Publications by authors named "Robert B West"

117 Publications

Unmasking the immune microecology of ductal carcinoma in situ with deep learning.

NPJ Breast Cancer 2021 Mar 1;7(1):19. Epub 2021 Mar 1.

Centre for Evolution and Cancer, Institute of Cancer Research, London, UK.

Despite increasing evidence supporting the clinical relevance of tumour infiltrating lymphocytes (TILs) in invasive breast cancer, TIL spatial variability within ductal carcinoma in situ (DCIS) samples and its association with progression are not well understood. To characterise tissue spatial architecture and the microenvironment of DCIS, we designed and validated a new deep learning pipeline, UNMaSk. Following automated detection of individual DCIS ducts using a new method IM-Net, we applied spatial tessellation to create virtual boundaries for each duct. To study local TIL infiltration for each duct, DRDIN was developed for mapping the distribution of TILs. In a dataset comprising grade 2-3 pure DCIS and DCIS adjacent to invasive cancer (adjacent DCIS), we found that pure DCIS cases had more TILs compared to adjacent DCIS. However, the colocalisation of TILs with DCIS ducts was significantly lower in pure DCIS compared to adjacent DCIS, which may suggest a more inflamed tissue ecology local to DCIS ducts in adjacent DCIS cases. Our study demonstrates that technological developments in deep convolutional neural networks and digital pathology can enable an automated morphological and microenvironmental analysis of DCIS, providing a new way to study differential immune ecology for individual ducts and identify new markers of progression.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41523-020-00205-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7921670PMC
March 2021

Somatostatin receptor 2 expression in nasopharyngeal cancer is induced by Epstein Barr virus infection: impact on prognosis, imaging and therapy.

Nat Commun 2021 01 5;12(1):117. Epub 2021 Jan 5.

Department of Nuclear Medicine, Medical University of Innsbruck, Innsbruck, Austria.

Nasopharyngeal cancer (NPC), endemic in Southeast Asia, lacks effective diagnostic and therapeutic strategies. Even in high-income countries the 5-year survival rate for stage IV NPC is less than 40%. Here we report high somatostatin receptor 2 (SSTR2) expression in multiple clinical cohorts comprising 402 primary, locally recurrent and metastatic NPCs. We show that SSTR2 expression is induced by the Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) via the NF-κB pathway. Using cell-based and preclinical rodent models, we demonstrate the therapeutic potential of SSTR2 targeting using a cytotoxic drug conjugate, PEN-221, which is found to be superior to FDA-approved SSTR2-binding cytostatic agents. Furthermore, we reveal significant correlation of SSTR expression with increased rates of survival and report in vivo uptake of the SSTR2-binding Ga-DOTA-peptide radioconjugate in PET-CT scanning in a clinical trial of NPC patients (NCT03670342). These findings reveal a key role in EBV-associated NPC for SSTR2 in infection, imaging, targeted therapy and survival.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-020-20308-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7785735PMC
January 2021

The Human Tumor Atlas Network: Charting Tumor Transitions across Space and Time at Single-Cell Resolution.

Cell 2020 04;181(2):236-249

Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Ludwig Center for Cancer Research and Laboratory of Systems Pharmacology, Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA.

Crucial transitions in cancer-including tumor initiation, local expansion, metastasis, and therapeutic resistance-involve complex interactions between cells within the dynamic tumor ecosystem. Transformative single-cell genomics technologies and spatial multiplex in situ methods now provide an opportunity to interrogate this complexity at unprecedented resolution. The Human Tumor Atlas Network (HTAN), part of the National Cancer Institute (NCI) Cancer Moonshot Initiative, will establish a clinical, experimental, computational, and organizational framework to generate informative and accessible three-dimensional atlases of cancer transitions for a diverse set of tumor types. This effort complements both ongoing efforts to map healthy organs and previous large-scale cancer genomics approaches focused on bulk sequencing at a single point in time. Generating single-cell, multiparametric, longitudinal atlases and integrating them with clinical outcomes should help identify novel predictive biomarkers and features as well as therapeutically relevant cell types, cell states, and cellular interactions across transitions. The resulting tumor atlases should have a profound impact on our understanding of cancer biology and have the potential to improve cancer detection, prevention, and therapeutic discovery for better precision-medicine treatments of cancer patients and those at risk for cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2020.03.053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7376497PMC
April 2020

Integrating genomic features for non-invasive early lung cancer detection.

Nature 2020 04 25;580(7802):245-251. Epub 2020 Mar 25.

Massachusetts General Hospital Cancer Center, Harvard Medical School, Boston, MA, USA.

Radiologic screening of high-risk adults reduces lung-cancer-related mortality; however, a small minority of eligible individuals undergo such screening in the United States. The availability of blood-based tests could increase screening uptake. Here we introduce improvements to cancer personalized profiling by deep sequencing (CAPP-Seq), a method for the analysis of circulating tumour DNA (ctDNA), to better facilitate screening applications. We show that, although levels are very low in early-stage lung cancers, ctDNA is present prior to treatment in most patients and its presence is strongly prognostic. We also find that the majority of somatic mutations in the cell-free DNA (cfDNA) of patients with lung cancer and of risk-matched controls reflect clonal haematopoiesis and are non-recurrent. Compared with tumour-derived mutations, clonal haematopoiesis mutations occur on longer cfDNA fragments and lack mutational signatures that are associated with tobacco smoking. Integrating these findings with other molecular features, we develop and prospectively validate a machine-learning method termed 'lung cancer likelihood in plasma' (Lung-CLiP), which can robustly discriminate early-stage lung cancer patients from risk-matched controls. This approach achieves performance similar to that of tumour-informed ctDNA detection and enables tuning of assay specificity in order to facilitate distinct clinical applications. Our findings establish the potential of cfDNA for lung cancer screening and highlight the importance of risk-matching cases and controls in cfDNA-based screening studies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41586-020-2140-0DOI Listing
April 2020

Origins and clonal convergence of gastrointestinal IgE B cells in human peanut allergy.

