Amy Ly

Whole exome sequencing of circulating tumor cells provides a window into metastatic prostate cancer

Comprehensive analyses of cancer genomes promise to inform prognoses and precise cancer treatments. A major barrier, however, is inaccessibility of metastatic tissue. A potential solution is to characterize circulating tumor cells (CTCs), but this requires overcoming the challenges of isolating rare cells and sequencing low-input material. Here we report an integrated process to isolate, qualify and sequence whole exomes of CTCs with high fidelity using a census-based sequencing strategy. Power calculations suggest that mapping of >99.995% of the standard exome is possible in CTCs. We validated our process in two patients with prostate cancer, including one for whom we sequenced CTCs, a lymph node metastasis and nine cores of the primary tumor. Fifty-one of 73 CTC mutations (70%) were present in matched tissue. Moreover, we identified 10 early trunk and 56 metastatic trunk mutations in the non-CTC tumor samples and found 90% and 73% of these mutations, respectively, in CTC exomes. This study establishes a foundation for CTC genomics in the clinic.

The Oxytricha trifallax Macronuclear Genome: A Complex Eukaryotic Genome with 16,000 Tiny Chromosomes

With more chromosomes than any other sequenced genome, the macronuclear genome of Oxytricha trifallax has a unique and complex architecture, including alternative fragmentation and predominantly single-gene chromosomes.

RNA Sequencing of Tumor-Associated Microglia Reveals Ccl5 as a Stromal Chemokine Critical for Neurofibromatosis-1 Glioma Growth

Solid cancers develop within a supportive microenvironment that promotes tumor formation and growth through the elaboration of mitogens and chemokines. Within these tumors, monocytes (macrophages and microglia) represent rich sources of these stromal factors. Leveraging a genetically engineered mouse model of neurofibromatosis type 1 (NF1) low-grade brain tumor (optic glioma), we have previously demonstrated that microglia are essential for glioma formation and maintenance. To identify potential tumor-associated microglial factors that support glioma growth (gliomagens), we initiated a comprehensive large-scale discovery effort using optimized RNA-sequencing methods focused specifically on glioma-associated microglia. Candidate microglial gliomagens were prioritized to identify potential secreted or membrane-bound proteins, which were next validated by quantitative real-time polymerase chain reaction as well as by RNA fluorescence in situ hybridization following minocycline-mediated microglial inactivation in vivo. Using these selection criteria, chemokine (C-C motif) ligand 5 (Ccl5) was identified as a chemokine highly expressed in genetically engineered Nf1 mouse optic gliomas relative to nonneoplastic optic nerves. As a candidate gliomagen, recombinant Ccl5 increased Nf1-deficient optic nerve astrocyte growth in vitro. Importantly, consistent with its critical role in maintaining tumor growth, treatment with Ccl5 neutralizing antibodies reduced Nf1 mouse optic glioma growth and improved retinal dysfunction in vivo. Collectively, these findings establish Ccl5 as an important microglial growth factor for low-grade glioma maintenance relevant to the development of future stroma-targeted brain tumor therapies.

cDNA Hybrid Capture Improves Transcriptome Analysis on Low-Input and Archived Samples

The use of massively parallel sequencing for studying RNA expression has greatly enhanced our understanding of the transcriptome through the myriad ways these data can be characterized. In particular, clinical samples provide important insights about RNA expression in health and disease, yet these studies can be complicated by RNA degradation that results from the use of formalin as a clinical preservative and by the limited amounts of RNA often available from these precious samples. In this study we describe the combined use of RNA sequencing with an exome capture selection step to enhance the yield of on-exon sequencing read data when compared with RNA sequencing alone. In particular, the exome capture step preserves the dynamic range of expression, permitting differential comparisons and validation of expressed mutations from limited and FFPE preserved samples, while reducing the data generation requirement. We conclude that cDNA hybrid capture has the potential to significantly improve transcriptome analysis from low-yield FFPE material.

