Pathologist Opinions about EPIC Beaker AP: a Multi-Institutional Survey of Early Adopters.

EPIC Systems Corporation provides a widely used electronic medical record. Beaker Anatomic Pathology is a newly developed laboratory information system (LIS) that has been implemented at a small number of academic pathology departments. Pathologist opinions of EPIC Beaker AP have not been well described in the literature. A 37-question survey was administered to pathologists and pathology trainees to assess overall satisfaction and efficiency of report generation using Beaker AP. Data about experience in pathology, signout responsibilities, Beaker AP usage, and the legacy LIS was also collected. Seventy-four pathologists (51 faculty, 23 residents) responded to the survey (overall response rate 29.7%). Overall pathologist satisfaction with Beaker AP showed high inter-institutional variability; institutions with legacy LISs with a graphical interface had a generally neutral to negative assessment of Beaker AP. The majority of respondents disagreed with the statement "Beaker AP is easy to use and designed for my needs". Pathologists felt that Beaker AP was useful for reviewing clinical information and billing; areas of weakness included searching for prior cases and grossing efficiency. Overall, pathologists had a neutral opinion of whether generating and signing out a complete report was faster in Beaker AP, with marked inter-institutional variation. This variability was likely due to a combination of the efficacy of the legacy LIS, familiarity with Beaker AP at the time of the survey, and institution-specific optimization efforts.

Synaptophysin-Ki67 double stain: a novel technique that improves interobserver agreement in the grading of well-differentiated gastrointestinal neuroendocrine tumors.

A common problem in the assessment of Ki67 proliferative index in well-differentiated gastrointestinal neuroendocrine tumors is distinguishing tumor from non-tumor. This is because background stromal lymphocytes, entrapped non-neoplastic glands, and the delicate vascular network characteristic of neuroendocrine tumors frequently contain a subset of proliferating cells. Furthermore, in small biopsies, crush and cautery artifact can alter the morphologic appearance of tumor cells, making the Ki67 proliferative index more difficult to assess. To address these issues, we developed a synaptophysin-Ki67 double stain using a commercially available immunohistochemistry kit, allowing simultaneous visualization of tumor and proliferating nuclei. To test this method, three gastrointestinal pathologists individually graded 50 gastrointestinal neuroendocrine tumors first using synaptophysin-Ki67 double-stained slides and then, after a washout period, using Ki67-only stained slides (along with routine hematoxylin- and eosin-stained slides). Interobserver agreement on Ki67 proliferative index was moderate using the Ki67-only stained slides (intraclass correlation 0.51, 95% confidence interval: 0.35-0.66) and improved using the synaptophysin-Ki67 double stain (intraclass correlation 0.79, 95% confidence interval: 0.69-0.86). Similarly, interobserver agreement on tumor grade was fair with Ki67-only stained slides (κ=0.39, P<0.001) and improved with the double stain (κ=0.58, P<0.001). Analysis of individual pathologists' scores revealed that fewer total number of tumor cells counted correlated with higher grade designation and appeared to contribute to grade discordance. In tumors cited as particularly challenging to assess by the pathologists, three of four tumors were grade discordant with the Ki67-only stain, whereas all four tumors were grade concordant with the synaptophysin-Ki67 stain. The synaptophysin-Ki67 double stain is the first technique to address specifically the histomorphologic challenges of evaluating Ki67 proliferative index in well-differentiated gastrointestinal neuroendocrine tumors. Although further validation is needed, this study provides evidence that the synaptophysin-Ki67 double stain can improve interobserver agreement.

Hirschsprung Disease for the Practicing Surgical Pathologist.

