Small Tissue Biopsies Research Articles

Pathogenesis and pathobiology of testicular germ cell tumours: a view from a developmental biological perspective with guidelines for pathological diagnostics.

Testicular germ cell tumours (GCT) are divided into three different subtypes (types I-III) regarding to their developmental origin, histological differences and molecular features. Type I GCT develop from disturbed primordial germ cells and most commonly occur in children and young adolescents, which is why they are referred to as prepubertal GCT. Type II GCT develop from a non-invasive germ cell neoplasia in situ (GCNIS) and show an isochromosome 12p (i12p) or gain of 12p material as a common and characteristic molecular alteration. Type III GCT originate from distorted postpubertal germ cells (e.g. spermatogonia) in adult patients and have changes on chromosome 9 with amplification of the DMRT1 gene. Type I GCT encompass prepubertal-type teratomas and yolk-sac tumours (YST). Type II GCT include seminoma, embryonal carcinoma, choriocarcinoma, postpubertal-type teratoma and postpubertal-type YST. Types I and II GCT both show similar morphology, but are separated from each other by the detection of a GCNIS and an i12p in type II GCT. For type II GCT it is especially important to detect non-seminomatous elements, as these tumours have a worse biological behaviour and need a different treatment to seminomas. In contrast to types I and II GCT, type III tumours are equivalent to spermatocytic tumours and usually occur in elderly men, with few exceptions in young adults. The development of types I and II GCT seems to depend not upon driver mutations, but rather on changes in the epigenetic landscape. Furthermore, different pluripotency associated factors (e.g. OCT3/4, SOX2, SOX17) play a crucial role in GCT development and can be used as immunohistochemical markers allowing to distinguish the different subtypes from each other in morphologically challenging tissue specimens. Especially in metastatic sites, a morphological and immunohistochemical diagnostic algorithm is important to detect small subpopulations of each non-seminomatous GCT subtype, which are associated with a poorer prognosis and need a different treatment. Furthermore, primary extragonadal GCT of the retroperitoneum or mediastinum develop from misguided germ cells during embryonic development, and might be challenging to detect in small tissue biopsies due to their rarity at corresponding sites. This review article summarises the pathobiological and developmental aspects of the three different types of testicular GCT that can be helpful in the histopathological examination of tumour specimens by pathologists.

Increasing Proteome Coverage Through a Reduction in Analyte Complexity in Single-Cell Equivalent Samples.

The advancement of sophisticated instrumentation in mass spectrometry has catalyzed an in-depth exploration of complex proteomes. This exploration necessitates a nuanced balance in experimental design, particularly between quantitative precision and the enumeration of analytes detected. In bottom-up proteomics, a key challenge is that oversampling of abundant proteins can adversely affect the identification of a diverse array of unique proteins. This issue is especially pronounced in samples with limited analytes, such as small tissue biopsies or single-cell samples. Methods such as depletion and fractionation are suboptimal to reduce oversampling in single cell samples, and other improvements on LC and mass spectrometry technologies and methods have been developed to address the trade-off between precision and enumeration. We demonstrate that by using a monosubstrate protease for proteomic analysis of single-cell equivalent digest samples, an improvement in quantitative accuracy can be achieved, while maintaining high proteome coverage established by trypsin. This improvement is particularly vital for the field of single-cell proteomics, where single-cell samples with limited number of protein copies, especially in the context of low-abundance proteins, can benefit from considering analyte complexity. Considerations about analyte complexity, alongside chromatographic complexity, integration with data acquisition methods, and other factors such as those involving enzyme kinetics, will be crucial in the design of future single-cell workflows.

Association of Clinical, Tumor, and Treatment Characteristics With Seizure Control in Patients With IDH1/2-Mutant Lower-Grade Glioma.

