Small Tissue Specimens Research Articles

Radiation damping at clinical field strength: Characterization and compensation in quantitative measurements.

In any MR experiment, the bulk magnetization acts on itself, caused by the induced current in the RF receiver circuit that generates an oscillating damping field. This effect, known as "radiation damping" (RD), is usually weak and, therefore, unconsidered in MRI, but can affect quantitative studies performed with dedicated coils that provide a high SNR. The current work examined RD in a setup for investigations of small tissue specimens including a quantitative characterization of the spin-coil system. A custom-made Helmholtz coil (radius and spacing 16 mm) was interfaced to a transmit-receive (Tx/Rx) switch with integrated passive feedback for modulation or suppression of RD similar to preamplifier decoupling. Pulse sequences included pulse-width arrays to demonstrate the absence/ presence of RD and difference techniques employing gradient pulses or composite RF pulses to quantify RD effects during free precession and transmission, respectively. Experiments were performed at 3T in small samples of MnCl2 solution. Significant RD effects may impact RF pulse application and evolution periods. Effective damping time constants were comparable to typical T2 * times or echo spacings in multi-echo sequences. Measurements of the phase relation showed that deviations from the commonly assumed 90° angle between the damping field and the transverse magnetization may occur. Radiation damping may affect the accuracy of quantitative MR measurements performed with dedicated RF coils. Efficient mitigation can be achieved hardware-based or by appropriate consideration in the pulse sequence.

Comparative analysis of genomic profiles between tissue-based and plasma-based next-generation sequencing in patients with non-small cell lung cancer

ObjectivesGenotype-guided personalized therapy has become an essential part of routine clinical care in non-small cell lung cancer (NSCLC) patients. However, small tissue specimens often yield inadequate molecular testing material. Plasma ctDNA-based liquid biopsy is an increasingly common non-invasive alternative to tissue biopsy. This study examined the similarities and differences in the molecular profiling of tissue and plasma samples to provide insight into sample selection in clinical practice. Materials and MethodsSequencing data from 190 NSCLC patients who underwent concurrent tissue-based next-generation sequencing (tissue-NGS) and plasma-based NGS (plasma-NGS) using a 168-gene panel were analyzed. ResultsTissue-NGS identified genomic alterations in 97.4% (185/190) of the enrolled patients and plasma-NGS identified genomic alterations in 72.1% (137/190) of the enrolled patients. Considering all NSCLC guideline-recommended biomarkers in the entire cohort of 190 cases, 81 patients had positive concordant mutations detected in both tissue and plasma samples, while 69 patients had no predefined alterations detected in either tissue or plasma samples. Additional mutations were found in the tissues of 34 patients and the plasma of six patients. The overall concordance rate between tissue and plasma samples was 78.9% (150/190). The tissue-NGS and plasma-NGS sensitivities were 95.0% and 71.9%, respectively. In the 137 patients with detectable ctDNA in plasma samples, the concordance rate between tissue and plasma samples reached 91.2%, and the sensitivity of plasma-NGS reached 93.5%. ConclusionOur findings indicate that plasma-NGS is less capable of detecting genetic alterations than tissue-NGS, especially for copy number variations and gene fusions. Tissue-NGS remains the preferred method for evaluating the molecular profile of NSCLC patients when tumor tissue is available. We suggest that the concurrent use of liquid biopsy and tissue biopsy is the optimal approach in clinical practice; alternatively, plasma can be used as substitute material when tissue is unavailable.