OBJECTIVES: Hirschsprung disease (HD) is a congenital condition defined by the absence of ganglion cells in the distal-most portion of the gastrointestinal tract. Biopsies and resections for HD can be adrenaline inducing for the general surgical pathologist because specimens are infrequent; HD is 1 of only a few neuroanatomic diseases that general surgical pathologists diagnose; numerous preanalytic factors (eg, biopsy adequacy, surgeon sampling protocol, processing artifacts) can affect histologic interpretation; and most importantly, the diagnosis has high stakes. METHODS: We provide a comprehensive overview of the background, relevant clinical procedures, and pathologic assessment of HD. Grossing and frozen section protocols, an algorithmic approach to diagnosis, and histologic pearls and pitfalls are also discussed. RESULTS: Evaluation and recognition of the features of HD have evolved significantly in the past 2 decades with the discovery of the value of calretinin immunohistochemistry in the late 2000s and the recent development of straightforward and reproducible histologic criteria for identification of the HD transition zone. CONCLUSIONS: These advancements have substantially improved the pathologist's ability to reliably evaluate for HD. Nonetheless, as with any high-stakes surgical pathology specimen, clear communication with the clinical team is essential.

Intra-ampullary papillary-tubular neoplasm.

Video 1Cholangioscopic examination of the ampullary channel and extrahepatic bile duct.

Anatomy-Specific Classification Model Using Label-Free FLIm to Aid Intraoperative Surgical Guidance of Head and Neck Cancer.

Intraoperative identification of head and neck cancer tissue is essential to achieve complete tumor resection and mitigate tumor recurrence. Mesoscopic fluorescence lifetime imaging (FLIm) of intrinsic tissue fluorophores emission has demonstrated the potential to demarcate the extent of the tumor in patients undergoing surgical procedures of the oral cavity and the oropharynx. Here, we report FLIm-based classification methods using standard machine learning models that account for the diverse anatomical and biochemical composition across the head and neck anatomy to improve tumor region identification. Three anatomy-specific binary classification models were developed (i.e., "base of tongue," "palatine tonsil," and "oral tongue"). FLIm data from patients (N = 85) undergoing upper aerodigestive oncologic surgery were used to train and validate the classification models using a leave-one-patient-out cross-validation method. These models were evaluated for two classification tasks: (1) to discriminate between healthy and cancer tissue, and (2) to apply the binary classification model trained on healthy and cancer to discriminate dysplasia through transfer learning. This approach achieved superior classification performance compared to models that are anatomy-agnostic; specifically, a ROC-AUC of 0.94 was for the first task and 0.92 for the second. Furthermore, the model demonstrated detection of dysplasia, highlighting the generalization of the FLIm-based classifier. Current findings demonstrate that a classifier that accounts for tumor location can improve the ability to accurately identify surgical margins and underscore FLIm's potential as a tool for surgical guidance in head and neck cancer patients, including those subjects of robotic surgery.

Analyzing Anatomic Pathology On-Call Summaries to Improve Resident Education.

CONTEXT.­: Pathology on-call experiences help prepare trainees for successful transition from residency to independent practice, and as such are an integral component of training. However, few data exist on anatomic pathology resident on-call workload and experience. OBJECTIVE.­: To obtain an overall picture of the anatomic pathology on-call experience to inform and improve resident education. DESIGN.­: Retrospective and prospective review of daily anatomic pathology on-call summaries from July 2016 to June 2020. RESULTS.­: During the first 2 years of the study (ie, retrospective portion), only 19% of on-call summaries (138 of 730) were available for review. After interventions, the on-call summary submission rate jumped to 98% (716 of 731). After-hours calls were most frequent on weekdays from 5 to 8 pm. The most frequent requests were for frozen sections (55%; 619 of 1125 calls), inquiries regarding disposition of fresh placentas (13%; 148 of 1125 calls), and inquiries regarding disposition of various other specimens (6%; 68 of 1125 calls). After-hours frozen section requests were most frequent for gynecologic and head and neck specimens. Notably, a significant number of after-hours calls were recurring preanalytic issues amenable to system-level improvements. We were able to eliminate the most common of these recurring preanalytic calls with stepwise interventions. CONCLUSIONS.­: To our knowledge, this is the first study analyzing the anatomic pathology resident on-call experience. In addition to obtaining a broad overview of the residents' clinical exposure on this service, we identified and resolved issues critical to optimal patient care (eg, inconsistent "patient hand-off") and improved the resident on-call experience (eg, fewer preanalytic calls increased resident time for other clinical, educational, or wellness activities).

