Breast MRI-detected cystic apocrine metaplasia: imaging features with microvessel analysis and histologic correlation.

OBJECTIVE: The purpose of this article is to characterize the histologic vascular features and distinguishing MRI features of cystic apocrine metaplasia to better understand imaging-pathology concordance. MATERIALS AND METHODS: Retrospective review of 261 consecutive MRI-guided biopsy cases was performed. Pathology results were reviewed for all biopsies; cystic apocrine metaplasia was identified as the predominant finding in 19 cases (7%). CD31 immunohistochemistry was subsequently performed on the most representative block of cystic apocrine metaplasia, and microvasculature was evaluated using computer-assisted image analysis. The contrast-enhanced MRI examinations correlating with the cystic apocrine metaplasia cases were independently reviewed by two radiologists specializing in breast imaging; lesions were analyzed for morphologic, kinetic, and T2 characteristics. RESULTS: On MRI review, 17 of 19 (89%) lesions were 10 mm or smaller. Washout kinetics were present in 11 of 19 (58%) lesions, and 14 of 19 (74%) lesions were at least partially hyperintense on T2-weighted sequences relative to adjacent glandular tissue. Cystic apocrine metaplasia had a higher percentage area (mean, 4.1%) of CD31-immunostained microvessels compared with background fibroglandular tissue (mean, 1.2%). CONCLUSION: Cystic apocrine metaplasia should be considered in the differential diagnosis of a T2-hyperintense enhancing focus or subcentimeter smoothly marginated mass, even if associated with washout kinetics. Cystic apocrine metaplasia contains a statistically significant increase in microvessel area compared with background fibroglandular tissue and fat and, therefore, may be considered a concordant result for this set of imaging findings.

Spectral discrimination of breast pathologies in situ using spatial frequency domain imaging.

INTRODUCTION: Nationally, 25% to 50% of patients undergoing lumpectomy for local management of breast cancer require a secondary excision because of the persistence of residual tumor. Intraoperative assessment of specimen margins by frozen-section analysis is not widely adopted in breast-conserving surgery. Here, a new approach to wide-field optical imaging of breast pathology in situ was tested to determine whether the system could accurately discriminate cancer from benign tissues before routine pathological processing. METHODS: Spatial frequency domain imaging (SFDI) was used to quantify near-infrared (NIR) optical parameters at the surface of 47 lumpectomy tissue specimens. Spatial frequency and wavelength-dependent reflectance spectra were parameterized with matched simulations of light transport. Spectral images were co-registered to histopathology in adjacent, stained sections of the tissue, cut in the geometry imaged in situ. A supervised classifier and feature-selection algorithm were implemented to automate discrimination of breast pathologies and to rank the contribution of each parameter to a diagnosis. RESULTS: Spectral parameters distinguished all pathology subtypes with 82% accuracy and benign (fibrocystic disease, fibroadenoma) from malignant (DCIS, invasive cancer, and partially treated invasive cancer after neoadjuvant chemotherapy) pathologies with 88% accuracy, high specificity (93%), and reasonable sensitivity (79%). Although spectral absorption and scattering features were essential components of the discriminant classifier, scattering exhibited lower variance and contributed most to tissue-type separation. The scattering slope was sensitive to stromal and epithelial distributions measured with quantitative immunohistochemistry. CONCLUSIONS: SFDI is a new quantitative imaging technique that renders a specific tissue-type diagnosis. Its combination of planar sampling and frequency-dependent depth sensing is clinically pragmatic and appropriate for breast surgical-margin assessment. This study is the first to apply SFDI to pathology discrimination in surgical breast tissues. It represents an important step toward imaging surgical specimens immediately ex vivo to reduce the high rate of secondary excisions associated with breast lumpectomy procedures.

Establishment of a CYP2C19 genotyping assay for clinical use.