The Ki67 index evaluation of pancreatic neuroendocrine tumors using 3D immunohistochemistry in small tissue specimens

Background/Objectives: This study aimed to evaluate the usefulness of three-dimensional (3D) immunohistochemistry for the Ki67 index of small tissue specimens of pancreatic neuroendocrine tumor (PanNET). MethodsClinicopathological materials from 17 patients with PanNET who underwent surgical resection at Jichi Medical University Hospital were analyzed. We compared the Ki67 index of endoscopic ultrasonography-fine-needle aspiration biopsy (EUS-FNAB) specimens, surgical specimens, and small tissue specimens hollowed from paraffin blocks of surgical specimens that were substituted for EUS-FNAB specimens (“sub-FNAB”). The sub-FNAB specimens were optically cleared using LUCID (IlLUmination of Cleared organs to IDentify target molecules) and analyzed using 3D immunohistochemistry. ResultsThe median Ki67 index in FNAB, sub-FNAB, and surgical specimens with conventional immunohistochemistry were 1.2% (0.7–5.0), 2.0% (0.5–14.6), and 5.4% (1.0–19.4), respectively. The median Ki67 index in sub-FNAB specimens with tissue clearing was calculated separately using the total number of cells on multiple images (“multiple slice”), with the image of the fewest positive cells (“coldspot”), and with the image of most positive cells (“hotspot”), which were 2.7% (0.2–8.2), 0.8% (0–4.8), and 5.5% (2.3–12.4), respectively. PanNET grade evaluated for the hotspot of the surgical specimens was significantly more consistent with those of the hotspot than multiple images of sub-FNAB specimens (16/17 vs. 10/17, p = 0.015). Hotspot evaluation using 3D immunohistochemistry of the sub-FNAB specimens showed agreement with the assessment of the surgical specimens (Kappa coefficient: 0.82). ConclusionsTissue clearing and 3D immunohistochemistry for the Ki67 index can potentially improve the preoperative evaluation of EUS-FNAB specimens of PanNET in routine clinical practice.

Abstract 985: An innovative spatial biology biomarker discovery approach to enable acceleration of drug development

Abstract Biomarker development in the clinical trial setting is frequently limited by the availability of small tissue specimens, such as core needle biopsies. To enable forward and reverse translational assays to best understand patient-specified biomarkers, high-plex assays which can be performed on individual slides are critical. We’ve developed the GeoMx® Digital Spatial Profiler (DSP) and CosMx™ Spatial Molecular Imager (SMI) to allow researchers to explore more hypotheses simultaneously from standard histopathology slides. We demonstrate on standard FFPE sections how the GeoMx and CosMx platforms integrate within the context of biomarker discovery. The GeoMx platform can resolve transcriptome-wide RNA expression profiles or 150+ protein targets from tissue structures selected by pathological review. The CosMx platform allows subcellular profiling of up to 1000 RNA transcripts or 64 proteins from a single slide. We demonstrate the integration of GeoMx and CosMx into a multi-modal approach to how high-plex spatial platforms can complement conventional diagnostic assays for translational research. This includes integration with downstream analytical tools such as digital pathology suites as well as machine learning approaches to model patient outcomes. Spatial technologies, such as GeoMx and CosMx, present groundbreaking opportunities for improving accuracy in biomarker discovery and for providing rich biological insight beyond low-plex profiling approaches. Citation Format: Jason W. Reeves, Esperanza Anguiano, Vikram Devgan, Nicholas Confuorto, Joachim Schmid, Yan Liang, Joseph Beechem. An innovative spatial biology biomarker discovery approach to enable acceleration of drug development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 985.

In Vivo Toxicity, Redox-Modulating Capacity and Intestinal Permeability of Novel Aroylhydrazone Derivatives as Anti-Tuberculosis Agents

The emergence and spread of Mycobacterium tuberculosis strains resistant to many or all anti-tuberculosis (TB) drugs require the development of new compounds both efficient and with minimal side effects. Structure-activity-toxicity relationships of such novel, structurally diverse compounds must be thoroughly elucidated before further development. Here, we present the aroylhydrazone compounds (3a and 3b) regarding their: (i) acute and subacute toxicity in mice; (ii) redox-modulating in vivo and in vitro capacity; (iii) pathomorphology in the liver, kidney, and small intestine tissue specimens; and (iv) intestinal permeability. The acute toxicity test showed that the two investigated compounds exhibited low toxicity by oral and intraperitoneal administration. Changes in behavior, food amount, and water intake were not observed during 14 days of the oral administration at two doses of 1/10 and 1/20 of the LD50. The histological examination of the different tissue specimens did not show toxic changes. The in vitro antioxidant assays confirmed the ex vivo results. High gastrointestinal tract permeability at all tested pH values were demonstrated for both compounds. To conclude, both compounds 3a and 3b are highly permeable with low toxicity and can be considered for further evaluation and/or lead optimization.