Sci Immunol 2020 03;5(45)

Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305, USA.

B cells in human food allergy have been studied predominantly in the blood. Little is known about IgE B cells or plasma cells in tissues exposed to dietary antigens. We characterized IgE clones in blood, stomach, duodenum, and esophagus of 19 peanut-allergic patients, using high-throughput DNA sequencing. IgE cells in allergic patients are enriched in stomach and duodenum, and have a plasma cell phenotype. Clonally related IgE and non-IgE-expressing cell frequencies in tissues suggest local isotype switching, including transitions between IgA and IgE isotypes. Highly similar antibody sequences specific for peanut allergen Ara h 2 are shared between patients, indicating that common immunoglobulin genetic rearrangements may contribute to pathogenesis. These data define the gastrointestinal tract as a reservoir of IgE B lineage cells in food allergy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/sciimmunol.aay4209DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7691169PMC
March 2020

Primary mammary angiosarcomas harbor frequent mutations in KDR and PIK3CA and show evidence of distinct pathogenesis.

Mod Pathol 2020 08 2;33(8):1518-1526. Epub 2020 Mar 2.

Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA.

Angiosarcoma (AS) is the most frequent primary sarcoma of the breast but nevertheless remains uncommon, accounting for <0.05% of breast malignancies. Secondary mammary AS arise following radiation therapy for breast cancer, in contrast to primary AS which occur sporadically. Essentially all show aggressive clinical behavior independent of histologic grade and most are treated by mastectomy. MYC amplification is frequently identified in radiation-induced AS but only rarely in primary mammary AS (PMAS). As a heterogeneous group, AS from various anatomic sites have been shown to harbor recurrent alterations in TP53, MAP kinase pathway genes, and genes involved in angiogenic signaling including KDR (VEGFR2) and PTPRB. In part due to its rarity, the pathogenesis of PMAS has not been fully characterized. In this study, we examined the clinical, pathologic, and genomic features of ten cases of PMAS, including one patient with bilateral disease. Recurrent genomic alterations were identified in KDR (70%), PIK3CA/PIK3R1 (70%), and PTPRB (30%), each at higher frequencies than reported in AS across all sites. Six tumors harbored a KDR p.T771R hotspot mutation, and all seven KDR-mutant cases showed evidence suggestive of biallelism (four with loss of heterozygosity and three with two aberrations). Of the seven tumors with PI3K alterations, six harbored pathogenic mutations other than in the canonical PIK3CA residues which are most frequent in breast cancer. Three AS were hypermutated (≥10 mutations/megabase (Mb)); hypermutation was seen concurrent with KDR or PIK3CA mutations. The patient with bilateral disease demonstrated shared alterations, indicative of contralateral metastasis. No MYC or TP53 aberrations were detected in this series. Immunohistochemistry for VEGFR2 was unable to discriminate between KDR-mutant tumors and benign vascular lesions of the breast. These findings highlight the underrecognized frequency of KDR and PIK3CA mutation in PMAS, and a significant subset with hypermutation, suggesting a pathogenesis distinct from other AS.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41379-020-0511-6DOI Listing
August 2020

HER2 Dual In Situ Hybridization: Correlations and Cautions.

Arch Pathol Lab Med 2020 12;144(12):1525-1534

From the Department of Pathology, Stanford University Medical Center, Stanford, California.

Context.—: Accurate HER2 testing in breast cancer is crucial for appropriate precision therapy. HER2 testing is most commonly accomplished by a combination of immunohistochemistry and in situ hybridization techniques, as gene amplification is closely tied to protein overexpression. During the last 5+ years, brightfield dual in situ hybridization (DISH) has replaced fluorescence methods (fluorescence in situ hybridization [FISH]) in some laboratories.

Objective.—: To analyze routine HER2 DISH performance in the field.

Design.—: We reviewed our experience with HER2 DISH performed at outside laboratories and referred for patient care.

Results.—: Of 273 identified retrospective DISH results, 55 had repeated FISH testing at our institution; 7 (13%) were discordant. Additional cases had technical flaws hampering appropriate scoring. In 23 cases (42%), HER2 DISH was performed without immunohistochemistry. Slide review of a prospective cohort of 42 consecutive DISH cases revealed 14 (33%) with technical or interpretative limitations potentially jeopardizing results. Commonly identified problems include lack of or weak signals in most tumor cells, and silver precipitate or red signals outside of nuclei, resulting in false-negative or false-positive interpretations, respectively. Further, 44% (24 of 55) of DISH reports lacked complete data, specifically average HER2 signals/cell.

Conclusions.—: While HER2 DISH can be an efficient and effective alternative to FISH, we illustrate pitfalls and reinforce that careful attention to slide quality and technical parameters are critically important. HER2 DISH cotesting with immunohistochemistry could help minimize false-negative or false-positive HER2 results.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.5858/arpa.2019-0510-OADOI Listing
December 2020

Gene Expression Profiling of Head and Neck Tumors Identifies FOXP1 and SOX10 Expression as Useful for Distinguishing Ameloblastoma From Basaloid Salivary Gland Tumors.

Am J Surg Pathol 2020 05;44(5):665-672

Department of Pathology, Stanford University School of Medicine, Stanford.