Intraoperative delineation of p16+ oropharyngeal carcinoma of unknown primary origin with fluorescence lifetime imaging: Preliminary report.

BACKGROUND: This study evaluated whether fluorescence lifetime imaging (FLIm), coupled with standard diagnostic workups, could enhance primary lesion detection in patients with p16+ head and neck squamous cell carcinoma of the unknown primary (HNSCCUP). METHODS: FLIm was integrated into transoral robotic surgery to acquire optical data on six HNSCCUP patients' oropharyngeal tissues. An additional 55-patient FLIm dataset, comprising conventional primary tumors, trained a machine learning classifier; the output predicted the presence and location of HNSCCUP for the six patients. Validation was performed using histopathology. RESULTS: Among the six HNSCCUP patients, p16+ occult primary was surgically identified in three patients, whereas three patients ultimately had no identifiable primary site in the oropharynx. FLIm correctly detected HNSCCUP in all three patients (ROC-AUC: 0.90 ± 0.06), and correctly predicted benign oropharyngeal tissue for the remaining three patients. The mean sensitivity was 95% ± 3.5%, and specificity 89% ± 12.7%. CONCLUSIONS: FLIm may be a useful diagnostic adjunct for detecting HNSCCUP.

Optimizing the Diagnosis and Biomarker Testing for Patients with Intrahepatic Cholangiocarcinoma: A Multidisciplinary Approach.

Cholangiocarcinoma (CCA) is a heterogenous group of malignancies originating in the biliary tree, and associated with poor prognosis. Until recently, treatment options have been limited to surgical resection, liver-directed therapies, and chemotherapy. Identification of actionable genomic alterations with biomarker testing has revolutionized the treatment paradigm for these patients. However, several challenges exist to the seamless adoption of precision medicine in patients with CCA, relating to a lack of awareness of the importance of biomarker testing, hurdles in tissue acquisition, and ineffective collaboration among the multidisciplinary team (MDT). To identify gaps in standard practices and define best practices, multidisciplinary hepatobiliary teams from the University of California (UC) Davis and UC Irvine were convened; discussions of the meeting, including optimal approaches to tissue acquisition for diagnosis and biomarker testing, communication among academic and community healthcare teams, and physician education regarding biomarker testing, are summarized in this review.

Pleiotrophin produced by multiple myeloma induces transdifferentiation of monocytes into vascular endothelial cells: a novel mechanism of tumor-induced vasculogenesis.

Enhanced angiogenesis is a hallmark of cancer. Pleiotrophin (PTN) is an angiogenic factor that is produced by many different human cancers and stimulates tumor blood vessel formation when it is expressed in malignant cancer cells. Recent studies show that monocytes may give rise to vascular endothelium. In these studies, we show that PTN combined with macrophage colony-stimulating factor (M-CSF) induces expression of vascular endothelial cell (VEC) genes and proteins in human monocyte cell lines and monocytes from human peripheral blood (PB). Monocytes induce VEC gene expression and develop tube-like structures when they are exposed to serum or cultured with bone marrow (BM) from patients with multiple myeloma (MM) that express PTN, effects specifically blocked with antiPTN antibodies. When coinjected with human MM cells into severe combined immunodeficient (SCID) mice, green fluorescent protein (GFP)-marked human monocytes were found incorporated into tumor blood vessels and expressed human VEC protein markers and genes that were blocked by anti-PTN antibody. Our results suggest that vasculogenesis in human MM may develop from tumoral production of PTN, which orchestrates the transdifferentiation of monocytes into VECs.

A Rare Cause of Gastric Variceal Bleeding.

