Diagnostic Tissue Research Articles

Diagnostic efficacy of three suction techniques for endoscopic ultrasound-guided fine-needle biopsy of solid pancreatic lesions: protocol for a multicenter randomized cross-over clinical trial

BackgroundHow suction technique affects endoscopic ultrasound-guided tissue acquisition (EUS-TA) remains unclear. A standardized protocol is currently lacking, with most previous studies being restricted to EUS-guided fine-needle aspiration (EUS-FNA). The research related to EUS-guided fine-needle biopsy (EUS-FNB) is sparse. AimsThe aim of this study is to evaluate the diagnostic efficacy, cellularity, tissue acquisition, blood contamination and adverse event rate of three common suction techniques (standard suction, slow-pull, and wet suction) used for EUS-FNB of solid pancreatic masses. MethodsThis is a multicenter single-blind randomized cross-over superiority trial. A total of 300 patients with suspected pancreatic malignancy will be enrolled from digestive endoscopic centers at five large tertiary hospitals in China. All three suction techniques will be performed on each patient using a 25G ProCore needle, with the sequence of suction techniques determined by randomization. Cytological and histological specimens obtained with each of the three techniques will be assessed independently. Outcomes among the three suction techniques will be compared. DiscussionTo the best of our knowledge, this is the largest multicenter randomized cross-over trial designed to determine the optimal suction technique for the diagnosis of solid pancreatic masses. This study may contribute to standardizing the suction technique for EUS-FNB.

Can pathology diagnostic services for cancer be stratified and serve global health?

Before initiating cancer therapy, a diagnostic tumor tissue sample evaluated within a pathology laboratory by a pathologist is essential to confirm the malignancy type and provide key prognostic factors that direct the treatment offered. Pathology evaluation includes multiple expensive reagents, complex equipment, and both laboratory and pathologist technical skills. By using breast cancer as an example, at a minimum, key tumor prognostic information required before the initiation of treatment includes subtype, tumor grade, tumor size, lymph node status when possible, and biomarker expression determined by immunohistochemistry for estrogen receptor. The additional determination of biomarker expression of progesterone receptor and human epidermal growth factor receptor (HER2) is the standard of care in high-resource settings, but assays may not be affordable in low-income and middle-income countries. With positive tests, patients are eligible for either tamoxifen (for estrogen receptor-positive/progesterone receptor-positive cancers) or monoclonal antibody therapy (for HER2-positive cancers). For settings in which endocrine therapy and/or HER2-targeted therapy is unavailable, biomarker studies have no utility, and high-resource setting standards for pathology evaluation and reporting are unachievable. Resource-stratified pathology evaluation guidelines in cancer diagnosis have not been developed, in contrast to excellent comprehensive, resource-stratified clinical guidelines for use in low-income and middle-income countries, and these are long overdue. The challenges of pathology evaluation in the context of global health are being met by innovative solutions, which may change the face of pathology practice.

Uterine cervical squamous cell carcinoma without p16 (CDKN2A) expression: Heterogeneous causes of an unusual immunophenotype.

Immunohistochemically p16 (CDKN2A)-negative uterine cervical squamous cell carcinoma (SCC) is uncommon, and there are few reports about its pathological features. This study explored the causes of p16 negativity in such cases. We analyzed diagnostic tissue samples of five cases of p16-negative cervical SCC among 107 patients who underwent hysterectomy at Kyoto University Hospital between January 2010 and December 2015. The samples were subjected to immunohistochemical staining, in situ hybridization and a genetic analysis. Two of five cases were positive for human papilloma virus (HPV) by genotyping. One was positive for HPV56 with promoter hypermethylation of CDKN2A and co-existing Epstein-Barr virus infection. Another was positive for HPV6 categorized as low-risk HPV with condylomatous morphology. Among the remaining three cases, one had amplification of the L1 gene of HPV with promoter hypermethylation of CDKN2A and TP53 mutation, and one of the other two HPV-negative cases had a homozygous CDKN2A deletion, while the other was positive for p53 and CK7. p16-negativity of cervical SCC is often associated with an unusual virus infection status and CDKN2A gene abnormality.