Patients with IDH1/2-mutant lower-grade glioma have a high frequency of seizures. We aimed to investigate the correlations between seizures and tumor/patient characteristics and the impact of surgery and adjuvant treatments (AT) on seizure control along the disease trajectory. We retrospectively included patients with IDH1/2-mutant lower-grade glioma who underwent surgery at the neurosurgery divisions of the University of Turin and Milan and were treated at the Division of Neuro-Oncology of Turin. Inclusion criteria were a diagnosis according to the 2021 WHO Classification and presentation with seizures; exclusion criteria were presence of CDKN2A/B homozygous deletion, intense/ring contrast enhancement on MRI at presentation, and small tissue biopsy. We evaluated seizure freedom for 2 months after surgery, 6 months from starting observation or AT, at recurrence, and for 6 months after treatments of recurrence. We included 150 patients. There were 77 (51%) and 31 (21%) patients with IDH-mutant/1p19q-codeleted grade 2 and 3 oligodendroglioma and 30 (20%) and 12 (8%) with IDH-mutant grade 2 and 3 astrocytoma, respectively. Total resection was accomplished in 68 (45%). Seventy-five patients (50%) received AT while the remaining 75 were observed with MRI. After 6 months after AT, 28 of 29 patients (96.5%) displayed seizure reduction, 5 of 28 (18%) being seizure-free. 66 of 124 patients (53%) had seizures at recurrence. After 6 months after second-line treatments, 60 of 66 patients (91%) had seizure reduction, 11 (17%) being seizure-free. In multivariable analyses, grade 3 histology positively correlated with seizure freedom at 2 months after surgery (OR 3.5, 1.4-8.9, p = 0.008), 6 months after AT (OR 9.0, 1.5-54.9, p = 0.017), and 6 months after treatment of recurrence (OR 4.9, 1.5-16.5, p = 0.009). Adjuvant radiotherapy reduced seizures at recurrence in a univariate analysis (OR 0.14, 0.03-0.7, p = 0.020). Patients with seizure freedom after surgery and AT displayed longer progression-free survival (PFS) (65, 24.5-105, vs 48 months, 32-63.5, p = 0.037). This study analyzed seizure control in patients with IDH1/2-mutant lower-grade glioma across multiple time points. Grade 3 correlated with better seizure control throughout the entire disease trajectory, and seizure freedom after surgery and AT correlated with a longer PFS regardless of tumor grade. These results could serve as an external control arm in clinical trials evaluating the efficacy on seizures of antitumor agents in patients with IDH-mutant lower-grade glioma.

Diagnosing liposarcoma on (peri)-renal mass biopsy: A clinicopathological study of 30 cases.

Classification of renal neoplasms on small tissue biopsies is in increasing demand, and maintaining broad differential diagnostic considerations in this setting is necessary. When evaluating a renal or perirenal tumour biopsy with sarcomatoid morphology, together with sarcomatoid renal cell carcinoma and sarcomatoid urothelial carcinoma as top diagnostic considerations, it is vital to additionally consider the possibility of well-differentiated and de-differentiated liposarcoma. This study reports a series of 30 biopsy samples from sites in or around the kidney collected from four institutions in which the correct diagnosis was either well-differentiated or de-differentiated liposarcoma. The majority (26 of 30, 87%) of lesions were accurately diagnosed on biopsy sampling, all of which incorporated testing for MDM2 by immunohistochemistry (IHC), fluorescence in-situ hybridisation (FISH) or a combination of the two as part of the diagnostic work-up. Tumour expression of MDM2 by IHC without confirmatory FISH analysis was sometimes (30%) sufficient to reach a diagnosis, but demonstration of MDM2 amplification by FISH was ascertained in the majority (57%) of biopsy samples. A diagnosis of de-differentiated liposarcoma was not definitively established until resection in four (13%) patients, as no MDM2 testing was performed on the corresponding pre-operative biopsies. When a retroperitoneal tumour is not clinically suspected, histological consideration of a liposarcoma diagnosis may be overlooked. Implementation of ancillary immunohistochemical and cytogenetic testing can ultimately lead to a definitive diagnosis in this potentially misleading anatomical location.