A 68-year-old woman with stage III colon cancer status after right hemicolectomy and adjuvant FOLFOX (5-fluorouracil/leucovorin/oxaliplatin) chemotherapy was hospitalized for melena and found to have new-onset esophageal and gastric varices on esophagogastroduodenoscopy. Her workup did not reveal an underlying liver disease, but her liver biopsy showed noncirrhotic portal hypertension from obliterative portal venopathy (OPV). The development of OPV is likely from her use of oxaliplatin-based chemotherapy.

Improving the accuracy of gastrointestinal neuroendocrine tumor grading with deep learning.

The Ki-67 index is an established prognostic factor in gastrointestinal neuroendocrine tumors (GI-NETs) and defines tumor grade. It is currently estimated by microscopically examining tumor tissue single-immunostained (SS) for Ki-67 and counting the number of Ki-67-positive and Ki-67-negative tumor cells within a subjectively picked hot-spot. Intraobserver variability in this procedure as well as difficulty in distinguishing tumor from non-tumor cells can lead to inaccurate Ki-67 indices and possibly incorrect tumor grades. We introduce two computational tools that utilize Ki-67 and synaptophysin double-immunostained (DS) slides to improve the accuracy of Ki-67 index quantitation in GI-NETs: (1) Synaptophysin-KI-Estimator (SKIE), a pipeline automating Ki-67 index quantitation via whole-slide image (WSI) analysis and (2) deep-SKIE, a deep learner-based approach where a Ki-67 index heatmap is generated throughout the tumor. Ki-67 indices for 50 GI-NETs were quantitated using SKIE and compared with DS slide assessments by three pathologists using a microscope and a fourth pathologist via manually ticking off each cell, the latter of which was deemed the gold standard (GS). Compared to the GS, SKIE achieved a grading accuracy of 90% and substantial agreement (linear-weighted Cohen's kappa 0.62). Using DS WSIs, deep-SKIE displayed a training, validation, and testing accuracy of 98.4%, 90.9%, and 91.0%, respectively, significantly higher than using SS WSIs. Since DS slides are not standard clinical practice, we also integrated a cycle generative adversarial network into our pipeline to transform SS into DS WSIs. The proposed methods can improve accuracy and potentially save a significant amount of time if implemented into clinical practice.

Digital Whole Slide Imaging Compared With Light Microscopy for Primary Diagnosis in Surgical Pathology.

CONTEXT.­: The adoption of digital capture of pathology slides as whole slide images (WSI) for educational and research applications has proven utility. OBJECTIVE.­: To compare pathologists' primary diagnoses derived from WSI versus the standard microscope. Because WSIs differ in format and method of observation compared with the current standard glass slide microscopy, this study is critical to potential clinical adoption of digital pathology. DESIGN.­: The study enrolled a total of 2045 cases enriched for more difficult diagnostic categories and represented as 5849 slides were curated and provided for diagnosis by a team of 19 reading pathologists separately as WSI or as glass slides viewed by light microscope. Cases were reviewed by each pathologist in both modalities in randomized order with a minimum 31-day washout between modality reads for each case. Each diagnosis was compared with the original clinical reference diagnosis by an independent central adjudication review. RESULTS.­: The overall major discrepancy rates were 3.64% for WSI review and 3.20% for manual slide review diagnosis methods, a difference of 0.44% (95% CI, -0.15 to 1.03). The time to review a case averaged 5.20 minutes for WSI and 4.95 minutes for glass slides. There was no specific subset of diagnostic category that showed higher rates of modality-specific discrepancy, though some categories showed greater discrepancy than others in both modalities. CONCLUSIONS.­: WSIs are noninferior to traditional glass slides for primary diagnosis in anatomic pathology.

Epigenetic regulation and molecular characterization of C/EBPalpha in pancreatic cancer cells.