Conversion of clopidogrel (Plavix) to its active metabolite is catalyzed largely by the P450 enzyme 2C19 (CYP2C19). Numerous allelic variants of CYP2C19 exist. The *1 allele is considered wild type, whereas the *2 and *3 alleles have no in vivo enzymatic activity. Conversely, the *17 allele has increased expression, resulting in increased clopidogrel activation. Poor metabolizers (*2/*2 and *2/*3 genotypes) experience higher rates of therapeutic failure. For this reason, we have validated a CYP2C19 genotyping assay for the *1, *2, *3, and *17 alleles. Genomic DNA extracted from 30 deidentified EDTA whole-blood samples from patients was analyzed at 2 independent facilities using specific TaqMan realtime polymerase chain reaction primers and probes. Concordant genotypes were generated on all samples tested. Of the 30 samples, 15 were CYP2C19*1/*1, 8 were CYP2C19*1/*17, 5 were CYP2C19*1/*2, and 2 were CYP2C19*2/*17. There were no CYP2C19*3 alleles or *2/*2 homozygous genotypes detected. This CYP2C19 genotyping assay is appropriate for clinical testing, demonstrating excellent interlaboratory concordance, enabling the selection of the most effective clopidogrel treatment regimen for patients undergoing percutaneous coronary intervention.

Cytomorphologic features of advanced lung adenocarcinomas tested for EGFR and KRAS mutations: a retrospective review of 50 cases.

Associations between bronchioloalveolar carcinoma (BAC), mucinous differentiation, and epidermal growth factor receptor (EGFR) and KRAS mutations have been previously reported in studies of surgical specimens. We present the cytomorphology of lung adenocarcinomas, including metastases that were diagnosed by cytologic methods and the relationship to both EGFR and KRAS mutational status. We retrospectively reviewed the clinical and cytomorphologic features of 50 lung adenocarcinomas that were tested for both EGFR and KRAS mutations. Cytomorphologic features evaluated included cell size, architectural pattern, nucleoli, intranuclear cytoplasmic inclusions (INCI), mucin, necrosis, squamoid features, lymphocytic response, and histologic features of BAC differentiation. DNA was extracted from a paraffin-embedded cell block or frozen needle core fragments. Exon 19 deletions and the L858R mutation in exon 21 of EGFR were detected using PCR followed by capillary electrophoresis for fragment sizing. KRAS mutational analysis was performed by real-time PCR using a set of seven different Taqman(r) allelic discrimination assays to detect six mutations in codon 12 and one mutation in codon 13. Six cases (12%) showed EGFR mutations, 12 (24%) showed KRAS mutations, and 38 (62%) contained neither EGFR nor KRAS mutations. The majority of patients had stage IV disease (78%); 20 samples (40%) were from metastatic sites. The presence of prominent INCI (P = 0.036), papillary fragments (P = 0.041), and histologic features of BAC on paraffin block (P = 0.039) correlated with the presence of EGFR mutations. The presence of necrosis (P = 0.030), squamoid features (P = 0.048), and poorly differentiated tumors (P = 0.025) were more likely to be identified in the KRAS positive group.

Scatter spectroscopic imaging distinguishes between breast pathologies in tissues relevant to surgical margin assessment.

PURPOSE: A new approach to spectroscopic imaging was developed to detect and discriminate microscopic pathologies in resected breast tissues; diagnostic performance of the prototype system was tested in 27 tissues procured during breast conservative surgery. EXPERIMENTAL DESIGN: A custom-built, scanning in situ spectroscopy platform sampled broadband reflectance from a 150-µm-diameter spot over a 1 × 1 cm(2) field using a dark field geometry and telecentric lens; the system was designed to balance sensitivity to cellular morphology and imaging the inherent diversity within tissue subtypes. Nearly 300,000 broadband spectra were parameterized using light scattering models and spatially dependent spectral signatures were interpreted using a cooccurrence matrix representation of image texture. RESULTS: Local scattering changes distinguished benign from malignant pathologies with 94% accuracy, 93% sensitivity, 95% specificity, and 93% positive and 95% negative predictive values using a threshold-based classifier. Texture and shape features were important to optimally discriminate benign from malignant tissues, including pixel-to-pixel correlation, contrast and homogeneity, and the shape features of fractal dimension and Euler number. Analysis of the region-based diagnostic performance showed that spectroscopic image features from 1 × 1 mm(2) areas were diagnostically discriminant and enabled quantification of within-class tissue heterogeneities. CONCLUSIONS: Localized scatter-imaging signatures detected by the scanning spectroscopy platform readily distinguished benign from malignant pathologies in surgical tissues and showed new spectral-spatial signatures of clinical breast pathologies.