Model Combining Tumor Molecular and Clinicopathologic Risk Factors Predicts Sentinel Lymph Node Metastasis in Primary Cutaneous Melanoma.

PURPOSEMore than 80% of patients who undergo sentinel lymph node (SLN) biopsy have no nodal metastasis. Here, we describe a model that combines clinicopathologic and molecular variables to identify patients with thin- and intermediate-thickness melanomas who may forgo the SLN biopsy procedure because of their low risk of nodal metastasis.PATIENTS AND METHODSGenes with functional roles in melanoma metastasis were discovered by analysis of next-generation sequencing data and case-control studies. We then used polymerase chain reaction to quantify gene expression in diagnostic biopsy tissue across a prospectively designed archival cohort of 754 consecutive thin- and intermediate-thickness primary cutaneous melanomas. Outcome of interest was SLN biopsy metastasis within 90 days of melanoma diagnosis. A penalized maximum likelihood estimation algorithm was used to train logistic regression models in a repeated cross-validation scheme to predict the presence of SLN metastasis from molecular, clinical, and histologic variables.RESULTSExpression of genes with roles in epithelial-to-mesenchymal transition (glia-derived nexin, growth differentiation factor 15, integrin-β3, interleukin 8, lysyl oxidase homolog 4, transforming growth factor-β receptor type 1, and tissue-type plasminogen activator) and melanosome function (melanoma antigen recognized by T cells 1) were associated with SLN metastasis. The predictive ability of a model that only considered clinicopathologic or gene expression variables was outperformed by a model that included molecular variables in combination with the clinicopathologic predictors Breslow thickness and patient age (area under the receiver operating characteristic curve, 0.82; 95% CI, 0.78 to 0.86; SLN biopsy reduction rate, 42%; negative predictive value, 96%).CONCLUSIONA combined model that included clinicopathologic and gene expression variables improved the identification of patients with melanoma who may forgo the SLN biopsy procedure because of their low risk of nodal metastasis.

Utilizing Automated Breast Cancer Detection to Identify Spatial Distributions of Tumor-Infiltrating Lymphocytes in Invasive Breast Cancer

Quantitative assessment of spatial relations between tumor and tumor-infiltrating lymphocytes (TIL) is increasingly important in both basic science and clinical aspects of breast cancer research. We have developed and evaluated convolutional neural network analysis pipelines to generate combined maps of cancer regions and TILs in routine diagnostic breast cancer whole slide tissue images. The combined maps provide insight about the structural patterns and spatial distribution of lymphocytic infiltrates and facilitate improved quantification of TILs. Both tumor and TIL analyses were evaluated by using three convolutional neural network networks (34-layer ResNet, 16-layer VGG, and Inception v4); the results compared favorably with those obtained by using the best published methods. We have produced open-source tools and a public data set consisting of tumor/TIL maps for 1090 invasive breast cancer images from The Cancer Genome Atlas. The maps can be downloaded for further downstream analyses.

Prognostic Impact of Tumor-Associated Macrophages on Survival Is Checkpoint Dependent in Classical Hodgkin Lymphoma.