Usefulness of percutaneous biopsy in diagnosing benign renal masses that mimic malignancy

Percutaneous biopsy has long been used to diagnose malignancies of the kidney. It is an established technique with multiple indications. Percutaneous biopsy now can be used to diagnose benign conditions that may mimic a malignancy and lead to unnecessary treatments. Advances in cytological techniques such as immunocytochemistry and cytogenetics have allowed for an increased diagnostic yield. In this review, various benign entities that may present as a renal mass are discussed and the vital role of percutaneous biopsy detailed.

Interobserver Concordance in Implementing the 2010 ASCO/CAP Recommendations for Reporting ER in Breast Carcinomas A Demonstration of the Difficulties of Consistently Reporting Low Levels of ER Expression by Manual Quantification

OBJECTIVES Endocrine therapy reduces recurrence risk by 30% to 50% in estrogen receptor (ER)-positive breast cancer. The ER-positive threshold recommended by the American Society of Clinical Oncology/College of American Pathologists is 1% based on studies using the ER-6F11 antibody. ER-SP1 antibody has a higher sensitivity and is more widely used. METHODS We report interobserver concordance manually measuring ER in 264 breast cancers using ER-SP1 and 1D5 and 2 scoring methods (H-score and Allred score). RESULTS With both antibodies, 3% to 4% of cases have a low level of ER expression (1%-10%), more than previously reported (<1%). We find a high level of paired observer concordance with both antibodies and scoring methods (κ = 0.892-0.943) with no significant difference with method of scoring. Despite excellent concordance, positive/negative discordance was almost 5% among 3 observers using either antibody, an underappreciated clinically significant rate. CONCLUSIONS Discordance overwhelmingly reflected differing opinions recording the proportion of tumor cells positive with low levels of expression (<10% staining; 12/13 cases).

Prognostic Factors for Patients with Breast Cancer: Traditional and New

At the time of breast cancer diagnosis, multiple features of the tumor are routinely assessed to evaluate for prognostic and predictive factors. Prognostic factors provide information about the patients likely clinical course and include tumor stage (composed of lymph node status, tumor size, and presence of chest wall involvement), tumor histologic type and grade, estrogen and progesterone receptor expression, and HER2 status. These traditional prognostic factors are reviewed with particular attention to problematic areas in classification. Several newer prognostic tests may be able to provide information beyond the traditional prognostic factors and are presented.

Prediction of primary breast cancer size and T-stage using micro-computed tomography in lumpectomy specimens

Background: Histopathology is the only accepted method to measure and stage the breast tumor size. However, there is a need to find another method to measure and stage the tumor size when the pathological assessment is not available. Micro-computed tomography. (micro-CT) has the ability to measure tumor in three dimensions in an intact lumpectomy specimen. In this study, we aimed to determine the accuracy of micro-CT to measure and stage the primary tumor size in breast lumpectomy specimens, as compared to the histopathology. Materials and Methods: Seventy-two women who underwent lumpectomy surgery at the Massachusetts General Hospital Department of Surgery from June 2011 to September 2011, and from August 2013 to December 2013 participated in this study. The lumpectomy specimens were scanned using micro-CT followed by routine pathological processing. The maximum dimension of the invasive breast tumor was obtained from the micro-CT image and was compared to the corresponding pathology report for each subject. Results: The invasive tumor size measurement by micro-CT was underestimated in 24. cases. (33%), overestimated in 37. cases. (51%), and matched it exactly in 11. cases. (15%) compared to the histopathology measurement for all the cases. However, micro-CT T.stage classification differed from histopathology in only 11. (15.2%) with 6. cases. (8.3%) classified as a higher stage by micro-CT, and 5. cases. (6.9%) classified as lower compared to histopathology. In addition, micro-CT demonstrated a statically significant strong agreement (κ =0.6, P < 0.05) with pathological tumor size and staging for invasive ductal carcinoma. (IDC) group. In contrast, there was no agreement. (κ = .2, P = 0.67) between micro-CT and pathology in estimating and staging tumor size for invasive lobular carcinoma. (ILC) group. This could be explained by a small sample size. (7) for ILC group. Conclusions: Micro-CT is a promising modality for measuring and staging the IDC.