Odontogenic tumors show considerable morphologic heterogeneity and at times the diagnosis can be challenging. Ameloblastoma, the most common odontogenic tumor, can have morphologic similarity to some salivary gland tumors and therefore we sought to identify biomarkers that might aid in the diagnosis by performing transcriptome wide gene expression profiling of 80 odontogenic and salivary gland neoplasms. These data identified the FOXP1/SOX10 expression profile as characteristic of many odontogenic tumors including ameloblastoma but largely absent in salivary gland tumors. We then assessed 173 salivary gland tumors and 108 odontogenic tumors by immunohistochemistry for FOXP1 and SOX10 expression and found that 34/35 (97%) cases of ameloblastomas were diffusely positive for FOXP1 but completely negative for SOX10. None of the basaloid salivary neoplasms (basal cell adenoma, adenoid cystic carcinoma, polymorphous adenocarcinoma, and myoepitheloma) demonstrated FOXP1/SOX10 expression pattern. Taken together, the results of this study suggest that the FOXP1/SOX10 immunophenotype is common in odontogenic tumors including ameloblastoma and might be useful distinguishing these from similar appearing basaloid salivary gland tumors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1097/PAS.0000000000001421DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7156310PMC
May 2020

Gene expression profiling of single cells from archival tissue with laser-capture microdissection and Smart-3SEQ.

Genome Res 2019 11 13;29(11):1816-1825. Epub 2019 Sep 13.

Department of Pathology, Stanford University School of Medicine, Stanford, California 94305, USA.

RNA sequencing (RNA-seq) is a sensitive and accurate method for quantifying gene expression. Small samples or those whose RNA is degraded, such as formalin-fixed paraffin-embedded (FFPE) tissue, remain challenging to study with nonspecialized RNA-seq protocols. Here, we present a new method, Smart-3SEQ, that accurately quantifies transcript abundance even with small amounts of total RNA and effectively characterizes small samples extracted by laser-capture microdissection (LCM) from FFPE tissue. We also obtain distinct biological profiles from FFPE single cells, which have been impossible to study with previous RNA-seq protocols, and we use these data to identify possible new macrophage phenotypes associated with the tumor microenvironment. We propose Smart-3SEQ as a highly cost-effective method to enable large gene expression profiling experiments unconstrained by sample size and tissue availability. In particular, Smart-3SEQ's compatibility with FFPE tissue unlocks an enormous number of archived clinical samples; combined with LCM it allows unprecedented studies of small cell populations and single cells isolated by their in situ context.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/gr.234807.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6836736PMC
November 2019

Cell cycle progression in confining microenvironments is regulated by a growth-responsive TRPV4-PI3K/Akt-p27 signaling axis.

Sci Adv 2019 08 7;5(8):eaaw6171. Epub 2019 Aug 7.

Department of Mechanical Engineering, Stanford University, CA, USA.

In tissues, cells reside in confining microenvironments, which may mechanically restrict the ability of a cell to double in size as it prepares to divide. How confinement affects cell cycle progression remains unclear. We show that cells progressed through the cell cycle and proliferated when cultured in hydrogels exhibiting fast stress relaxation but were mostly arrested in the G/G phase of the cell cycle when cultured in hydrogels that exhibit slow stress relaxation. In fast-relaxing gels, activity of stretch-activated channels (SACs), including TRPV4, promotes activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, which in turn drives cytoplasmic localization of the cell cycle inhibitor p27, thereby allowing S phase entry and proliferation. Cell growth during G activated the TRPV4-PI3K/Akt-p27 signaling axis, but growth is inhibited in the confining slow-relaxing hydrogels. Thus, in confining microenvironments, cells sense when growth is sufficient for division to proceed through a growth-responsive signaling axis mediated by SACs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/sciadv.aaw6171DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6685709PMC
August 2019

Genomic landscape of ductal carcinoma in situ and association with progression.

Breast Cancer Res Treat 2019 Nov 17;178(2):307-316. Epub 2019 Aug 17.

Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA.

Purpose: The detection rate of breast ductal carcinoma in situ (DCIS) has increased significantly, raising the concern that DCIS is overdiagnosed and overtreated. Therefore, there is an unmet clinical need to better predict the risk of progression among DCIS patients. Our hypothesis is that by combining molecular signatures with clinicopathologic features, we can elucidate the biology of breast cancer progression, and risk-stratify patients with DCIS.

Methods: Targeted exon sequencing with a custom panel of 223 genes/regions was performed for 125 DCIS cases. Among them, 60 were from cases having concurrent or subsequent invasive breast cancer (IBC) (DCIS + IBC group), and 65 from cases with no IBC development over a median follow-up of 13 years (DCIS-only group). Copy number alterations in chromosome 1q32, 8q24, and 11q13 were analyzed using fluorescence in situ hybridization (FISH). Multivariable logistic regression models were fit to the outcome of DCIS progression to IBC as functions of demographic and clinical features.

Results: We observed recurrent variants of known IBC-related mutations, and the most commonly mutated genes in DCIS were PIK3CA (34.4%) and TP53 (18.4%). There was an inverse association between PIK3CA kinase domain mutations and progression (Odds Ratio [OR] 10.2, p < 0.05). Copy number variations in 1q32 and 8q24 were associated with progression (OR 9.3 and 46, respectively; both p < 0.05).

Conclusions: PIK3CA kinase domain mutations and the absence of copy number gains in DCIS are protective against progression to IBC. These results may guide efforts to distinguish low-risk from high-risk DCIS.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s10549-019-05401-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6800639PMC
November 2019

Genomic analysis of benign prostatic hyperplasia implicates cellular re-landscaping in disease pathogenesis.

JCI Insight 2019 05 16;5. Epub 2019 May 16.

Department of Pathology.