Artificial Intelligence Optical Biopsy for Evaluating the Functional State of Wounds

IntroductionThe clinical characterization of the functional status of active wounds in terms of their driving cellular and molecular biology remains a considerable challenge that currently requires excision via a tissue biopsy. In this pilot study, we use convolutional Siamese neural network (SNN) architecture to predict the functional state of a wound using digital photographs of wounds in a canine model of volumetric muscle loss (VML). MethodsDigital images of VML injuries and tissue biopsies were obtained in a standardized fashion from an established canine model of VML. Gene expression profiles for each biopsy site were obtained using RNA sequencing. These profiles were converted to functional profiles by a manual review of validated gene ontology databases in which we determined a hierarchical representation of gene functions based on functional specificity. An SNN was trained to regress functional profile expression values, informed by an image segment showing the surface of a small tissue biopsy. ResultsThe SNN was able to predict the functional expression of a range of functions based with error ranging from ∼5% to ∼30%, with functions that are most closely associated with the early state of wound healing to be those best-predicted. ConclusionsThese initial results suggest promise for further research regarding this novel use of machine learning regression on medical images. The regression of functional profiles, as opposed to specific genes, both addresses the challenge of genetic redundancy and gives a deeper insight into the mechanistic configuration of a region of tissue in wounds.

The effectiveness of using dye models for small tissue biopsies in the surgical pathology laboratory.

Losing of small tissues during tissue preparatory steps may seriously affect pathological diagnostic performance. Using an appropriate tissue marking dye could be an alternative solution. Therefore, the aim of the study was to find a suitable tissue marking dye to enhance the observable ability of various types of small-size tissues during several steps of tissue preparation. Various small-size samples of various organs and tissues (0.2 to 0.3 cm), including breast, endometrial, and cervical tissue, stomach, small and large intestine, lung, and kidney, were marked with different dyes such as merbromin, hematoxylin, eosin, crystal violet, and alcian blue prior to tissue processing step and their colored-observable ability was evaluated by pathology assistants. Moreover, the diagnostic interfering effect of each tissue marking dye was determined by pathologists. Merbromin, hematoxylin, and alcian blue increased the colored-observable ability of small tissue samples. We suggest using hematoxylin as a tissue marking dye over merbromin and alcian blue because of less toxicity and no interference effect in the step of routine pathological slide examination. Hematoxylin could be a suitable tissue marking dye for small-size samples and may improve the preanalytical process of tissue preparation in pathological laboratories.

One procedure—one report: the Re-Imagine Cytopathology Task Force position paper on small tissue biopsy triage in anatomic pathology

Endoscopic biopsy procedures increasingly generate multiple tissue samples from multiple sites, and frequently retrieve concurrent cytologic specimens and small core needle biopsies. There is currently lack of consensus in subspecialized practices as to whether cytopathologists or surgical pathologists should review such samples, and whether the pathology findings should be reported together or separately. In December 2021, the American Society of Cytopathology convened the Re-Imagine Cytopathology Task Force to examine various workflows that would facilitate unified pathology reporting of concurrently obtained biopsies and improve clinical care. This position paper summarizes the key points and highlights the advantages, challenges, and resources available to support the implementation of such workflows that result in "one procedure-one report".

DNA methylation-based biomarkers for ageing long-lived cetaceans.

Epigenetic approaches for estimating the age of living organisms are revolutionizing studies of long-lived species. Molecular biomarkers that allow age estimates from small tissue biopsies promise to enhance studies of long-lived whales, addressing a fundamental and challenging parameter in wildlife management. DNA methylation (DNAm) can affect gene expression, and strong correlations between DNAm patterns and age have been documented in humans and nonhuman vertebrates and used to construct "epigenetic clocks". We present several epigenetic clocks for skin samples from two of the longest-lived cetaceans, killer whales and bowhead whales. Applying the mammalian methylation array to genomic DNA from skin samples we validate four different clocks with median errors of 2.3-3.7 years. These epigenetic clocks demonstrate the validity of using cytosine methylation data to estimate the age of long-lived cetaceans and have broad applications supporting the conservation and management of long-lived cetaceans using genomic DNA from remote tissue biopsies.