Tumor microenvironment and immune escape affect pathogenesis and survival in classical Hodgkin lymphoma (cHL). While tumor-associated macrophage (TAM) content has been associated with poor outcomes, macrophage-derived determinants with clinical impact have remained undefined. Here, we have used multiplex immunohistochemistry and digital image analysis to characterize TAM immunophenotypes with regard to expression of checkpoint molecules programmed cell death ligand 1 (PD-L1) and indoleamine 2,3-dioxygenase 1 (IDO-1) from the diagnostic tumor tissue samples of 130 cHL patients, and correlated the findings with clinical characteristics and survival. We show that a large proportion of TAMs express PD-L1 (CD68+, median 32%; M2 type CD163+, median 22%), whereas the proportion of TAMs expressing IDO-1 is lower (CD68+, median 5.5%; CD163+, median 1.4%). A high proportion of PD-L1 and IDO-1 expressing TAMs from all TAMs (CD68+), or from CD163+ TAMs, is associated with inferior outcome. In multivariate analysis with age and stage, high proportions of PD-L1+ and IDO-1+ TAMs remain independent prognostic factors for freedom from treatment failure (PD-L1+CD68+/CD68+, HR = 2.63, 95% CI 1.17–5.88, p = 0.019; IDO-1+CD68+/CD68+, HR = 2.48, 95% CI 1.03–5.95, p = 0.042). In contrast, proportions of PD-L1+ tumor cells, all TAMs or PD-L1− and IDO-1− TAMs are not associated with outcome. The findings implicate that adverse prognostic impact of TAMs is checkpoint-dependent in cHL.

Onscreen-guided resection of extra-axial and intra-axial forebrain masses through registration of a variable-suction tissue resection device with a neuronavigation system.

To describe a novel surgical technique in which neuronavigation is used to guide a tissue resection device during excision of forebrain masses in locations difficult to visualize optically. Short case series. Six dogs and one cat with forebrain masses (five neoplastic, two nonneoplastic) undergoing excision with a novel tissue resection device and veterinary neuronavigation system. The animals and resection instrument were coregistered to the neuronavigation system. Surgery was guided by real-time onscreen visualization of the resection instrument position relative to the preoperative MR images. Surgical outcome was evaluated by calculating residual tumor volume according to postoperative MRI. The technique was technically simple and led to the collection of diagnostic tissue samples in all cases. Postoperative MRI was available in six cases, two with gross-total resection, three with near-total resection, and one with subtotal resection. Neuronavigation-guided resection of intra-axial and extra-axial brain masses with the resection device resulted in gross-total or near-total resection in five of six animals with tumors otherwise difficult to visualize. Risk of brain shift limited absolute reliance on navigation images. Real-time neuronavigation assistance is a feasible method for guidance and successful resection of brain masses that are poorly visualized because of intra-axial or deep location, tumor appearance, or hemorrhage.

Stereotactic Brainstem Biopsy: Technical Aspects with Emphasis on Role of Awake Craniotomy

Abstract Introduction The role of frame-based stereotactic biopsy in brainstem lesions has been well established in literature. Transfrontal, transtentorial, and transcerebellar routes are used to access various targets within the brainstem. While the transfrontal approach is preferable in midbrain lesions, a transcerebellar approach via the middle cerebellar peduncle forms the shortest possible trajectory for pontine and medullary lesions. Objective Authors to describe the technical nuances of frame-based stereotactic biopsy of lower brainstem lesions to increase the procedural safety and efficacy. Materials and Methods Technical modifications in frame fixation were done to acheive the desired trajectory to the target. In adult cases biopsy was performed with patient awake during the procedure. Results Total of five patients underwent biopsy with the technical modifications. Three patients were adults and two were in pediatric age group. Their age ranged from 12 to 50 years. No complications were encountered. Two of the biopsies showed demyelination and the other three turned out to be low-grade glioma, pilocytic astrocytoma, and lymphoma, respectively. Conclusion Despite obvious inherent risks, the transcerebellar biopsy in semi-sitting position is a well-tolerated and effective method of obtaining a diagnostic tissue sample in brainstem lesions. We have noted that adoption of the technical modifications described in this article has aided in improving the safety and ease of the procedure.