Benign prostatic hyperplasia (BPH) is the most common cause of lower urinary tract symptoms in men. Current treatments target prostate physiology rather than BPH pathophysiology and are only partially effective. Here, we applied next-generation sequencing to gain new insight into BPH. By RNAseq, we uncovered transcriptional heterogeneity among BPH cases, where a 65-gene BPH stromal signature correlated with symptom severity. Stromal signaling molecules BMP5 and CXCL13 were enriched in BPH while estrogen regulated pathways were depleted. Notably, BMP5 addition to cultured prostatic myofibroblasts altered their expression profile towards a BPH profile that included the BPH stromal signature. RNAseq also suggested an altered cellular milieu in BPH, which we verified by immunohistochemistry and single-cell RNAseq. In particular, BPH tissues exhibited enrichment of myofibroblast subsets, whilst depletion of neuroendocrine cells and an estrogen receptor (ESR1)-positive fibroblast cell type residing near epithelium. By whole-exome sequencing, we uncovered somatic single-nucleotide variants (SNVs) in BPH, of uncertain pathogenic significance but indicative of clonal cell expansions. Thus, genomic characterization of BPH has identified a clinically-relevant stromal signature and new candidate disease pathways (including a likely role for BMP5 signaling), and reveals BPH to be not merely a hyperplasia, but rather a fundamental re-landscaping of cell types.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/jci.insight.129749DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6629137PMC
May 2019

YAP-independent mechanotransduction drives breast cancer progression.

Nat Commun 2019 04 23;10(1):1848. Epub 2019 Apr 23.

Department of Mechanical Engineering, Stanford University, Stanford, CA, 94305, USA.

Increased tissue stiffness is a driver of breast cancer progression. The transcriptional regulator YAP is considered a universal mechanotransducer, based largely on 2D culture studies. However, the role of YAP during in vivo breast cancer remains unclear. Here, we find that mechanotransduction occurs independently of YAP in breast cancer patient samples and mechanically tunable 3D cultures. Mechanistically, the lack of YAP activity in 3D culture and in vivo is associated with the absence of stress fibers and an order of magnitude decrease in nuclear cross-sectional area relative to 2D culture. This work highlights the context-dependent role of YAP in mechanotransduction, and establishes that YAP does not mediate mechanotransduction in breast cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-019-09755-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6478686PMC
April 2019

Increased Galectin-1 Expression in Thymic Epithelial Tumors.

Clin Lung Cancer 2019 05 21;20(3):e356-e361. Epub 2018 Dec 21.

Department of Radiation Oncology, Stanford University School of Medicine Stanford Cancer Institute, Stanford, CA.

Introduction: Thymic epithelial tumors (TET) are rare malignancies with a paucity of data on biology and therapeutics. Galectin-1 is a member of the β-galactoside binding protein family and has been shown to mediate tumor growth via modulation of immune cell function. This study examined galectin-1 expression in TET.

Patients And Methods: A tissue microarray of 68 patients with TET and 8 benign thymus controls were stained for galectin-1 expression and scored by a pathologist blinded to patient clinical and pathologic data. Galectin-1 expression +1 or greater staining intensity was considered positive. Clinical and pathologic data were abstracted from institutional databases. Expression of galectin-1 in thymic tumor was compared to benign thymus controls and correlated with pertinent clinical and pathologic data.

Results: Galectin-1 expression was higher in TET compared to benign thymus controls (65% vs. 0%). No significant association between galectin-1 expression and the development of recurrent disease, paraneoplastic syndromes, or overall survival was noted.

Conclusion: Galectin-1 is overexpressed in the majority of TET. Detection of galectin-1 may differentiate benign from neoplastic thymic processes. Additional studies are needed to assess the role of galectin-1 in the development of TET.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cllc.2018.12.005DOI Listing
May 2019

Most canine ameloblastomas harbor HRAS mutations, providing a novel large-animal model of RAS-driven cancer.

Oncogenesis 2019 Feb 11;8(2):11. Epub 2019 Feb 11.

Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA.

Canine acanthomatous ameloblastomas (CAA), analogs of human ameloblastoma, are oral tumors of odontogenic origin for which the genetic drivers have remained undefined. By whole-exome sequencing, we have now discovered recurrent HRAS and BRAF activating mutations, respectively, in 63% and 8% of CAA. Notably, cell lines derived from CAA with HRAS mutation exhibit marked sensitivity to MAP kinase (MAPK) pathway inhibitors, which constrain cell proliferation and drive ameloblast differentiation. Our findings newly identify a large-animal spontaneous cancer model to study the progression and treatment of RAS-driven cancer. More broadly, our study highlights the translational potential of canine cancer genome sequencing to benefit both humans and their companion animals.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41389-019-0119-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6370874PMC
February 2019

The HTN3-MSANTD3 Fusion Gene Defines a Subset of Acinic Cell Carcinoma of the Salivary Gland.

Am J Surg Pathol 2019 04;43(4):489-496

Department of Pathology, Stanford University School of Medicine, Stanford, CA.