Molecular-targeted therapy is a hopeful approach for pancreatic cancer. Silencing of tumor suppressor genes can occur by histone deacetylation and/or DNA methylation in the promoter. Here, we identified epigenetically silenced genes in pancreatic cancer cells. Pancreatic cancer cell line, PANC-1 cells were treated either with or without 5Aza-dC (a DNA methyltransferase inhibitor) and suberoylanilide hydroxamic acid (SAHA, a histone deacetylase inhibitor), and mRNA was isolated from these cells. Oligonucleotide microarray analysis revealed that 30 genes including UCHL1, C/EBPalpha, TIMP2 and IRF7 were up-regulated after treatment with 5Aza-dC and SAHA in PANC-1. The induction of these 4 genes was validated by real-time PCR in several pancreatic cancer cell lines. Interestingly, expression of C/EBPalpha was significantly restored in 6 of 6 pancreatic cancer cell lines. Chromatin immunoprecipitation assay revealed that histone H3 of the promoter region of C/EBPalpha was acetylated in PANC-1 treated with SAHA; and bisulfate sequencing showed methylation of its promoter region in several pancreatic cancer cell lines. Forced expression of C/EBPalpha markedly suppressed clonal proliferation of PANC-1 cells. Co-immunoprecipitation assay showed the interaction of C/EBPalpha and E2F1; and the interaction caused the inhibition of E2F1 transcriptional activity. Immunohistochemical analysis revealed that C/EBPalpha localized in the cytoplasm in pancreatic adenocarcinoma cells, whereas it localized predominantly in the nucleus in normal pancreatic cells. Our data demonstrated that aberrant silencing, as well as, inappropriate cytoplasmic localization of C/EBPalpha causes dysregulation of its function, suggesting that C/EBPalpha is a novel candidate tumor suppressor gene in pancreatic cancer cells.

An Introduction to Hardware, Software, and Other Information Technology Needs of Biomedical Biobanks.

Biobanks support medical research by facilitating access to biospecimens. Biospecimens that are linked to clinical and molecular information are particularly useful for translational biomedical research. Tracking and managing the biospecimens and their associated data are therefore crucial tasks in the functioning of a biobank. Adequate computing hardware, efficient and comprehensive biobanking software, and cost-effective data storage are needed for proper management of biospecimens. As biobanks build up extensive stores of specimens and patient data, ethical considerations also inevitably arise. Herein, we describe some basic considerations for establishing a biobanking information technology infrastructure that a beginning biobanker needs. Finally, we also discuss trends and future needs in biobanking informatics.

Comparison of Endoscopic Ultrasound Biopsy Needles for Endoscopic Ultrasound-Guided Liver Biopsy.

BACKGROUND/AIMS: To compare the performance of latest commercially available endoscopic ultrasound biopsy needles. METHODS: Six latest commercially available needles were tested on a freshly harvested bovine liver; the tested needles included three 19 G, one 20 G, and two 22 G needles. Five biopsies were performed per needle with 10 mL of wet suction. The primary outcome was the number of complete portal tracts (CPTs) per needle aspirate. The secondary outcomes were the mean specimen length and mean fragment length. Analysis of variance and Tukey's test were applied. RESULTS: All 19 G needles and the 20 G needle yielded similar mean CPTs and were superior to the SharkCore 22 G needle (p<0.001 adjusted for multiplicity). There was no statistically significant difference in total specimen length among the three 19 G needles and the 20 G needle tested. The two 22 G needles performed similarly with respect to the number of CPTs, mean fragment length, and mean specimen length (adjusted p=0.07, p=0.59, and p=0.10, respectively). CONCLUSION: The specimen adequacy was similar among the 3 latest commercially available 19 G needles. The endoscopist may choose a larger-bore needle based on availability without concerns of specimen adequacy. Further studies are needed to assess the ease of needle use in various anatomical locations and to confirm the optimal needle design.