Diagnostic Challenges in Primary Sacral Tumors and the Yield of Computed Tomography-Guided Needle Biopsy in the Modern Era

Primary sacral tumors pose unique challenges because of their complex radiographic appearances, diverse pathologic entities, and dramatically different treatment paradigms based on tumor type. Magnetic resonance imaging and computed tomography (CT) can provide valuable information; however, sacral lesions can possess unique radiographic features and pose diagnostic dilemmas. CT-guided percutaneous needle biopsy is a critical component of the diagnostic workup. However, limited data are available on its efficacy for primary sacral tumors. The data from patients with newly diagnosed primary sacral lesions during a 12-year period at our hospital were analyzed. The preoperative magnetic resonance imaging findings, biopsy results, and pathological data for patients who required surgery were analyzed. Unique cases in which the final pathologic result was unexpected from the preoperative imaging findings have been highlighted. Of 38 patients who underwent percutaneous needle biopsy, diagnostic tissue was obtained on the first attempt for 31 (82%). Five of the remaining 7 obtained diagnostic tissue on the second attempt, yielding 95% diagnosis, with only two requiring open biopsies. In 2 patients with diagnostic tissue on CT-guided biopsy, an open biopsy was still recommended because of the clinical scenario. In both patients, the open biopsy results matched those of the CT-guided biopsy. For the 18 patients who required surgery, we found 100% correlation between the percutaneous needle biopsy findings and the final pathological diagnosis. No biopsy-induced complications or extraspinal tumor seeding occurred. CT-guided biopsy is a safe and effective technique. It represents a critical component of the diagnostic algorithm, given the diverse pathological findings of primary sacral lesions and dramatic differences in treatment.

17-Gene Genomic Prostate Score Test Results in the Canary Prostate Active Surveillance Study (PASS) Cohort.

The 17-gene Oncotype DX Genomic Prostate Score (GPS) test predicts adverse pathology (AP) in patients with low-risk prostate cancer treated with immediate surgery. We evaluated the GPS test as a predictor of outcomes in a multicenter active surveillance cohort. Diagnostic biopsy tissue was obtained from men enrolled at 8 sites in the Canary Prostate Active Surveillance Study. The primary endpoint was AP (Gleason Grade Group [GG] ≥ 3, ≥ pT3a) in men who underwent radical prostatectomy (RP) after initial surveillance. Multivariable regression models for interval-censored data were used to evaluate the association between AP and GPS. Inverse probability of censoring weighting was applied to adjust for informative censoring. Predictiveness curves were used to evaluate how models stratified risk of AP. Association between GPS and time to upgrade on surveillance biopsy was evaluated using Cox proportional hazards models. GPS results were obtained for 432 men (median follow-up, 4.6 years); 101 underwent RP after a median 2.1 years of surveillance, and 52 had AP. A total of 167 men (39%) upgraded at a subsequent biopsy. GPS was significantly associated with AP when adjusted for diagnostic GG (hazards ratio [HR]/5 GPS units, 1.18; 95% CI, 1.04 to 1.44; P = .030), but not when also adjusted for prostate-specific antigen density (PSAD; HR, 1.85; 95% CI, 0.99 to 4.19; P = .066). Models containing PSAD and GG, or PSAD, GG, and GPS may stratify risk better than a model with GPS and GG. No association was observed between GPS and subsequent biopsy upgrade (P = .48). In our study, the independent association of GPS with AP after initial active surveillance was not statistically significant, and there was no association with upgrading in surveillance biopsy. Adding GPS to a model containing PSAD and diagnostic GG did not significantly improve stratification of risk for AP over the clinical variables alone.

Utilizing genomic profiling in a patient with a thoracic malignancy of uncertain primary: case report

Carcinoma of unknown primary (CUP) is estimated to be 2% of diagnosed cancers. In the era of precision medicine, we are able to use more tools to identify a possible source of primary, direct potential treatments as well as provide prognostic information by use of molecular characteristics of the tumor. Here we provide a case of a 62-year-old woman with a remote stage I left sided colon adenocarcinoma s/p hemicolectomy who presented predominantly with a lung mass. Immunohistochemistry of the diagnostic tissue was suggestive of a primary but with the help of molecular profiling, more information was obtained and patient is being currently treated with a durable response. With our case as an example, we review some of the literature on additional molecular tests available to better define CUP and suggest some changes to European Society for Medical Oncology (ESMO) algorithm for evaluation of CUP.