The spectrum of tumors arising in the salivary glands is wide and has recently been shown to harbor a network of tumor-specific fusion genes. Acinic cell carcinoma (AciCC) is one of the more frequently encountered types of salivary gland carcinoma, but it has remained a genetic orphan until recently when a fusion between the HTN3 and MSANTD3 genes was described in one case. Neither of these 2 genes is known to be implicated in any other malignancy. This study was undertaken to investigate whether the HTN3-MSANTD3 fusion is a recurrent genetic event in AciCC and whether it is a characteristic of one of its histological variants. Of the 273 AciCCs screened, 9 cases showed rearrangement of MSANTD3 by break-apart fluorescence in situ hybridization, 2 had 1 to 2 extra signals, and 1 had gain, giving a total of 4.4% with MSANTD3 aberrations. In 6 of 7 available cases with MSANTD3 rearrangement, the HTN3-MSANTD3 fusion transcript was demonstrated with real-time polymerase chain reaction. Histologically, all fusion-positive cases were predominantly composed of serous tumor cells growing in solid sheets, with serous tumor cells expressing DOG-1 and the intercalated duct-like cell component being CK7 positive and S-100 positive in 6/9 cases. All but one case arose in the parotid gland, and none of the patients experienced a recurrence during follow-up. In contrast, the case with MSANTD3 gain metastasized to the cervical lymph nodes and lungs. In conclusion, we find the HTN3-MSANTD3 gene fusion to be a recurrent event in AciCC with prominent serous differentiation and an indolent clinical course.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1097/PAS.0000000000001200DOI Listing
April 2019

Matrix mechanical plasticity regulates cancer cell migration through confining microenvironments.

Nat Commun 2018 10 8;9(1):4144. Epub 2018 Oct 8.

Department of Mechanical Engineering, Stanford University, Stanford, CA, 94305, USA.

Studies of cancer cell migration have found two modes: one that is protease-independent, requiring micron-sized pores or channels for cells to squeeze through, and one that is protease-dependent, relevant for confining nanoporous matrices such as basement membranes (BMs). However, many extracellular matrices exhibit viscoelasticity and mechanical plasticity, irreversibly deforming in response to force, so that pore size may be malleable. Here we report the impact of matrix plasticity on migration. We develop nanoporous and BM ligand-presenting interpenetrating network (IPN) hydrogels in which plasticity could be modulated independent of stiffness. Strikingly, cells in high plasticity IPNs carry out protease-independent migration through the IPNs. Mechanistically, cells in high plasticity IPNs extend invadopodia protrusions to mechanically and plastically open up micron-sized channels and then migrate through them. These findings uncover a new mode of protease-independent migration, in which cells can migrate through confining matrix if it exhibits sufficient mechanical plasticity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-018-06641-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6175826PMC
October 2018

-Expressing Cancer-Associated Fibroblasts Mediate Metabolic Reprogramming in Human Lung Adenocarcinoma.

Cancer Res 2018 07 14;78(13):3445-3457. Epub 2018 May 14.

Department of Radiology, Stanford University School of Medicine, Stanford, California.

Metabolic reprogramming of the tumor microenvironment is recognized as a cancer hallmark. To identify new molecular processes associated with tumor metabolism, we analyzed the transcriptome of bulk and flow-sorted human primary non-small cell lung cancer (NSCLC) together with FDG-PET scans, which provide a clinical measure of glucose uptake. Tumors with higher glucose uptake were functionally enriched for molecular processes associated with invasion in adenocarcinoma and cell growth in squamous cell carcinoma (SCC). Next, we identified genes correlated to glucose uptake that were predominately overexpressed in a single cell-type comprising the tumor microenvironment. For SCC, most of these genes were expressed by malignant cells, whereas in adenocarcinoma, they were predominately expressed by stromal cells, particularly cancer-associated fibroblasts (CAF). Among these adenocarcinoma genes correlated to glucose uptake, we focused on glutamine-fructose-6-phosphate transaminase 2 (), which codes for the glutamine-fructose-6-phosphate aminotransferase 2 (GFAT2), a rate-limiting enzyme of the hexosamine biosynthesis pathway (HBP), which is responsible for glycosylation. was predictive of glucose uptake independent of GLUT1, the primary glucose transporter, and was prognostically significant at both gene and protein level. We confirmed that normal fibroblasts transformed to CAF-like cells, following TGFβ treatment, upregulated HBP genes, including , with less change in genes driving glycolysis, pentose phosphate pathway, and TCA cycle. Our work provides new evidence of histology-specific tumor stromal properties associated with glucose uptake in NSCLC and identifies as a critical regulator of tumor metabolic reprogramming in adenocarcinoma. These findings implicate the hexosamine biosynthesis pathway as a potential new therapeutic target in lung adenocarcinoma. .
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/0008-5472.CAN-17-2928DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6030462PMC
July 2018

Higher Absolute Lymphocyte Counts Predict Lower Mortality from Early-Stage Triple-Negative Breast Cancer.

Clin Cancer Res 2018 06 26;24(12):2851-2858. Epub 2018 Mar 26.

Department of Health Research and Policy, Stanford University School of Medicine, Stanford, California.

Tumor-infiltrating lymphocytes (TIL) in pretreatment biopsies are associated with improved survival in triple-negative breast cancer (TNBC). We investigated whether higher peripheral lymphocyte counts are associated with lower breast cancer-specific mortality (BCM) and overall mortality (OM) in TNBC. Data on treatments and diagnostic tests from electronic medical records of two health care systems were linked with demographic, clinical, pathologic, and mortality data from the California Cancer Registry. Multivariable regression models adjusted for age, race/ethnicity, socioeconomic status, cancer stage, grade, neoadjuvant/adjuvant chemotherapy use, radiotherapy use, and germline mutations were used to evaluate associations between absolute lymphocyte count (ALC), BCM, and OM. For a subgroup with TIL data available, we explored the relationship between TILs and peripheral lymphocyte counts. A total of 1,463 stage I-III TNBC patients were diagnosed from 2000 to 2014; 1,113 (76%) received neoadjuvant/adjuvant chemotherapy within 1 year of diagnosis. Of 759 patients with available ALC data, 481 (63.4%) were ever lymphopenic (minimum ALC <1.0 K/μL). On multivariable analysis, higher minimum ALC, but not absolute neutrophil count, predicted lower OM [HR = 0.23; 95% confidence interval (CI), 0.16-0.35] and BCM (HR = 0.19; CI, 0.11-0.34). Five-year probability of BCM was 15% for patients who were ever lymphopenic versus 4% for those who were not. An exploratory analysis ( = 70) showed a significant association between TILs and higher peripheral lymphocyte counts during neoadjuvant chemotherapy. Higher peripheral lymphocyte counts predicted lower mortality from early-stage, potentially curable TNBC, suggesting that immune function may enhance the effectiveness of early TNBC treatment. .
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/1078-0432.CCR-17-1323DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6366842PMC
June 2018

Local estrogen axis in the human bone microenvironment regulates estrogen receptor-positive breast cancer cells.