Pulmonary embolism as the presenting symptom and a confounder in ACTH-secreting bronchial carcinoid

Summary: Ectopic ACTH-secreting pulmonary neuroendocrine tumors are rare and account for less than 5% of endogenous Cushing's syndrome cases. We describe an unusual case of metastatic bronchial carcinoid tumor in a young woman presenting with unprovoked pulmonary emboli, which initially prevented the detection of the primary tumor on imaging. The source of ectopic ACTH was ultimately localized by a Gallium-DOTATATE scan, which demonstrated increased tracer uptake in a right middle lobe lung nodule and multiple liver nodules. The histological diagnosis was established based on a core biopsy of a hepatic lesion and the patient was started on a glucocorticoid receptor antagonist and a somatostatin analog. This case illustrates that hypercogulability can further aggravate the diagnostic challenges in ectopic ACTH syndrome. We discuss the literature on the current diagnosis and management strategies for ectopic ACTH syndrome. Learning Points: In a young patient with concurrent hypokalemia and uncontrolled hypertension on multiple antihypertensive agents, secondary causes of hypertension should be evaluated. Patients with Cushing's syndrome can develop an acquired hypercoagulable state leading to spontaneous and postoperative venous thromboembolism. Pulmonary emboli may complicate the imaging of the bronchial carcinoid tumor in ectopic ACTH syndrome. Imaging with Gallium-68 DOTATATE PET/CT scan has the highest sensitivity and specificity in detecting ectopic ACTH-secreting tumors. A combination of various noninvasive biochemical tests can enhance the diagnostic accuracy in differentiating Cushing's disease from ectopic ACTH syndrome provided they have concordant results. Bilateral inferior petrosal sinus sampling remains the gold standard.

Noncirrhotic Portal Hypertension Secondary to Nodular Regenerative Hyperplasia Postrenal Transplant.

Noncirrhotic portal hypertension (NCPH) is a well-known clinical entity, but often underdiagnosed. One of the common causes of NCPH is nodular regenerative hyperplasia (NRH) that presents as nodularity with features of portal hypertension and thus often diagnosed as cirrhosis. Although NRH has no histologic fibrosis, the liver synthetic function remains intact; thus, clinical diagnosis is essential because management may differ from cirrhosis. We were asked to consult in this series of 4 patients who had new-onset ascites after kidney transplantation and were diagnosed with NCPH from NRH.

The California Telepathology Service: UCLA's Experience in Deploying a Regional Digital Pathology Subspecialty Consultation Network.

BACKGROUND: The need for extending pathology diagnostic expertise to more areas is now being met by the maturation of technology that can effectively deliver this level of care. The experience and lessons learned from our successfully deployed International Telepathology Service (ITS) to a hospital system in China guided us in starting a domestic telepathology network, the California Telepathology Service (CTS). Many of the lessons learned from the ITS project informed our decision-making for the CTS. New challenges were recognized and overcome, such as addressing the complexity and cost-benefit tradeoffs involved in setting up a digital consultation system that competes with an established conventional glass slide delivery system. METHODS: The CTS is based on a hub-and-spoke telepathology network using Leica Biosystems whole-slide image scanners and the eSlide Manager (eSM Version 12.3.3.7055, Leica Biosystems) digital image management software solution. The service currently comprises six spoke sites (UC San Diego [UCSD], UC Irvine [UCI], UC Davis, Northridge Hospital Medical Center [NHMC], Olive View Medical Center [OVMC], and Children's Hospital Los Angeles) and one central hub site (UCLA Medical Center). So far, five sites have been validated for telepathology case consultations following established practice guidelines, and four sites (UCI, UCSD, NHMC, and OVMC) have activated the service. RESULTS: For the active spoke sites, we reviewed the volume, turnaround time (TAT), and case types and evaluated for utility and value. From May 2017 to July 2018, a total of 165 cases were submitted. Of note, digital consultations were particularly advantageous for preliminary kidney biopsy diagnoses (avg TAT 0.7 day). CONCLUSION: For spoke sites, telepathology provided shortened TAT and significant financial savings over hiring faculty with expertise to support a potentially low-volume service. For the hub site, the value includes exposure to educationally valuable cases, additional caseload volume to support specialized services, and improved communication with referring facilities over traditional carrier mail. The creation of a hub-and-spoke telepathology network is an expensive undertaking, and careful consideration needs to be given to support the needs of the clinical services, acquisition and effective deployment of the appropriate equipment, network requirements, and laboratory workflows to ensure a successful and cost-effective system.