Chordoma: analysis of 47 fine-needle aspiration biopsy, cytologic imprint, and small biopsy specimens.

Fine-needle aspiration (FNA) and small tissue biopsy of chordoma have been reported in several small series, but no large series exists. We undertook an examination of 47 cases (with concurrent core needle biopsy in a subset) to analyze diagnostic accuracy, cytomorphology, and immunohistochemistry. Our cytopathology files were searched for examples of chordoma with histopathologic verification. FNA biopsy smears and core needle were performed using standard techniques. Forty-seven cases of chordoma were retrieved from 44 patients [M:F; 1.8:1; age range 5-81 years; mean age 55 years]. Twenty-seven presented with primary, 10 with locally recurrent, and 7 with metastatic tumors. Two aspirates were from the appendicular skeleton, 2 from the trunk, 1 from neck lymph node, and 42 aspirates (89%) from axial and peri-axial skeleton and surrounding soft tissues. Four were cytologic touch imprints while the remainder were FNA biopsy specimens. Specific cytologic diagnoses were chordoma/consistent with chordoma (44 cases, 94%), suspicious for chordoma (2), and malignant neoplasm (1). Along with a single case of benign notochordal tumor misdiagnosed as chordoma, our diagnostic accuracy was 91%. Concurrent tissue biopsy was performed in 51% of cases. Immunohistochemical staining of tumor in 29 (62%) cases showed expression of brachyury in 23 of 24 (96%) instances. Cytopathology consisted of cellular smears populated by large cells possessing enormous amounts of vacuolated and non-vacuolated cytoplasm with an abundant background myxoid/chondromyxoid stroma. FNA and small tissue biopsy specimens show a very high degree of diagnostic accuracy in recognition of chordoma.

Pseudogout Causing Thoracic Outlet Syndrome

Abstract Introduction/Objective Calcium pyrophosphate dihydrate (CPPD) crystal deposition or pseudogout is a common entity encountered in routine practice. However, when a common entity presents in uncommon locations with unusual clinical manifestations, it creates a diagnostic conundrum. Pseudogout commonly occurs in the joint extremities including knee, wrist, shoulder, hip, and ankle joints. The sternoclavicular joint is a rare site for pseudogout with very low incidence. To the best of our knowledge, there are only two other cases of pseudogout mimicking a tumor causing vascular obstruction in the literature. Methods/Case Report A 63 year old male presented with an enlarging supraclavicular mass. A computed tomography angiography scan of the chest revealed an irregular soft tissue mass located behind the medial clavicle measuring up to 4 cm and was noted to compress and partially occlude the right subclavian vein. Partial occlusion of right axillary vein, right internal jugular vein, and right brachiocephalic vein were also noted on imaging. The venous compression led to outflow obstruction and resultant recurrent deep vein thrombosis of the right upper extremity. Due to compression by the mass, he required stenting of the right subclavian vein which unfortunately reoccluded. Subsequently, the patient underwent excision of the mass and partial resection of the first rib with decompression of the thoracic outlet. The excisional biopsy revealed dense rhomboid-shaped CPPD crystal depositions in irregular nodules within a background of fibrinoid material and fragments of unremarkable cartilaginous tissue. The patients compressive symptoms were relieved following the excision. While clinically and radiologically the mass was concerning for a malignant tumor, histological examination provided a definitive diagnosis of a benign process. Results (if a Case Study enter NA) NA. Conclusion Pseudogout is a rare mimicker of bone and soft tissue tumors and differentiation from an enchondroma, or grade 1 chondrosarcoma becomes a challenge especially on small tissue biopsies and is a possible diagnostic pitfall.

CelLock TM: an innovative standardized cell-block preparation procedure

ABSTRACT The CelLock™ procedure kit is used to collect and prepare cellular specimens such as fine needle aspirates (FNA), cytology specimens, cultured cells, small tissue biopsies, and samples with scant tissue fragments or cells into a paraffin cell-block. This cell-block can be used for subsequent microtomy and staining using hematoxylin and eosin (H&E), special stains, immunohistochemistry (IHC), and applicable molecular techniques such as in situ hybridization (ISH). CelLock is a standardized method that provides optimal receipt, preservation, preparation, and processing of cell-blocks which, contain virtually all of the submitted specimens and are able to be embedded and sectioned in a reproducible fashion. The specimen contained within the cell-block is preserved such that all the cellular protein and genetic information is available for histological and ancillary testing.