3:36 PM Abstract No. 203 Safety and efficacy of percutaneous computed tomography–guided transplant pancreas biopsy

Percutaneous computed tomographic (CT)-guided biopsy is a minimally invasive alternative to open biopsy, not frequently described in the literature. The purpose is to describe and assess technique, complications, and diagnostic yield of CT-guided percutaneous transplant pancreas biopsy. This is a single institution retrospective review of 75 patients (47 women, 28 men, mean age 47) who underwent CT-guided transplant pancreas biopsy (n = 149) between April 2005 and October 2018.Three cases (2%) were aborted due to inadequate safe percutaneous approach. Biopsy results were classified based on final pathology as diagnostic (with the subcategory partially diagnostic used if the sample was adequate for the diagnosis of cell mediated rejection, but not humoral rejection) and nondiagnostic. Diagnostic yield and complication rates were evaluated. 149 percutaneous CT-guided biopsies of transplant pancreas were performed. 98% (n =143) of the biopsies used 17/18 Gauge coaxial core biopsy system, and 2% (n = 3) used a 19/20 Gauge coaxial device. On-site pathologic touch prep assessment was utilized for 91% of biopsies. Diagnostic tissue was obtained 94% of the time, of which 9% were considered partially diagnostic. 6% of the samples were deemed non-diagnostic. Of the diagnostic biopsies, 7 (51%) were positive for rejection (24% grade I, 16% grade II, 4% grade III, and 7% chronic) and 49% were negative for rejection. On-site pathologic touch prep did not appear to have an effect on sample adequacy (P = 0.9) nor did the number of cores (P =.28) or graft location (P = 1.0). The most common complication was minor bleeding (9%), pancreatitis (1%) and sedation related adverse events (1%); there was 1 moderate complication (hemorrhage requiring overnight observation), and no major complications. No significant difference in the INR (P = 0.68) or platelet count (P = 0.07) between patients who had bleeding complications and those who did not have bleeding complications. Image-guided percutaneous CT-guided transplant pancreas biopsies have high diagnostic yield with low complication rates.

Multiparametric in situ imaging of NPM1-mutated acute myeloid leukemia reveals prognostically-relevant features of the marrow microenvironment.

Ancillary testing during the initial workup of acute myeloid leukemia (AML) is largely performed using aspirated materials. We utilized multiplex immunofluorescence (MIF) imaging with digital image analysis to perform an in situ analysis of the microenvironment in NPM1-mutated AML using diagnostic bone marrow biopsy tissues (N = 17) and correlated these findings with diagnostic next-generation sequencing (NGS, N = 17), flow cytometry (FC, N = 14), and first remission (CR1) NPM1-specific molecular MRD (n = 16) data. The total CD3-positive T-cell percentages correlated positively between FC and MIF (r = 0.53, p = 0.05), but were significantly lower by MIF (1.62% vs. 3.4%, p = 0.009). The percentage of mutant NPM1-positive (NPM1c+) cells ranged from 9.7 to 90.8% (median 45.4%) and did not correlate with the NPM1 mutant allele fraction by NGS (p > 0.05). The percentage of CD34+/NPM1c+ cells ranged from 0 to 1.8% (median 0.07%). The percentage of NPM1c+ cells correlated inversely (34% vs. 62%, p = 0.03), while the percentages of CD3-/NPM1c- cells (64% vs. 35%, p = 0.03), and specifically CD3-/CD4-/NPM1c- cells (26% vs. 13%, p = 0.04), correlated positively with subsequent MRD. Discordances between MIF and FC/NGS data suggest that aspirate materials are likely an imperfect reflection of the core biopsy tissue. Furthermore, increased numbers of NPM1 wild-type cells within the microenvironment at diagnosis correlate with the subsequent presence of MRD.