In vivo efficacy of laparoscopic assisted percutaneous renal cryotherapy: evidence based guidelines for the practicing urologist.

PURPOSE: The treatment of small renal tumors continues to evolve in parallel with advances in ablative technology. We compared the lesion geometry of 3, 17 gauge cryoneedles to determine the most effective distance and configuration of the cryoneedles in an in vivo porcine kidney model. MATERIALS AND METHODS: Argon gas based renal cryoablation was performed in 6 pigs using a laparoscopically assisted percutaneous approach. Cryoablation using a single cryoneedle and a template of 3 cryoneedles with various ice ball shapes, including elliptical, bulb-shaped and standard 17 gauge cryoneedles (Galil Medical, Plymouth Meadow, Pennsylvania) was performed in 3 pigs. Three additional pigs underwent renal cryoablation using elliptical cryoneedles in 3 triangular template configurations with the cryoneedles spaced 1, 1.5 and 2 cm apart, respectively. The animals were sacrificed a minimum of 2 weeks following treatment. RESULTS: Elliptical cryoneedles achieved the largest area of necrosis when used in single and template configurations. When used in a template configuration of 3 needles 1, 1.5 and 2 cm apart from each other the calculated volume of necrosis was 4.3 x 4.5 x 2.5, 4.9 x 4.1 x 2.5 and 4.0 x 4.5 x 2.5 cm, respectively. CONCLUSIONS: Using a single 17 gauge cryoneedle is inadequate for treating most small renal tumors. Cryoneedles with an elliptical ice ball are most effective for achieving consistent and reliable tissue destruction. The 1.5 cm template configuration generated the largest area of necrosis. Our data suggest that with the current technology renal cryoablation should be limited to lesions not greater than 4 cm.

Functional domains of CCN1 (Cyr61) regulate breast cancer progression.

CCN1 plays diverse roles in cellular proliferation, survival, migration and angiogenesis. We determined the relationship between CCN1 protein expression and clinical factors that are important for the classification of breast cancer. CCN1 contains four functional domains; the contribution of each of the structural domains to the biological properties of CCN1 in breast cancer was investigated. We performed immunohistochemistry for CCN1 on a breast cancer tissue array, and conducted a detailed statistical analysis on the relationship between CCN1 protein expression and clinical factors that are important for the classification of breast cancer. The structure-function relationship was examined using four mutant constructs in which one of the modules (DM1-DM4) had been deleted. MCF-7 breast cancer cells were stably transfected with these constructs and their biological activity was tested in comparison to full-length CCN1. Staining of CCN1 in tumors was positively correlated with AJCC disease stage. A strong association also was found between lymph node involvement and high CCN1 expression in patients with invasive breast cancer; there was a significant increase in the breast cancer expression of CCN1 in patients with positive lymph nodes (P=0.004), and the levels of CCN1 correlated with the number of positive lymph nodes (P=0.0006). Deletion of module 4 rendered CCN1 unable to either bind heparin or associate with the extracellular matrix. Furthermore, MCF-7/DM4 cells demonstrated reduced cell spreading, migration and proliferation, indicating that module 4 of the protein is important for its ability to promote these activities. These findings indicate that CCN1 is involved throughout the clinical progression of breast cancer to an invasive phenotype. The multimodular structure of CCN1 enables it to fulfill multiple functions that may contribute to the different stages of cancer development, raising the prospect that specific regions of CCN1 could be targeted for therapeutic benefit to inhibit particular aspects of malignancy in breast cancer.