A 1-min double embedding method for small tissue specimens preserves comedone histology and eliminates the need for punch biopsies.

It is difficult to preserve the structure and microbial distribution inside comedonal plugs during routine processing. The objective of this study is to determine the optimal method to preserve the comedonal corneum plug structure and inherent microorganisms thereby eliminating the need to perform punch biopsies in relevant studies. Corneum plugs were extracted from comedones of acne vulgaris patients. Primary embedding using either a 2% agarose, 2% agar, 25% gelatin, or 2% agar + 2.5% gelatin solution was subsequently performed and the results compared. The specimens were then fixed, waxed, sectioned, and examined by light, fluorescence, and scanning electron microscopies to observe the structures and microorganisms within the plugs. Both the 25% gelatin and 2% agarose solutions successfully preserved the structural integrity of corneum plugs and the inherent microorganisms. When considering other factors such as thermostability, reusability, and convenience, the 25% gelatin solution was the superior choice among the four materials. We report a simple and effective method for double embedding comedonal plugs and other small tissue specimens. The technique preserves the structure and microbial distribution in situ within comedonal corneum plugs, eliminates the need for punch biopsies. This method may also be applied to other tiny and fragile tissue specimens, thereby enabling a potentially wide array of future large-scale investigations and alleviated patients' pain.

Adequacy of small biopsy and cytology specimens for comprehensive genomic profiling of patients with non-small-cell lung cancer to determine eligibility for immune checkpoint inhibitor and targeted therapy

AimsIn advanced-stage non-small-cell lung cancer (NSCLC), incomplete genotyping for guideline-recommended genomic biomarkers poses a significant challenge to making informed and timely clinical decisions. We report our institution’s experience in assessing...

A method to prepare large resin sections for counting myelinated axons in rodent CNS and PNS structures

We present a method that allows preparing histological sections from large blocks of nervous tissue embedded in epoxy resin. Resin-embedding provides excellent resolution especially for the myelin-rich white matter and is often being used for visualizing the myelinated axons in peripheral nerves. However, because of the limited penetration of the reagents, only very small tissue specimens can be processed in this way. Here, we describe a method that enables to embed large specimens and their sectioning on a standard sliding microtome. To process the large specimens, modifications in several steps of the processing technique had to be made. In this paper we demonstrate, that with this technique 1−3 μm thick transversal sections can be prepared from the resin-embedded specimens as large as rat brain hemisphere. Such a large section allows simultaneously: 1.) overviewing and delineating the gross anatomical structures, and 2.) observing the subcellular details at the highest possible optical magnifications. Such a large section with excellent resolution allows application of unbiased stereological methods and reliable quantification of very small objects within the area of interest.

Larger Nephron Size and Nephrosclerosis Predict Progressive CKD and Mortality after Radical Nephrectomy for Tumor and Independent of Kidney Function.