Breast Cancer Res 2017 Nov 15;19(1):121. Epub 2017 Nov 15.

Department of Pediatrics, Stanford University School of Medicine, 150E Clark Center, 318 Campus Drive, Stanford, CA, 94305-5427, USA.

Background: Approximately 70% of all breast cancers express the estrogen receptor, and are regulated by estrogen. While the ovaries are the primary source of estrogen in premenopausal women, most breast cancer is diagnosed following menopause, when systemic levels of this hormone decline. Estrogen production from androgen precursors is catalyzed by the aromatase enzyme. Although aromatase expression and local estrogen production in breast adipose tissue have been implicated in the development of primary breast cancer, the source of estrogen involved in the regulation of estrogen receptor-positive (ER+) metastatic breast cancer progression is less clear.

Methods: Bone is the most common distant site of breast cancer metastasis, particularly for ER+ breast cancers. We employed a co-culture model using trabecular  bone tissues obtained from total hip replacement (THR) surgery specimens to study ER+ and estrogen receptor-negative (ER-) breast cancer cells within the human bone microenvironment. Luciferase-expressing ER+ (MCF-7, T-47D, ZR-75) and ER- (SK-BR-3, MDA-MB-231, MCF-10A) breast cancer cells were cultured directly on bone tissue fragments or in bone tissue-conditioned media, and monitored over time with bioluminescence imaging (BLI). Bone tissue-conditioned media were generated in the presence vs. absence of aromatase inhibitors, and testosterone. Bone tissue fragments were analyzed for aromatase expression by immunohistochemistry.

Results: ER+ breast cancer cells were preferentially sustained in co-cultures with bone tissues and bone tissue-conditioned media relative to ER- cells. Bone fragments analyzed by immunohistochemistry revealed expression of the aromatase enzyme. Bone tissue-conditioned media generated in the presence of testosterone had increased estrogen levels and heightened capacity to stimulate ER+ breast cancer cell proliferation. Pretreatment of cultured bone tissues with aromatase inhibitors, which inhibited estrogen production, reduced the capacity of conditioned media to stimulate ER+ cell proliferation.

Conclusions: These results suggest that a local estrogen signaling axis regulates ER+ breast cancer cell viability and proliferation within the bone metastatic niche, and that aromatase inhibitors modulate this axis. Although endocrine therapies are highly effective in the treatment of ER+ breast cancer, resistance to these treatments reduces their efficacy. Characterization of estrogen signaling networks within the bone microenvironment will identify new strategies for combating metastatic progression and endocrine resistance.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13058-017-0910-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5688761PMC
November 2017

Early Detection of Molecular Residual Disease in Localized Lung Cancer by Circulating Tumor DNA Profiling.

Cancer Discov 2017 12 24;7(12):1394-1403. Epub 2017 Sep 24.

Department of Radiation Oncology, Stanford University, Stanford, California.

Identifying molecular residual disease (MRD) after treatment of localized lung cancer could facilitate early intervention and personalization of adjuvant therapies. Here, we apply cancer personalized profiling by deep sequencing (CAPP-seq) circulating tumor DNA (ctDNA) analysis to 255 samples from 40 patients treated with curative intent for stage I-III lung cancer and 54 healthy adults. In 94% of evaluable patients experiencing recurrence, ctDNA was detectable in the first posttreatment blood sample, indicating reliable identification of MRD. Posttreatment ctDNA detection preceded radiographic progression in 72% of patients by a median of 5.2 months, and 53% of patients harbored ctDNA mutation profiles associated with favorable responses to tyrosine kinase inhibitors or immune checkpoint blockade. Collectively, these results indicate that ctDNA MRD in patients with lung cancer can be accurately detected using CAPP-seq and may allow personalized adjuvant treatment while disease burden is lowest. This study shows that ctDNA analysis can robustly identify posttreatment MRD in patients with localized lung cancer, identifying residual/recurrent disease earlier than standard-of-care radiologic imaging, and thus could facilitate personalized adjuvant treatment at early time points when disease burden is lowest. .
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/2159-8290.CD-17-0716DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5895851PMC
December 2017

Genome-wide reconstruction of complex structural variants using read clouds.

Nat Methods 2017 Sep 17;14(9):915-920. Epub 2017 Jul 17.

Joint Initiative for Metrology in Biology, Stanford, California, USA.

In read cloud approaches, microfluidic partitioning of long genomic DNA fragments and barcoding of shorter fragments derived from these fragments retains long-range information in short sequencing reads. This combination of short reads with long-range information represents a powerful alternative to single-molecule long-read sequencing. We develop Genome-wide Reconstruction of Complex Structural Variants (GROC-SVs) for SV detection and assembly from read cloud data and apply this method to Illumina-sequenced 10x Genomics sarcoma and breast cancer data sets. Compared with short-fragment sequencing, GROC-SVs substantially improves the specificity of breakpoint detection at comparable sensitivity. This approach also performs sequence assembly across multiple breakpoints simultaneously, enabling the reconstruction of events exhibiting remarkable complexity. We show that chromothriptic rearrangements occurred before copy number amplifications, and that rates of single-nucleotide variants and SVs are not correlated. Our results support the use of read cloud approaches to advance the characterization of large and complex structural variation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nmeth.4366DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5578891PMC
September 2017

Recurrent rearrangements of the Myb/SANT-like DNA-binding domain containing 3 gene (MSANTD3) in salivary gland acinic cell carcinoma.