A Novel Breast Cancer Index for Prediction of Distant Recurrence in HR+ Early-Stage Breast Cancer with One to Three Positive Nodes

Purpose: The study objective was to characterize the prognostic performance of a novel Breast Cancer Index model (BCIN+), an integration of BCI gene expression, tumor size, and grade, specifically developed for assessment of distant recurrence (DR) risk in HR+ breast cancer patients with one to three positive lymph nodes (pN1). Experimental Design: Analysis was conducted in a well-annotated retrospective series of pN1 patients (N = 402) treated with adjuvant endocrine therapy with or without chemotherapy using a prespecified model. The primary endpoint was time-to-DR. Results were determined blinded to clinical outcome. Kaplan-Meier estimates of overall (0–15 years) and late (≥5 years) DR, HRs, and 95% confidence interval (CIs) were estimated. Likelihood ratio statistics assessed relative contributions of prognostic information. Results: BCIN+ classified 81 patients (20%) as low risk with a 15-year DR rate of 1.3% (95% CI, 0.0%–3.7%) versus 321 patients as high risk with a DR rate of 29.0% (95% CI, 23.2%–34.4%). In patients DR-free for ≥5 years (n = 349), the late DR rate was 1.3% (95% CI, 0.0%–3.7%) and 16.1% (95% CI, 10.6%–21.3%) in low- and high-risk groups, respectively. BCI gene expression alone was significantly prognostic (ΔLR-χ2 = 20.12; P < 0.0001). Addition of tumor size (ΔLR-χ2 = 13.29, P = 0.0003) and grade (ΔLR-χ2 = 12.72; P = 0.0004) significantly improved prognostic performance. BCI added significant prognostic information to tumor size (ΔLR-χ2 = 17.55; P < 0.0001); addition to tumor grade was incremental (ΔLR-χ2 = 2.38; P = 0.1) with considerable overlap between prognostic values (ΔLR-χ2 = 17.74). Conclusions: The integrated BCIN+ identified 20% of pN1 patients with limited risk of recurrence over 15 years, in whom extended endocrine treatment may be spared. Ongoing studies will characterize combined clinical-genomic risk assessment in node-positive patients. Clin Cancer Res; 23(23); 7217–24. ©2017 AACR.

A Study of the Growth Patterns of Breast Carcinoma Using 3D Reconstruction: A Pilot Study

Lumpectomy with microscopically clear margins is a safe and effective approach for surgical management of breast carcinoma. Margins are positive for tumor in 18–50% of lumpectomies, as it is not possible to accurately determine the shape or microscopic borders of a tumor preoperatively or intraoperatively. We examined the 3D microanatomy and growth patterns of common breast carcinoma subtypes to provide guidance for lumpectomy surgery. Prospective consent was obtained for the use of excess tissue from patients undergoing lumpectomy or mastectomy for breast carcinoma. Tissue blocks from nine breast carcinomas were serially sectioned. Hematoxylin and eosin‐stained slides at 100 μm intervals were scanned using a Nanozoomer (Hamamatsu, Japan) microscopic‐resolution scanner. Three‐dimensional reconstructions of tumors were created from scanned images using Reconstruct, open‐access software. Breast carcinoma subtypes demonstrated characteristic growth patterns within breast tissue, which may have implications for lumpectomy surgery. Invasive ductal carcinomas showed a spherical shape, with a spiculated surface representing tumor cells infiltrating into surrounding parenchyma. Ductal carcinoma in situ appeared to spread along the duct system, creating dilated, tortuous, tumor‐filled ducts. The invasive lobular carcinomas examined had a haphazard, linear, infiltrative growth pattern, different from the shape seen in ductal carcinomas. Our preliminary work suggests that invasive ductal and invasive lobular carcinomas appear to have distinct growth patterns in three dimensions and ductal carcinoma in situ appears to grow in a linear fashion along the duct network. The microanatomy studies described have the potential to guide refinements in breast lumpectomy technique.