Nephron hypertrophy and nephrosclerosis may be important determinants of CKD and mortality. However, studies of outcomes associated with these microstructural features have been limited to small tissue specimens from patients selected for either good kidney health or known kidney disease. To determine whether microstructural features are predictive of progressive CKD and mortality outcomes, we studied patients who underwent a radical nephrectomy for a tumor. Large wedge sections of renal parenchyma distal to the tumor were stained and scanned into high-resolution images; we annotated the cortex and all glomeruli to calculate glomerular volume, cortex volume per glomerulus, and percentage of globally sclerotic glomeruli. Morphometric measurements also included percentages of artery luminal stenosis and interstitial fibrosis/tubular atrophy (IF/TA) of the cortex. At follow-up visits every 6-12 months, we determined which patients experienced progressive CKD (defined as dialysis, kidney transplantation, or a 40% decline from postnephrectomy eGFR). Cox models for these outcomes were adjusted for age, sex, body mass index, hypertension, diabetes, smoking, eGFR, and proteinuria. Among 936 patients (mean age, 64 years; postnephrectomy baseline eGFR, 48 ml/min per 1.73 m2), 117 progressive CKD events, 183 noncancer deaths, and 116 cancer deaths occurred during a median follow-up of 6.4 years. Larger glomerular volume, larger cortex per glomerulus, and higher percentage of globally sclerotic glomeruli or IF/TA predicted progressive CKD. Higher percentage IF/TA also predicted noncancer mortality. Microstructural features did not predict cancer mortality or recurrence. After a radical nephrectomy, larger nephrons and nephrosclerosis predicted progressive CKD, and IF/TA predicted noncancer mortality. Morphometric analysis of renal parenchyma can predict noncancer clinical events in patients long after their radical nephrectomy.

Chromogenic immunohistochemical quadruplex provides accurate diagnostic differentiation of non-small cell lung cancer

Lung cancer is the most common cancer worldwide and has the highest mortality rate. Carcinomas comprise 95% of all lung malignancies, the vast majority of which are non-small cell lung carcinomas (NSCLC). Increasingly, the diagnosis of lung cancer is established by examination of small tissue specimens obtained by minimally invasive techniques. It is critical to employ these tissues at maximum efficiency in order to render an accurate pathologic diagnosis and to perform theranostic studies, either genomic or by immunohistochemistry, to demonstrate genetic mutations that make patients eligible for molecularly targeted agents. Currently Thyroid Transcription Factor-1 (TTF-1) and Napsin A are the most commonly used immunohistochemical (IHC) stains to identify primary lung adenocarcinoma, and p40 and cytokeratin 5/6 (CK5/6) are used for squamous cell carcinoma. IHC stains for these markers, are performed either individually (IHC brown staining) or in combination as dual immunostains (i.e. TTF-1 + Napsin A and p40 + CK5/6, utilizing brown and red chromogens). Here we present a novel, truly multiplex immunohistochemical approach that combines staining with the above four antibodies on a single tissue section utilizing four different chromogens to accurately diagnose primary lung adenocarcinomas, squamous cell carcinomas, and combined adenosquamous carcinomas of the lung. Each marker is represented by a distinct color that can be read by a pathologist, using standard, bright field microscopy. We evaluated the ability of pathologists to differentiate NSCLCs using the multiplexed assay as compared to standard, single marker per slide diaminobenzidine (DAB)-based IHC. All cases in a cohort of 264 NSCLCs showed concordance of information (including positivity of stain, intensity of stain and coverage) between single IHC stains and the multiplex assay. This new multiplex IHC offers the capability to accurately diagnose and sub-classify primary lung NSCLCs, while conserving precious tissue for additional testing.

Accurate Technique for Electrical Permittivity Measurement of Biological Tissue Specimens at Low Frequencies

This paper proposes a technique aimed at accurate measurement of electrical permittivity (EP) of small tissue specimens at low frequencies. Accurate measurement of EP of healthy and pathological tissues can facilitate the establishment of many medical applications such as electrical impedance imaging. The proposed novel technique employs hardware with high precision for impedance measurement of capacitive structures formed by the tissue specimen as its dielectric combined with two conductive plates placed at two ends of the specimen. To determine the EP of the tissue specimen, the capacitance part of the measured impedance is processed using an inverse finite-element framework. In this framework, accurate geometry and boundary conditions of the specimen are considered. The proposed technique was validated before it was employed to measure EP of several tissue specimens including bovine heart, liver, and bone. Results obtained in this investigation indicate that the proposed technique is reliable as it provides improved EP measurement accuracy and repeatability of homogeneous or multilayered tissue specimens, especially at low-frequency range.