Evaluation of P40 and Napsin A in the Differential Diagnosis of Non Small Cell Bronchogenic Carcinoma on Small Lung Biopsies

BACKGROUND :- Lung cancer is the leading cause of cancer-related mortality over word wide, Although the pathological diagnosis of lung carcinoma is limited as only small specimen available for diagnosis.the availability of targeted therapies has created a need for precise subtyping of non-small cell lung carcinoma . Several recent studies have demonstrated that the use of Immunohistochemical markers can be helpful in differentiating lung squamous cell carcinoma (LSCC) from lung adenocarcinoma (LAC) not on surgically resected material but also on small biopsy samples and cytology.
 AIM (1) To classify the non small cell lung carcinoma into major categories like squamous cell carcinoma (LSCC) and adenocarcinoma (LAC) and other categories by applying immunohistochemicalmarker like p40 (truncated p63) and Napsin A
 (2) To analyse the sensitivity and specificity of p40 and Napsin A in light of histomorphology and/or other relevant immunohistochemical markers available, using appropriate statistical tests.
 Material and methods:- This study was a one and half year (18 months) prospective study from Jan 2017 to June 2018, conducted in department of pathology on patients attending the outpatient and inpatient department of TB and respiratory disease, a total of 210 bronchoscopic guided biopsies / transthoracic (CT/MRI /guided) small tissue biopsies from the patients suspected of lung malignancy were incorporated in the study. 20 corresponding resection specimens (wedge resection and lobectomy) were also included in the study for correlation of morphology and immunohistochemical findings on small biopsies.
 RESULTS:-In our study IHC for both p40 and napsin –A aided in subtyping of 71.9% cases of non small cell lung carcinoma and this diagnostic accuracy was found to be statistically significant with p-value < 0.05.,on statistical analysis we found that napsin-A had a sensitivity of 90% and specificity of 80%. Also, positive predictive value and negative predictive value were seen to be 88.0% and 81.8% respectively.
 

Analysis of Organization and Activity of Mitochondrial Respiratory Chain Complexes in Primary Fibroblasts Using Blue Native PAGE.

Blue Native polyacrylamide gel electrophoresis (BN-PAGE) is a well-established technique for the isolation and separation of mitochondrial membrane protein complexes in a native conformation with high resolution. In combination with histochemical staining methods, BN-PAGE has been successfully used as clinical diagnostic tool for the detection of oxidative phosphorylation (OXPHOS) defects from small tissue biopsies from patients with primary mitochondrial disease. However, its application to patient-derived primary fibroblasts is difficult due to limited proliferation and high background staining. Here, we describe a rapid and convenient method to analyze the organization and activity of OXPHOS complexes from cultured skin fibroblasts.

Microdissection Methods Utilizing Single-Cell Subtype Analysis and the Impact on Precision Medicine.

Improving the utilization of tumor tissue from diagnostic biopsies is an unmet medical need. This is especially relevant today in the rapidly evolving precision oncology field where tumor genotyping is often essential for the indication of many advanced and targeted therapies. National Comprehensive Cancer Network (NCCN) guidelines now mandate molecular testing for clinically actionable targets in certain malignancies. Utilizing advanced stage lung cancer as an example, an improved genotyping approach for solid tumors is possible. The strategy involves optimization of the microdissection process and analysis of a large number of identical target cells from formalin-fixed paraffin-embedded (FFPE) specimens sharing similar characteristics, in other words, single-cell subtype analysis. The shared characteristics can include immunostaining status, cell phenotype, and/or spatial location within a histological section. Synergy between microdissection and droplet digital PCR (ddPCR) enhances the molecular analysis. We demonstrate here a methodology that illustrates genotyping of a solid tumor from a small tissue biopsy sample in a time- and cost-efficient manner, using immunostain targeting as an example.