PLoS One 2017 17;12(2):e0171265. Epub 2017 Feb 17.

Department of Pathology, Stanford University School of Medicine, Stanford, California, United States of America.

Pathogenic gene fusions have been identified in several histologic types of salivary gland neoplasia, but not previously in acinic cell carcinoma (AcCC). To discover novel gene fusions, we performed whole-transcriptome sequencing surveys of three AcCC archival cases. In one specimen we identified a novel HTN3-MSANTD3 gene fusion, and in another a novel PRB3-ZNF217 gene fusion. The structure of both fusions was consistent with the promoter of the 5' partner (HTN3 or PRB3), both highly expressed salivary gland genes, driving overexpression of full-length MSANTD3 or ZNF217. By fluorescence in situ hybridization of an expanded AcCC case series, we observed MSANTD3 rearrangements altogether in 3 of 20 evaluable cases (15%), but found no additional ZNF217 rearrangements. MSANTD3 encodes a previously uncharacterized Myb/SANT domain-containing protein. Immunohistochemical staining demonstrated diffuse nuclear MSANTD3 expression in 8 of 27 AcCC cases (30%), including the three cases with MSANTD3 rearrangement. MSANTD3 displayed heterogeneous expression in normal salivary ductal epithelium, as well as among other histologic types of salivary gland cancer though without evidence of translocation. In a broader survey, MSANTD3 showed variable expression across a wide range of normal and neoplastic human tissue specimens. In preliminary functional studies, engineered MSANTD3 overexpression in rodent salivary gland epithelial cells did not enhance cell proliferation, but led to significant upregulation of gene sets involved in protein synthesis. Our findings newly identify MSANTD3 rearrangement as a recurrent event in salivary gland AcCC, providing new insight into disease pathogenesis, and identifying a putative novel human oncogene.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0171265PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5315303PMC
August 2017

'Non-classical' HER2 FISH results in breast cancer: a multi-institutional study.

Mod Pathol 2017 02 14;30(2):227-235. Epub 2016 Oct 14.

Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA.

The 2013 CAP/ASCO HER2 Testing Guidelines Update modified HER2 FISH categories such that some cases with 'monosomy', 'co-amplification/polysomy', low-level increased HER2 signals or clustered heterogeneity now are considered amplified or equivocal. This study examines the frequency and clinico-pathologic characteristics of breast cancers with equivocal or 'non-classical' HER2 FISH results. Breast cancers (2001-2014) with HER2 FISH results, HER2 immunohistochemistry, ER, grade, and age from three institutions (Stanford, UCSF, UWMC) were collected. HER2 FISH was interpreted using the updated recommendations. Amplified cases with non-classical results were grouped into the following categories: (1) 'monosomy' (ratio ≥2.0, mean HER2/cell<4.0); (2) 'co-amplified' (ratio<2.0, mean HER2/cell ≥6.0); (3) 'low amplified' (ratio ≥2.0, mean HER2/cell 4.0-5.9). Heterogeneous cases with clustered HER2-positive cells were also included. Of 8068 cases, 5.2% were equivocal and 4.6% had a 'non-classical' HER2 amplified result; 1.4% 'monosomy', 0.8% 'co-amplified', 2.1% 'low amplified', and 0.3% clustered heterogeneity. These cancers had a high frequency of ER positive (80.4%), Nottingham grade 3 (52.1%) results. The highest percentage of grade 3 cancers (66.7%) and positive HER2 immunohistochemistry (31.7%) was in the 'co-amplified' group. The 'monosomy' group had the highest percent grade 1 cancers (13.3%) and was most frequently HER2 immunohistochemistry negative (30.1%). Equivocal cases had very similar characteristics to the 'low-amplified' category. Cases with non-classical HER2 amplification or equivocal results are typically ER positive, higher grade cancers. 'Co-amplified' cases have the highest frequencies of aggressive characteristics and 'monosomy' cases the highest frequencies of lower risk features. With little clinical outcomes data currently available on these non-classical HER2 results, these results support the current classification scheme for HER2 FISH, with case-by-case correlation with additional clinical-pathologic factors when evaluating whether to offer HER2-targeted therapies in these non-classical cases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/modpathol.2016.175DOI Listing
February 2017

Role of KEAP1/NRF2 and TP53 Mutations in Lung Squamous Cell Carcinoma Development and Radiation Resistance.

Cancer Discov 2017 01 23;7(1):86-101. Epub 2016 Sep 23.

Stanford Cancer Institute, Stanford University School of Medicine, Stanford, California.

Lung squamous cell carcinoma (LSCC) pathogenesis remains incompletely understood, and biomarkers predicting treatment response remain lacking. Here, we describe novel murine LSCC models driven by loss of Trp53 and Keap1, both of which are frequently mutated in human LSCCs. Homozygous inactivation of Keap1 or Trp53 promoted airway basal stem cell (ABSC) self-renewal, suggesting that mutations in these genes lead to expansion of mutant stem cell clones. Deletion of Trp53 and Keap1 in ABSCs, but not more differentiated tracheal cells, produced tumors recapitulating histologic and molecular features of human LSCCs, indicating that they represent the likely cell of origin in this model. Deletion of Keap1 promoted tumor aggressiveness, metastasis, and resistance to oxidative stress and radiotherapy (RT). KEAP1/NRF2 mutation status predicted risk of local recurrence after RT in patients with non-small lung cancer (NSCLC) and could be noninvasively identified in circulating tumor DNA. Thus, KEAP1/NRF2 mutations could serve as predictive biomarkers for personalization of therapeutic strategies for NSCLCs.