Evaluation of a DNA Extraction and Purification Protocol Using Archived Formalin-fixed Paraffin-embedded Tissues for BRAF Mutations Analysis in Papillary Thyroid Microcarcinomas.

The isolation of good quality genomic DNA from formalin-fixed, paraffin-embedded tissues is challenging, especially in cases of small tissue specimens. The aim of our study was to evaluate a DNA extraction protocol using formalin-fixed, paraffin-embedded tissues in our laboratory and apply this method to a series of papillary thyroid microcarcinomas (PTMCs). A total of 25 PTMCs and 3 papillary thyroid carcinoma control cases were included in the study. We assessed a DNA extraction protocol on the basis of a precipitation method (MasterPure DNA purification kit, Epicentre), according to the manufacturer's instructions. All PTMCs were subject to real-time polymerase chain reaction (PCR) amplification targeting the BRAF gene and a housekeeping gene (GAPDH). BRAF gene mutations were then assessed by high-resolution melting analysis and confirmed by sequencing of the PCR products. Using this extraction method, we produced good yields of DNA (mean concentration, 147.4±77.8 ng/µL), in addition to high levels of purity (mean A260/A280 ratio: 1.63±0.1). We successfully assessed the BRAF mutation status in 24 cases (16 BRAF-negative; 8 BRAF positive), although 1 case revealed an inconclusive pattern following high-resolution melting analysis and sequencing of the PCR products. We observed no differences in the tumor size (P=0.693), storage period of the tumor block (P=0.282), DNA concentration (P=0.243), DNA purity (P=0.458), CpGAPDH (P=0.173), or CpBRAF (P=0.217) values between the BRAF-mutated and nonmutated group of PTMCs. Our findings demonstrate the importance of a reliable, reproducible DNA extraction technique for efficient PCR amplification, uniformly applied to all cases in this study, regardless of the BRAF mutation status.

Utility of near infrared spectrophotometry in mesenteric ischemia: An experimental study

This study investigated the effectiveness of near infrared (NIR) spectrophotometry for measuring abdominal saturation in a rat model of induced mesenteric ischemia. This was a randomized, controlled, non-blinded interventional animal study. Twenty-eight male Sprague Dawley rats were randomly allocated into four groups. Following induction of anesthesia, the abdominal region was shaved and intra-abdominal access was achieved through a 4-cm midline incision and basal measurements were performed using an INVOS-5100c device. The rats were grouped on the basis of rSO2 measurement times. Mesenteric artery ligation was not performed on the control group rats. From the rest of the mesenteric artery ligated rats, measurements were taken at 30 min, at 2 h and at 6 h. Measurements were repeated using NIR spectrophotometry probes after the abdomen was surgically closed. Following the measurements, the abdomen was re-opened and small intestine tissue specimens were taken for histopathological examination. Initial and final NIR spectrophotometry measurements were compared. No statistically significant difference was found in the group 1 between basal rSO2 values and post-incision values ( p = 0.748). There was a significant variation between the initial and final measurements in groups 2, 3, and 4 ( p = 0.018). Although various radiological or biochemical markers are available for detecting mesenteric ischemia, the use of NIR spectrophotometry can be considered a good non-invasive alternative method for early diagnosis of acute mesenteric ischemia.

Programmed cell death-ligand 1 (PD-L1) expression is associated with RAS/TP53 mutations in lung adenocarcinoma