Targeted Protein Profiling of In Vivo NIPP-Treated Tissues Using DigiWest Technology

Non-invasive physical plasma (NIPP) is a novel therapeutic tool, currently being evaluated for the treatment of cancer and precancerous lesions in gynecology and other disciplines. Additionally, patients with cervical intraepithelial neoplasia (CIN) may benefit from NIPP treatment due to its non-invasive, side-effect-free, and tissue-sparing character. However, the molecular impact of in vivo NIPP treatment needs to be further investigated. For this purpose, usually only very small tissue biopsies are available after NIPP treatment. Here, we adapted DigiWest technology, a high-throughput bead-based Western blot, for the analysis of formalin-fixed paraffin-embedded (FFPE) cervical punch biopsies with a minimal sample amount. We investigated the molecular effects of NIPP treatment directly after (0 h) and 24 h after in vivo application. Results were compared to in vitro NIPP-treated human malignant cervical cells. NIPP effects were primarily based on an inhibitory impact on the cell cycle and cell growth factors. DigiWest technology was suitable for detailed protein profiling of small, primary FFPE biopsies.

SomaMutDB: a database of somatic mutations in normal human tissues.

De novo mutations, a consequence of errors in DNA repair or replication, have been reported to accumulate with age in normal tissues of humans and model organisms. This accumulation during development and aging has been implicated as a causal factor in aging and age-related pathology, including but not limited to cancer. Due to their generally very low abundance mutations have been difficult to detect in normal tissues. Only with recent advances in DNA sequencing of single-cells, clonal lineages or ultra-high-depth sequencing of small tissue biopsies, somatic mutation frequencies and spectra have been unveiled in several tissue types. The rapid accumulation of such data prompted us to develop a platform called SomaMutDB (https://vijglab.einsteinmed.org/SomaMutDB) to catalog the 2.42 million single nucleotide variations (SNVs) and 0.12 million small insertions and deletions (INDELs) thus far identified using these advanced methods in nineteen human tissues or cell types as a function of age or environmental stress conditions. SomaMutDB employs a user-friendly interface to display and query somatic mutations with their functional annotations. Moreover, the database provides six powerful tools for analyzing mutational signatures associated with the data. We believe such an integrated resource will prove valuable for understanding somatic mutations and their possible role in human aging and age-related diseases.

Consequences of Splitting Small Tissue Biopsies from Cytology Specimens

Consequences of Splitting Small Tissue Biopsies from Cytology Specimens

A modified density gradient proteomic-based method to analyze endolysosomal proteins in cardiac tissue

SummaryThe importance of lysosomes in cardiac physiology and pathology is well established, and evidence for roles in calcium signaling is emerging. We describe a label-free proteomics method suitable for small cardiac tissue biopsies based on density-separated fractionation, which allows study of endolysosomal (EL) proteins. Density gradient fractions corresponding to tissue lysate; sarcoplasmic reticulum (SR), mitochondria (Mito) (1.3 g/mL); and EL with negligible contamination from SR or Mito (1.04 g/mL) were analyzed using Western blot, enzyme activity assay, and liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis (adapted discontinuous Percoll and sucrose differential density gradient). Kyoto Encyclopedia of Genes and Genomes, Reactome, Panther, and Gene Ontology pathway analysis showed good coverage of RAB proteins and lysosomal cathepsins (including cardiac-specific cathepsin D) in the purified EL fraction. Significant EL proteins recovered included catalytic activity proteins. We thus present a comprehensive protocol and data set of guinea pig atrial EL organelle proteomics using techniques also applicable for non-cardiac tissue.