Fluorescence-guided stereotactic biopsy: a proof-of-concept study

OBJECTIVEAccurate histopathological diagnoses are often necessary for treating neuro-oncology patients. However, stereotactic biopsy (STB), a common method for obtaining suspicious tissue from deep or eloquent brain regions, fails to yield diagnostic tissue in some cases. Failure to obtain diagnostic tissue can delay initiation of treatment and may result in further invasive procedures for patients. In this study, the authors sought to determine if the coupling of in vivo optical imaging with an STB system is an effective method for identification of diagnostic tissue at the time of biopsy.METHODSA minimally invasive fiber optic imaging system was developed by coupling a 0.65-mm-diameter coherent fiber optic fluorescence microendoscope to an STB system. Human U251 glioma cells were transduced for stable expression of blue fluorescent protein (BFP) to produce U251-BFP cells that were utilized for in vitro and in vivo experiments. In vitro, blue fluorescence was confirmed, and tumor cell delineation by fluorescein sodium (FNa) was quantified with fluorescence microscopy. In vivo, transgenic athymic rats implanted with U251-BFP cells (n = 4) were utilized for experiments. Five weeks postimplantation, the rats received 5–10 mg/kg intravenous FNa and underwent craniotomies overlying the tumor implantation site and contralateral normal brain. A clinical STB needle containing our 0.65-mm imaging fiber was passed through each craniotomy and images were collected. Fluorescence images from regions of interest ipsilateral and contralateral to tumor implantation were obtained and quantified.RESULTSLive-cell fluorescence imaging confirmed blue fluorescence from transduced tumor cells and revealed a strong correlation between tumor cells quantified by blue fluorescence and FNa contrast (R2 = 0.91, p < 0.001). Normalized to background, in vivo FNa-mediated fluorescence intensity was significantly greater from tumor regions, verified by blue fluorescence, compared to contralateral brain in all animals (301.7 ± 34.18 relative fluorescence units, p < 0.001). Fluorescence intensity measured from the tumor margin was not significantly greater than that from normal brain (p = 0.89). Biopsies obtained from regions of strong fluorescein contrast were histologically consistent with tumor.CONCLUSIONSThe authors found that in vivo fluorescence imaging with an STB needle containing a submillimeter-diameter fiber optic fluorescence microendoscope provided direct visualization of neoplastic tissue in an animal brain tumor model prior to biopsy. These results were confirmed in vivo with positive control cells and by post hoc histological assessment. In vivo fluorescence guidance may improve the diagnostic yield of stereotactic biopsies.

An Isothermal, Multiplex Amplification Assay for Detection and Genotyping of Human Papillomaviruses in Formalin-Fixed, Paraffin-Embedded Tissues

Rapid and accurate identification of human papillomavirus (HPV) is important for both clinical management and population screening. Analytic validation of Atila AmpFire Multiplex HPV assays on formalin-fixed, paraffin-embedded (FFPE) cervix/vulva and oropharynx diagnostic tissue samples was performed. The AmpFire assay incorporates a novel isothermal multiplex amplification coupled with real-time fluorescent detection to detect and genotype 15 high-risk (HR) HPV genotypes. Limits of detection determined by plasmids cloned with HPV genotype-specific sequences were 2 copies/reaction for HPV16, HPV18, and some HR HPV genotypes, and 20 copies/reaction for the remaining HR HPV genotypes. The performance of the AmpFire assays in clinical samples was evaluated using 214 FFPE specimens. The AmpFire assay failed in one clinical specimen for an invalid rate of 0.5%. The AmpFire assay detected HPV in clinical samples with positive percent agreements of 100.0% for HPV16, 100.0% for HPV18, and 94.7% for non-16/18 HR HPV, and 100% negative percent agreements for HPV16, HPV18, and non-16/18 HR HPV. Qualitative detection agreement was obtained in the reproducibility study. In summary, the Atila AmpFire HPV assay demonstrated excellent analytic sensitivity and specificity for detection and genotyping of 15 HR HPV genotypes. Assay parameters of simple specimen processing, small sample size requirement, rapid turnaround time, and being near instrument-free render it well suited for HPV detection and genotyping in FFPE specimens.