IntroductionThe systematic assessment of anti-programmed cell death ligand 1 (PD-L1) expression by immunohistochemistry (IHC) in lung adenocarcinomas is becoming standard practice. However, the assessment of PD-L1 expression on small tissue specimens needs to be evaluated and the association with other features more thoroughly analyzed. MethodsThis retrospective single center study evaluated the immunohistochemical expression of the SP263 anti-PD-L1 antibody on tissue microarrays (TMA) of 152 surgically resected lung adenocarcinomas, using a 25% positivity threshold. The positive cases and 50 randomly chosen negative cases in tissue microarray (TMA) were reassessed on whole tissue sections. The results were correlated to clinical, histopathological and to molecular data obtained through the screening of 214 mutations in 26 genes (LungCarta panel, Agena Biosciences). ResultsAmong 152 primary lung adenocarcinomas, 19 cases (13%) showed PD-L1 expression. The agreement between TMA and whole tissue sections was 89%, specificity was 97%. PD-L1 expression was correlated to RAS mutations (p = .04), RAS/TP53 co-mutations (p = .01) and to the solid or acinar subtype (p = .048). ConclusionsWith the SP263 PD-L1 antibody, small samples appear as a reliable means to evaluate the PD-L1 status in lung adenocarcinoma. The association between PD-L1 expression and RAS/TP53 mutations may have clinical relevance to predict the efficacy of PD-1/PD-L1 immune checkpoints inhibitors.

Immunoexpression of P63 and SOX2 in triple-negative breast cancers, Indonesia.

Background: Using immunohistochemical stains to target specific breast cancer markers has become indispensable for evaluation of small diagnostic tissue specimens, and therefore novel marker cocktails for specific breast cancers are required. This study was conducted to assess the immunoexpression of P63 and SOX2 in triple negative breast cancer (TNBC), and to evaluate the predictive diagnostic value of these markers for specific types of TNBC. Methods: Histological slides and paraffin blocks of TNBC cases were collected from Dr. Hasan Sadikin Hospital, Bandung, Indonesia from 5-years period (2011-2015). Each histological slide was subjected to immunohistochemical staining for P63 (nucleus and cytoplasm) and SOX2 (nucleus), with specific primer antibodies. Immunoexpression of P63 and SOX2 was evaluated using immunoreactivity scoring. Associations between P63 and SOX2 immunoexpression and TNBC types were assessed using Mann Whitney tests. In addition, the predictive diagnostic values of these markers were assessed. Results: Forty TNBC histological slides were included, and 23 (57.5%) were Basal-like type TNBC and 17 (42.5%) were Non basal-like type TNBC. Immunoexpression of P63 nucleus and SOX2 was not different between types of TNBC. However, immunoexpression of P63 in the cytoplasm in Basal-like type TNBC was significantly higher than in Non basal-like type TNBC ( p=0.021). Predictor diagnostic value analysis suggested that immunoexpression of P63 in cytoplasm had 56.5% sensitivity and 70.6% specificity for diagnosing Basal-like type TNBC, with area under curve of 0.64. Conclusions: Immunoexpression of P63 in the cytoplasm has a relatively weak diagnostic value to discriminate Basal-like and Non basal-like types of TNBC.

Immunoexpression of P63 and SOX2 in triple-negative breast cancers, Indonesia

Background: Using immunohistochemical stains to target specific breast cancer markers has become indispensable for evaluation of small diagnostic tissue specimens, and therefore novel marker cocktails for specific breast cancers are required. This study was conducted to assess the immunoexpression of P63 and SOX2 in triple negative breast cancer (TNBC), and to evaluate the predictive diagnostic value of these markers for specific types of TNBC. Methods: Histological slides and paraffin blocks of TNBC cases were collected from Dr. Hasan Sadikin Hospital, Bandung, Indonesia from 5-years period (2011-2015). Each histological slide was subjected to immunohistochemical staining for P63 (nucleus and cytoplasm) and SOX2 (nucleus), with specific primer antibodies. Immunoexpression of P63 and SOX2 was evaluated using immunoreactivity scoring. Associations between P63 and SOX2 immunoexpression and TNBC types were assessed using Mann Whitney tests. In addition, the predictive diagnostic values of these markers were assessed. Results: Forty TNBC histological slides were included, and 23 (57.5%) were Basal-like type TNBC and 17 (42.5%) were Non basal-like type TNBC. Immunoexpression of P63 nucleus and SOX2 was not different between types of TNBC. However, immunoexpression of P63 in the cytoplasm in Basal-like type TNBC was significantly higher than in Non basal-like type TNBC ( p=0.021). Predictor diagnostic value analysis suggested that immunoexpression of P63 in cytoplasm had 56.5% sensitivity and 70.6% specificity for diagnosing Basal-like type TNBC, with area under curve of 0.64. Conclusions: Immunoexpression of P63 in the cytoplasm has a relatively weak diagnostic value to discriminate Basal-like and Non basal-like types of TNBC.

