BRISK)-as a general technique for analyzing a defined representation of all DNA present in a sample. BRISK utilizes a Type IIB DNA restriction enzyme to create a defined representation of 27-mer DNAs in a sample. Massively parallel sequencing of this representation allows for construction of high-resolution karyotypes and identification of multiple species within a biome. Application to normal human tissue demonstrated linear recovery of tags by chromosome. We apply this technique to the biome of the oral mucosa and find that greater than 25% of recovered DNA is nonhuman. DNA from 41 microbial species could be identified from oral mucosa of two subjects. Of recovered nonhuman sequences, fewer than 30% are currently annotated. We characterized seven prevalent unknown sequences by chromosome walking and find these represent novel microbial sequences including two likely derived from novel phage genomes. Application of BRISK to archival tissue from a nasopharyngeal carcinoma resulted in identification of Epstein-Barr virus infection. These results suggest that BRISK is a powerful technique for the analysis of complex microbiomes and potentially for pathogen discovery.

Lower Gastrointestinal Tract

Publisher Summary The primary function of the large intestine in both species humans and mice is to dehydrate and store fecal material. Extensive reabsorption of water and salt occurs in the right/proximal colon and continues throughout. Movement of the increasingly solid colonic contents from proximal to distal is supported by abundant mucus-secreting goblet cells that serve to lubricate and protect the colonic mucosa from trauma. Both species have abundant bacterial flora within the colon, which carry out a rumen-like function that is more prominent in mice.

Nose, Sinus, Pharynx, and Larynx

Abstract For all mammalian species, the nose serves as the portal of entry for the air they breathe. This complex organ of the respiratory system is important not only for smell but also for conditioning the inhaled air before it enters the lungs. The nose is well structured to perform these functions, but to differing degrees in rodents and humans. Despite obvious postural and body size differences, the rodent and human pharynx and larynx share many functional and structural similarities. These include reasonable consistency in the types of covering epithelium, including the presence of intermediate zones, and subepithelial structures. Differences of note involve the portions of the structure of the Eustachian tube, some laryngeal and vocal cord components, submucosal lymphoid elements, and taste buds. This chapter provides a detailed description of the macro- and microscopic anatomy of the rodent nose, sinuses, larynx, and pharynx, and it highlights similarities and differences in structure and function between rodents and humans.

8 – Salivary Glands

Publisher Summary In mice and humans, there are three pairs of major salivary glands and numerous minor glands. Salivary secretions may be serous, mucous, or mixed, and they serve similar functions in mice and humans—to aid in digestion, taste, protection, and lubrication of the teeth and oral cavity. Mice and humans have three paired major salivary glands that are visible grossly—the parotid, submandibular, and sublingual—and numerous microscopic minor glands. Of the major glands in the mouse, the submandibular (submaxillary) is the largest. This gland is multilobed and lobulated and is located on the ventral midline of the neck, where the left and right glands are opposed and extend toward the sternum. The submandibular gland is rostrally bordered by the submandibular lymph node, where it also abuts the sublingual gland, extending dorsally to the parotid gland. The sublingual gland is much smaller and consists of a single lobe located between the submandibular lymph nodes and the submandibular salivary gland.

22 – Special Senses: Ear

The ear of mice, rats, and humans detects sound and maintains balance. In mammals, this “stato-acoustic” system is traditionally divided into three structural units: the external, middle, and inner ear. The external and middle ear are designed to receive, transmit, and amplify sound. The sensory receptors for both hearing and balance are located in a membranous portion of the inner ear. Sensory areas in all three species have similar histologic design with some modifications.