Biological responses to physicochemical properties of biomaterial surface.

Biomedical scientists use chemistry-driven processes found in nature as an inspiration to design biomaterials as promising diagnostic tools, therapeutic solutions, or tissue substitutes. While substantial consideration is devoted to the design and validation of biomaterials, the nature of their interactions with the surrounding biological microenvironment is commonly neglected. This gap of knowledge could be owing to our poor understanding of biochemical signaling pathways, lack of reliable techniques for designing biomaterials with optimal physicochemical properties, and/or poor stability of biomaterial properties after implantation. The success of host responses to biomaterials, known as biocompatibility, depends on chemical principles as the root of both cell signaling pathways in the body and how the biomaterial surface is designed. Most of the current review papers have discussed chemical engineering and biological principles of designing biomaterials as separate topics, which has resulted in neglecting the main role of chemistry in this field. In this review, we discuss biocompatibility in the context of chemistry, what it is and how to assess it, while describing contributions from both biochemical cues and biomaterials as well as the means of harmonizing them. We address both biochemical signal-transduction pathways and engineering principles of designing a biomaterial with an emphasis on its surface physicochemistry. As we aim to show the role of chemistry in the crosstalk between the surface physicochemical properties and body responses, we concisely highlight the main biochemical signal-transduction pathways involved in the biocompatibility complex. Finally, we discuss the progress and challenges associated with the current strategies used for improving the chemical and physical interactions between cells and biomaterial surface.

Fluorescein-guided resection of gliomas

Standard of care in the management of high-grade gliomas includes gross total resection (GTR) followed by treatment with radiation and temozolomide. GTR remains one of the few independent prognostic factors for improved survival in this disease. Sodium fluorescein is an organic fluorophore that has been studied as a surgical adjunct to improve the likelihood of achieving GTR in gliomas. Though sodium fluorescein does not selectively accumulate in glioma cells, it allows for real-time identification of regions of blood brain barrier breakdown, corresponding to the contrast-enhancing cores of high-grade gliomas. In addition to its high predictive value for identifying pathologic tissue, use of fluorescein has been shown to improve rates of GTR. In stereotactic needle biopsies, it helps reduce procedure time by rapidly confirming the presence of diagnostic tissue. Furthermore, in non-enhancing, low-grade gliomas, it labels focal regions of vascular dysregulation that have been correlated with high-grade features. Fluorescein has also been shown to be significantly less expensive than other contemporary surgical adjuncts such as intraoperative ultrasound, intraoperative MRI, and the recently FDA approved fluorophore, 5-aminolevulinic acid (5-ALA). Here, we review the current literature on the effectiveness of fluorescein as a surgical tool in the resection of gliomas.

Assessment of mitochondrial respiratory chain enzymes in cells and tissues.

Measurement of the individual enzymes involved in mitochondrial oxidative phosphorylation (OXPHOS) forms a key part of diagnostic investigations in patients with suspected mitochondrial disease, and can provide crucial information on mitochondrial OXPHOS function in a variety of cells and tissues that are applicable to many research investigations. In this chapter, we present methods for analysis of mitochondrial respiratory chain enzymes in cells and tissues based on assays performed in two geographically separate diagnostic referral centers, as part of clinical diagnostic investigations. Techniques for sample preparation from cells and tissues, and spectrophotometric assays for measurement of the activities of OXPHOS complexes I-V, the combined activity of complexes II+III, and the mitochondrial matrix enzyme citrate synthase, are provided. The activities of mitochondrial respiratory chain enzymes are often expressed relative to citrate synthase activity, since these ratios may be more robust in accounting for variability that may arise due to tissue quality, handling and storage, cell growth conditions, or any mitochondrial proliferation that may be present in tissues from patients with mitochondrial disease. Considerations for adaption of these techniques to other cells, tissues, and organisms are presented, as well as comparisons to alternate methods for analysis of respiratory chain function. In this context, a quantitative immunofluorescence protocol is also provided that is suitable for measurement of the amount of multiple respiratory chain complexes in small diagnostic tissue samples. The analysis and interpretation of OXPHOS enzyme activities are then placed in the context of mitochondrial disease tissue pathology and diagnosis.