MS 18.04 PD-L1 Expression in Early Stage Lung Cancer

The significant activity of programmed cell death 1 (PD-1)/PD-1 ligand 1 (PD-L1) checkpoint inhibitors in heavily pre-treated patients with advanced non–small-cell lung cancer (NSCLC) marked the beginning of a new era of immunotherapy. Recently published randomized clinical trials’ data have led to the approval of 3 PD-1/PD-L1 inhibitors—nivolumab (Opdivo; Bristol-Myers Squibb Company), pembrolizumab (Keytruda; Merck Sharp & Dohme Corp), and atezolizumab (Tecentriq, Genentech/Roche)—for the treatment of advanced NSCLC after first-line therapy. Furthermore, pembrolizumab was recently approved by the FDA as a first-line therapy for patients with advanced NSCLC. However, the overall response rates to these agents in an unselected population are reportedly low, thus emphasising the need for predictive biomarkers that identify beneficial candidates. The recently approved tests for anti-PD-1/PD-L1 therapy in NSCLC include the assessment of PD-L1 expression using immunohistochemistry (IHC) as a companion diagnostic test (22C3 for pembrolizumab) and 2 complementary diagnostic tests (28-8 for nivolumab and SP142 for atezolizumab). Another PD-L1 assay is being currently tested in clinical trials (e.g. SP263). In addition to commercial assays, laboratories and research institutions may establish their own laboratory-developed tests (LDTs) using various antibodies available, most notably the E1L3N clone. Hence, the PD-L1 expression status, as well as its predictive and prognostic value, differ considerably based on the antibody clones, platforms, and interpretation criteria used. However, the current assays evaluating the predictive role of tumor PD-L1 expression remain without harmonization in terms of the staining analysis and scoring system. The intratumoral heterogeneity in PD-L1 expression is another important issue. At present, PD-L1 testing is mainly conducted on biopsy specimens, which may not represent the tumor as a whole, and it may lead to false results, particularly in cases where testing is conducted using small tissue specimens, such as bronchial or transthoracic biopsy specimens. The resulting false-negative results could lead to the under-treatment of patients. In this presentation, I’d like to introduce 1) the results of comparison study between 4 different PD-L1 IHC and scoring systems in the surgically resected early stage lung cancer specimen 2) the correlation of PD-L1 expression between TMA specimens and the corresponding resected specimen to better understand the intratumoral heterogeneity. PD-L1, immunohistochemistry, lung cancer

Immunoexpression of P63 and SOX2 in triple-negative breast cancers, Indonesia.

Using immunohistochemical stains to target specific breast cancer markers has become indispensable for evaluation of small diagnostic tissue specimens, and therefore novel marker cocktails for specific breast cancers are required. This study was conducted to assess the immunoexpression of P63 and SOX2 in triple negative breast cancer (TNBC), and to evaluate the predictive diagnostic value of these markers for specific types of TNBC. Histological slides and paraffin blocks of TNBC cases were collected from Dr. Hasan Sadikin Hospital, Bandung, Indonesia from 5-years period (2011-2015). Each histological slide was subjected to immunohistochemical staining for P63 (nucleus and cytoplasm) and SOX2 (nucleus), with specific primer antibodies. Immunoexpression of P63 and SOX2 was evaluated using immunoreactivity scoring. Associations between P63 and SOX2 immunoexpression and TNBC types were assessed using Mann Whitney tests. In addition, the predictive diagnostic values of these markers were assessed. Forty TNBC histological slides were included, and 23 (57.5%) were Basal-like type TNBC and 17 (42.5%) were Non basal-like type TNBC. Immunoexpression of P63 nucleus and SOX2 was not different between types of TNBC. However, immunoexpression of P63 in the cytoplasm in Basal-like type TNBC was significantly higher than in Non basal-like type TNBC ( p=0.021). Predictor diagnostic value analysis suggested that immunoexpression of P63 in cytoplasm had 56.5% sensitivity and 70.6% specificity for diagnosing Basal-like type TNBC, with area under curve of 0.64. Conclusions: Immunoexpression of P63 in the cytoplasm has a relatively weak diagnostic value to discriminate Basal-like and Non basal-like types of TNBC.