Correlation of polypoid colorectal adenocarcinoma with pre-existing adenomatous polyps and KRAS mutation.

Cetuximab is an anti-epidermal growth factor receptor that helps effectively treat patients with advanced colorectal adenocarcinoma without KRAS activating mutations. KRAS mutations are associated with 16% to 50% of isolated villous adenomas and approximately 30% of colorectal cancer. Correlation between the gross and histological subset of colorectal adenocarcinoma with KRAS mutation is unknown. Archived surgical resection specimens of colorectal adenocarcinoma (n = 42) and villous adenoma (n = 9) were collected. The gross appearance and histopathological features of these lesions were thoroughly reviewed, including the presence of a pre-existing adenomatous polyp. DNA was extracted from formalin-fixed, paraffin-embedded tissue sections and then subjected to TaqMan real-time polymerase chain reaction to detect the seven most common KRAS mutations. KRAS mutations were found in 13 of 42 cases (31%) of colorectal adenocarcinoma and 7 of 9 cases (78%) of villous adenoma. All 13 cases of colorectal carcinoma with a KRAS mutation showed a gross polypoid configuration, compared to no KRAS mutation in the colorectal carcinomas with ulcerative configuration. In addition, 13 of 17 of these cases (76%) had histological features of adenocarcinoma with a persistent preexisting adenomatous polyp with villous architecture. In summary, grossly polypoid colorectal adenocarcinomas with a persistent pre-existing adenomatous polyp with villous architecture are strongly associated with KRAS mutations.

Tumor angiogenesis change estimated by using diffuse optical spectroscopic tomography: demonstrated correlation in women undergoing neoadjuvant chemotherapy for invasive breast cancer?

PURPOSE: To investigate if changes in tumor angiogenesis associated with complete pathologic response (pCR) or partial pathologic response (pPR) to treatment can be demonstrated by using diffuse optical spectroscopic (DOS) tomography. MATERIALS AND METHODS: All participants in this prospective, HIPAA-compliant, institutional review board-approved study provided written informed consent. Eleven women with invasive breast carcinoma were imaged with DOS tomography prior to, during, and at completion of neoadjuvant chemotherapeutic regimens. By using region of interest (ROI) analysis, the DOS measure of total tissue hemoglobin (Hb(T)) was temporally correlated with quantitative measures of existing (CD31-expressing) and tumor-induced (CD105-expressing) vessels, in pretreatment and posttreatment tissue specimens, to assess change. RESULTS: Quantified angiogenesis alone in pretreatment core biopsy specimens did not predict treatment response, but mean vessel density (MVD) and mean vessel area (MVA) of CD105-expressing vessels were significantly decreased in women with pCR (n = 7) (P < .001 and P = .003, respectively). MVA of CD105-expressing vessels was also significantly reduced at comparison of pre- and posttreatment residual tumor for women with pPR (n = 4) (P = .033). A longitudinal analysis showed significant decreases (P = .001) in mean Hb(T) levels during neoadjuvant chemotherapy in breast abnormality ROIs for women with pCR but not women with pPR. For women with pCR, but not women with pPR, pretreatment MVD of CD105-expressing vessels correlated with pretreatment Hb(T) (P ≤ .001). CONCLUSION: DOS tomographic examinations in women with breast cancer who are receiving neoadjuvant chemotherapy show a mean decrease in Hb(T) with time in patients with pCR only. Observed pretreatment and posttreatment correlates with quantified angiogenesis markers confirm the likely biologic origin for this DOS signature and support its potential to predict angiogenic tissue response early in the treatment cycle. SUPPLEMENTAL MATERIAL: http://radiology.rsna.org/lookup/suppl/doi:10.1148/radiol.11100699/-/DC1.

Warfarin genotyping using three different platforms.