Significance: We developed an LSCC mouse model involving Trp53 and Keap1, which are frequently mutated in human LSCCs. In this model, ABSCs are the cell of origin of these tumors. KEAP1/NRF2 mutations increase radioresistance and predict local tumor recurrence in radiotherapy patients. Our findings are of potential clinical relevance and could lead to personalized treatment strategies for tumors with KEAP1/NRF2 mutations. Cancer Discov; 7(1); 86-101. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 1.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/2159-8290.CD-16-0127DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5222718PMC
January 2017

BRAF inhibitor therapy of primary ameloblastoma.

Oral Surg Oral Med Oral Pathol Oral Radiol 2016 Oct 5;122(4):518-9. Epub 2016 Jul 5.

Stanford University School of Medicine, Stanford, CA, USA.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.oooo.2016.05.017DOI Listing
October 2016

Loss of Expression of AZGP1 Is Associated With Worse Clinical Outcomes in a Multi-Institutional Radical Prostatectomy Cohort.

Prostate 2016 11 21;76(15):1409-19. Epub 2016 Jun 21.

Department of Urologic Sciences and Vancouver Prostate Centre, Vancouver, British Columbia, Canada.

Background: Given the uncertainties inherent in clinical measures of prostate cancer aggressiveness, clinically validated tissue biomarkers are needed. We tested whether Alpha-2-Glycoprotein 1, Zinc-Binding (AZGP1) protein levels, measured by immunohistochemistry, and RNA expression, by RNA in situ hybridization (RISH), predict recurrence after radical prostatectomy independent of clinical and pathological parameters.

Methods: AZGP1 IHC and RISH were performed on a large multi-institutional tissue microarray resource including 1,275 men with 5 year median follow-up. The relationship between IHC and RISH expression levels was assessed using the Kappa analysis. Associations with clinical and pathological parameters were tested by the Chi-square test and the Wilcoxon rank sum test. Relationships with outcome were assessed with univariable and multivariable Cox proportional hazards models and the Log-rank test.

Results: Absent or weak expression of AZGP1 protein was associated with worse recurrence free survival (RFS), disease specific survival, and overall survival after radical prostatectomy in univariable analysis. AZGP1 protein expression, along with pre-operative serum PSA levels, surgical margin status, seminal vesicle invasion, extracapsular extension, and Gleason score predicted RFS on multivariable analysis. Similarly, absent or low AZGP1 RNA expression by RISH predicted worse RFS after prostatectomy in univariable and multivariable analysis.

Conclusions: In our large, rigorously designed validation cohort, loss of AZGP1 expression predicts RFS after radical prostatectomy independent of clinical and pathological variables. Prostate 76:1409-1419, 2016. © 2016 Wiley Periodicals, Inc.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/pros.23225DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5557496PMC
November 2016

BRAF inhibitor treatment of primary BRAF-mutant ameloblastoma with pathologic assessment of response.

Oral Surg Oral Med Oral Pathol Oral Radiol 2016 Jul 23;122(1):e5-7. Epub 2016 Feb 23.

Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA. Electronic address:

Objective: Molecular characterization of ameloblastoma has indicated a high frequency of driver mutations in BRAF and SMO. Preclinical data suggest that Food and Drug Administration-approved BRAF-targeted therapies may be immediately relevant for patients with ameloblastoma positive for the BRAF V600E mutation.

Methods: A neoadjuvant treatment regime of dabrafenib was given to a patient with recurrent BRAF-mutant mandibular ameloblastoma. The patient subsequently underwent left mandible composite resection of the tumor and pathologic evaluation of treatment response.

Results: The ameloblastoma had a slow but dramatic response with >90% tumor volume reduction. The inner areas of the tumor underwent degeneration and squamous differentiation, and intact ameloblastoma was present in the outer areas associated with bone.

Conclusions: Targeted neoadjuvant therapy for ameloblastoma may be useful in certain clinical settings of primary ameloblastoma. These might include tumors of advanced local stage when a neoadjuvant reduction could alter the extent of surgery and instances of local recurrence when surgical options are limited.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.oooo.2015.12.016DOI Listing
July 2016

Integrated digital error suppression for improved detection of circulating tumor DNA.

Nat Biotechnol 2016 05 28;34(5):547-555. Epub 2016 Mar 28.

Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, California, USA.

High-throughput sequencing of circulating tumor DNA (ctDNA) promises to facilitate personalized cancer therapy. However, low quantities of cell-free DNA (cfDNA) in the blood and sequencing artifacts currently limit analytical sensitivity. To overcome these limitations, we introduce an approach for integrated digital error suppression (iDES). Our method combines in silico elimination of highly stereotypical background artifacts with a molecular barcoding strategy for the efficient recovery of cfDNA molecules. Individually, these two methods each improve the sensitivity of cancer personalized profiling by deep sequencing (CAPP-Seq) by about threefold, and synergize when combined to yield ∼15-fold improvements. As a result, iDES-enhanced CAPP-Seq facilitates noninvasive variant detection across hundreds of kilobases. Applied to non-small cell lung cancer (NSCLC) patients, our method enabled biopsy-free profiling of EGFR kinase domain mutations with 92% sensitivity and >99.99% specificity at the variant level, and with 90% sensitivity and 96% specificity at the patient level. In addition, our approach allowed monitoring of NSCLC ctDNA down to 4 in 10(5) cfDNA molecules. We anticipate that iDES will aid the noninvasive genotyping and detection of ctDNA in research and clinical settings.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nbt.3520DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4907374PMC
May 2016