Utility of Endoscopic Ultrasound-Guided Fine Needle Aspiration in the Diagnosis of Local Recurrence of Pancreaticobiliary Cancer after Surgical Resection.

Background/AimsEndoscopic ultrasound (EUS)-guided fine needle aspiration (FNA; EUS-FNA) allows for diagnostic tissue specimens from various regions to be analyzed. However, diagnosing recurrent pancreaticobiliary cancer after surgery is sometimes difficult. We evaluated the efficacy of EUS-FNA in the diagnosis of local recurrence of pancreaticobiliary cancer and analyzed the factors associated with false-negative results.MethodsFifty-one consecutive patients who underwent EUS-FNA due to suspected recurrence of pancreaticobiliary cancer after surgery in an academic center were retrospectively analyzed. The criteria for EUS-FNA were a resected margin or remnant pancreas mass, round swollen lymph node (≥10 mm in diameter), and soft-tissue enhancement around a major artery. Patients with suspected liver metastasis or malignant ascites were excluded.ResultsThirty-nine of the 51 patients had pancreatic cancer; the remaining 12 had biliary cancer. The target sites for EUS-FNA were the soft tissue around a major artery (n=22, 43%), the resected margin or remnant pancreas (n=12, 24%), and the lymph nodes (n=17, 33%). The median size of the suspected recurrent lesions was 15 mm (range, 8 to 40 mm). The overall sensitivity, specificity and accuracy of EUS-FNA for the diagnosis of recurrence was 84% (32/38), 100% (13/13), and 88% (45/51), respectively. FNA of the soft tissue around major arteries (odds ratio, 8.23; 95% confidence interval, 1.2 to 166.7; p=0.033) was significantly associated with a false-negative diagnosis in the multivariate analysis.ConclusionsEUS-FNA is useful for diagnosing recurrent cancer, even after pancreaticobiliary surgery. The diagnoses of recurrence at soft-tissue sites should be interpreted with caution.

Molecular features of a large cohort of primary central nervous system lymphoma using tissue microarray

AbstractThe objective of this study was to evaluate the distribution and prognostic impact of a broad range of molecular attributes in a large cohort of immunocompetent patients with primary central nervous system lymphoma (PCNSL) by using tissue microarray. Patients diagnosed with PCNSL were initially identified in the BC Cancer Lymphoid Cancer clinical and pathology databases. Tissue microarrays were constructed by using archival formalin-fixed paraffin-embedded diagnostic biopsy tissue. Immunohistochemistry and fluorescent in situ hybridization studies were performed. A total of 115 patients with PCNSL with diffuse large B-cell lymphoma (DLBCL) histology were identified. The majority of cases (≥75%) had a non–germinal center B-cell phenotype according to immunohistochemistry algorithms, but cell of origin did not affect progression-free or overall survival. MYC (40%), BCL2 (75%), and programmed death-ligand 1 (29%) protein expression were common, but their corresponding gene rearrangements were rare (≤1% each), suggesting that alternate mechanisms were driving expression. There were no dual rearrangements involving MYC and BCL2. Only 22% of cases had membranous expression of major histocompatibility complex class II, suggesting a mechanism for escape from immune surveillance. Epstein-Barr virus–encoded RNA was positive in 1 immunocompetent patient. BCL6 protein expression (77%) and BCL6 rearrangements (31%) were frequent; the latter was the only factor associated with a poor prognosis in the overall cohort and in the subgroup of 52 patients treated with high-dose methotrexate–based regimens. This large population-based study shows that prominent molecular features of PCNSL are unique and different from those of systemic DLBCL. These results may better inform drug development in PCNSL.