Application of Immunohistochemistry in Undifferentiated Neoplasms: A Practical Approach.

- Advances in interventional technology have enhanced the ability to safely sample deep-seated suspicious lesions by fine-needle aspiration procedures. These procedures often yield scant amounts of diagnostic material, yet there is an increasing demand for the performance of more ancillary tests, especially immunohistochemistry and, not infrequently, molecular assays, to increase diagnostic sensitivity and specificity. A systematic approach to conserving diagnostic material is the key, and our previously proposed algorithm can be applied aptly in this context. - To elaborate a simple stepwise approach to the evaluation of cytology fine-needle aspiration specimens and small biopsy tissue specimens, illustrating the algorithmic application of small panels of immunohistochemical stains in providing an accurate diagnosis with scant amounts of tissue, including the potential pitfalls that may arise while using immunohistochemical staining on small quantities of tissue. - The sources include literature (PubMed), the first Chinese American Pathologists Association Diagnostic Pathology Course material, and the review authors' research data as well as practice experience. Seven examples selected from the CoPath database at Geisinger Medical Center (Danville, Pennsylvania) are illustrated. - A stepwise approach to the evaluation of fine-needle aspiration and small biopsy tissue specimens in conjunction with a small panel of select immunohistochemical stains has been successful in accurately assessing the lineage/origin of the metastatic tumors of unknown primaries. The awareness of the common pitfalls of these biomarkers is essential in many instances.

Abstract A19: Identification of Heregulin (HRG) expression as a driver of a difficult-to-treat cancer phenotype and development of a companion diagnostic for the HRG-ErbB3 targeting drug seribantumab

Abstract Heregulin (HRG) is the cognate ligand of the ErbB3 receptor and has been identified as a potent driver of a distinct tumor cell phenotype characterized by enhanced survival that appears to be inherently more resistant to standard of care therapies. Seribantumab (MM-121) is a fully human monoclonal antibody designed to block HRG from binding to ErbB3 and to prevent the establishment of HRG-driven cancer cell survival in response to cytotoxic therapies. Clinical evidence from randomized studies suggests that patients who receive standard therapy have shorter progression-free survival (PFS) when their tumors are positive for HRG RNA expression compared to patients receiving standard therapy with HRG-negative tumors. The addition of seribantumab was able to enhance the anti-tumor effect of these respective standard therapies and prolong PFS in patients with HRG-positive tumors. Profiling data indicates that positive HRG expression can be found in more than a third of most solid tumors identifying a high unmet medical need. In order to identify HRG-driven tumors with a more difficult-to-treat phenotype, an RNA in situ hybridization (ISH) diagnostic assay has been developed. Details will be presented illustrating the benefits of RNA ISH over alternate detection methods for HRG. This includes the visualization of tumor cell HRG expression with high sensitivity and specificity in small tissue specimens such as fine needle aspirates and core needle biopsies. This assay is currently being used to select patients for inclusion into diagnostic-driven clinical studies. Citation Format: Sara Mathews, Gregory Finn, Arthur J. Kudla, Victoria Rimkunas, Peter Laivins, Gavin MacBeath, Akos Czibere, Jason Baum. Identification of Heregulin (HRG) expression as a driver of a difficult-to-treat cancer phenotype and development of a companion diagnostic for the HRG-ErbB3 targeting drug seribantumab. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr A19.