Genetic testing for common variants in the CYP2C9 and VKORC1 genes may provide useful clinical information to guide dosing patients receiving oral warfarin. Specifically, the CYP2C9*2, CYP2C9*3 and either the VKORC1-1639 G>A or VKORC1 1173C>T polymorphisms can be used to help predict an approximate warfarin maintenance dose needed for a particular patient. Although clinical uptake and use of this genotyping has been slow, an increasing body of literature provides evidence of the clinical utility of supplementing traditional warfarin dosing algorithms with a pharmacogenetic approach. The availability of multiple methods for clinical genotyping provides the opportunity for molecular diagnostic laboratories to introduce genotyping assays tailored to their specific needs based on variables such as testing volumes, staffing, available instrumentation and needed turnaround times. Three assays (Invader, Verigene and TaqMan) designed to detect three genetic variations associated with warfarin dosing are evaluated and compared as potential clinical tests to assist in patient care. Identical genotypes were reported by each assay for all samples tested but the assays were found to differ in turnaround time, approval status by the U.S. Food and Drug Administration (FDA), requirements for amount of input genomic DNA and other logistical factors that might make each assay more favorable in different settings.

A validation study of a new molecular diagnostic assay: the Dartmouth-Hitchcock Medical Center experience with the GeneSearch BLN assay in breast sentinel lymph nodes.

BACKGROUND: Sentinel lymph node (SLN) processing remains variable in terms of performing multiple tissue levels and immunohistochemical (IHC) or PCR-based assays. A rapid and reliable molecular pathology assay, as an adjunct to routine SLN processing, could minimize and standardize the histologic evaluation needed for an accurate and clinically significant diagnosis. We compared the recently FDA-approved Veridex GeneSearch Breast Lymph Node (BLN) Assay (Veridex, LLC; Warren, NJ), a real time reverse transcriptase-polymerase chain reaction assay that is designed to detect metastases >0.2 mm, with our standard lymph node processing. MATERIALS AND METHODS: The GeneSearch BLN assay evaluates RNA expression data for three target genes (mammaglobin, cytokeratin 19, and internal control porphobilinogen deaminase), and provides a qualitative (positive/negative) result. In 59 patients, the assay was performed on SLN tissue that would normally be deep within the tissue block and not routinely evaluated histologically. Two 1 -mm slices from the outer node portions were submitted fresh for RNA extraction; the remaining tissue was submitted for routine histology. RESULTS: Of the 59 patients, the assay determined 43 as true negative, eight as true positive, one as false-negative, three as false-positive, and four as invalid. Assay sensitivity was 88.9%, specificity 93.5%. DISCUSSION: The sensitivity of the assay sampling from the outer node tissue was high (88.9%) and identical to that validated in the large registration study in which half of the node was assessed as alternate slices (87.6%). Our protocol uses this assay as an adjunct to traditional histologic evaluation, to reduce and standardize the number of tissue sections needed for thorough SLN evaluation, and to enhance our ability to bank RNA.

Heterogeneous patterns of pH regulation in glial cells in the dorsal and ventral medulla.

We examined pH regulation in two chemosensitive areas of the brain, the retrotrapezoid nucleus (RTN) and the nucleus tractus solitarius (NTS), to identify the proton transporters involved in regulation of intracellular pH (pHi) in medullary glia. Transverse brain slices from young rats [postnatal day 8 (P8) to P20] were loaded with the pH-sensitive probe 2',7'-bis (2-carboxyethyl)-5,6-carboxyfluorescein after kainic acid treatment removed neurons. Cells were alkalinized when they were depolarized (extracellular K+ increased from 6.24 to 21.24 mM) in the RTN but not in the NTS. This alkaline shift was inhibited by 0.5 mM DIDS. Removal of CO2/HCO3- or Na+ from the perfusate acidified the glial cells, but the acidification after Na+ removal was greater in the RTN than in the NTS. Treatment of the slice with 5-(N-ethyl-N-isopropyl)amiloride (100 microM) in saline containing CO2/HCO3- acidified the cells in both nuclei, but the acidification was greater in the NTS. Restoration of extracellular Cl- after Cl- depletion during the control condition acidified the cells. Immunohistochemical studies of glial fibrillary acid protein demonstrated much denser staining in the RTN compared with the NTS. We conclude that there is evidence of Na+-HCO3- cotransport and Na+/H+ exchange in glia in the RTN and NTS, but the distribution of glia and the distribution of these pH-regulatory functions are not identical in the NTS and RTN. The differential strength of glial pH regulatory function in the RTN and NTS may also alter CO2 chemosensory neuronal function at these two chemosensitive sites in the brain stem.