Abstract 373: Advancing precision oncology by synergizing ddPCR with microdissection

Abstract Background - The domain of precision oncology includes improving patient treatment outcomes based on the application of advanced molecular profiling methodologies to pathological specimens. To this end, optimal utilization of tumor tissue from diagnostic biopsies is an unmet medical need. This is especially relevant today since precision oncology is a rapidly evolving field where timely tumor genotyping is essential for the indication of many advanced and targeted therapies. National Comprehensive Cancer Network (NCCN) guidelines now mandate molecular testing for clinically actionable targets in certain malignancies. Patients diagnosed with advanced non-small cell lung cancer (NSCLC) are commonly of an older age and have significant co-morbidities. This frequently causes clinical dilemmas regarding the ability to obtain adequate amounts of tissue for tumor genotyping. In these cases, the tumor tissue may have been obtained by an image-guided biopsy, and the diagnosis of NSCLC proper determined via cytology. In certain instances, adequate tissue for tumor genotyping and/or a more advanced mutational analysis to identify oncogenic drivers may not be available. Methods - Formalin fixed paraffin embedded (FFPE) specimens were examined using advanced immuno-based laser capture microdissection (LCM), following a formal pathology review. In preparation for droplet digital PCR (ddPCR), DNA was extracted from samples and run with a series of positive and negative controls. Results - Utilizing lung cancer as an example, an improved genotyping approach for NSCLC solid tumors was developed and tested. The strategy involves optimization of the microdissection process and analysis of a large number of identical target cells from FFPE specimens sharing similar characteristics, in other words, single-cell subtype analysis. Immunostaining status, cell phenotype, and spatial location within a histological section are examples of shared characteristics that can guide cell procurement. Conclusions - Synergy between microdissection and ddPCR enhances molecular analysis. Demonstrated is a methodology that illustrates genotyping of a solid tumor from a small tissue biopsy sample in a time and cost-efficient manner, using immunohistochemistry directed LCM and ddPCR detection. Citation Format: Donald J. Johann, Ikjae Shin, Erich Peterson, Mathew Steliga, Jason Muesse, Katy Marino, Sarah Laun, Adam Roberge, Robert Weigman, Michael Emmert-Buck, Michael Tangrea. Advancing precision oncology by synergizing ddPCR with microdissection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 373.

Synergizing microdissection with ddPCR to advance precision oncology.

e15083 Background: Improving patient treatment outcomes based on the application of advanced molecular profiling methodologies applied to pathological specimens is the purview of precision oncology. Optimal utilization of tumor tissue from diagnostic biopsies remains an unmet medical need. This is especially relevant today since precision oncology is a rapidly evolving field where timely tumor genotyping is essential for the indication of many advanced and targeted therapies. National Comprehensive Cancer Network (NCCN) guidelines now mandate molecular testing for clinically actionable targets in certain malignancies. Patients diagnosed with advanced non-small cell lung cancer (NSCLC) are commonly of an older age and have significant co-morbidities. Clinical dilemmas regarding the ability to obtain adequate amounts of tissue for tumor genotyping frequently occur. In these cases, the tumor tissue may have been obtained by an image-guided biopsy, and the diagnosis of NSCLC proper determined via cytology. Adequate tissue for tumor genotyping and/or a more advanced mutational analysis to identify oncogenic drivers may not be available, and the opportunity to explore more optimal therapy options (eg, TKI in lieu of chemotherapy) is lost. Methods: Following a formal pathology review formalin fixed paraffin embedded (FFPE) specimens were examined using advanced immuno-based laser capture microdissection (LCM). In preparation for droplet digital PCR (ddPCR), nucleic acids were extracted from samples and run with a series of positive and negative controls. Results: Utilizing lung cancer as an example, an improved genotyping approach for NSCLC solid tumors was developed and tested. The strategy involves optimization of the microdissection process and analysis of a large number of identical target cells from FFPE specimens sharing similar characteristics, in other words, single-cell subtype analysis. Immunostaining status, cell phenotype, and spatial location within a histological section are examples of shared characteristics that can guide cell procurement. Conclusions: With this approach, tumor molecular analysis is enhanced through the synergy between microdissection and ddPCR. Here we demonstrate a methodology that illustrates genotyping of a solid tumor from a small tissue biopsy sample in a time and cost-efficient manner, using immunohistochemistry-directed LCM along with the detection and absolute quantitation of mutations by ddPCR.