Molecular testing techniques can be used on cytology specimens to facilitate cancer patient management. Fluorescence in situ hybridization (FISH) is well-suited for detecting genomic abnormalities in cytology specimens. FISH involves hybridizing fluorescent probes to target sequences to visualize locations. It can detect gains, losses, amplifications, and rearrangements. A variety of cytology specimens can be used for FISH, including smears, cell blocks, and liquid-based preparations. FISH has applications in detecting abnormalities in cancers like urothelial carcinoma, breast cancer, and lymphoma.
This document discusses patterns of fine needle aspiration cytology (FNAC) findings in benign and malignant breast lesions. It provides classifications of breast lesions, descriptions of cytology findings for specific lesions such as fibroadenomas, papillomas, cysts, fat necrosis, and various types of breast carcinomas. It notes that while FNAC is useful for evaluating breast lesions, its ability to distinguish between some proliferative benign lesions and low-grade carcinomas is limited. Histopathological examination is often needed for definitive diagnosis.
The Paris System for Reporting Urinary CytologyRawa Muhsin
The Paris System for Reporting Urinary Cytology provides standardized diagnostic categories for urine cytology specimens. It divides results into negative for high-grade urothelial carcinoma, positive for high-grade urothelial carcinoma, atypical urothelial cells, and suspicious for high-grade urothelial carcinoma based on the number and features of abnormal cells seen. The system aims to determine whether high-grade urothelial carcinoma is present or not, as this has important implications for patient management and prognosis. Risk of malignancy increases from negative to atypical to suspicious to positive categories.
The document discusses the development and benefits of the Milan System for Reporting Salivary Gland Cytopathology. It aims to standardize terminology for salivary gland FNA reports which previously lacked uniformity. The system categorizes specimens as non-diagnostic, non-neoplastic, atypia of undetermined significance, neoplastic (benign or uncertain malignant potential), suspicious for malignancy, or malignant. It is intended to improve communication between pathologists and clinicians, enhance patient care, and facilitate research by allowing standardized data collection across institutions. While validation is ongoing, the system provides a practical framework for uniform reporting of salivary gland cytology.
Cell blocks provide tissue fragments from FNA specimens that are processed into paraffin blocks. This allows examination of histological structure and use of ancillary tests like immunohistochemistry. Cell blocks increase diagnostic sensitivity and specificity compared to cytology alone. They require minimal effort and preserve tissue for second opinions without losing the original smears. The document discusses FNAC and cell block techniques, materials used, advantages like increased cellularity and diagnostic yield, and importance of clinical information for optimal diagnosis.
The bethesda system for reporting thyroid cytopathology dhanya89
The Bethesda System for Reporting Thyroid Cytopathology document outlines the standardized categories for reporting thyroid fine needle aspiration biopsy results. The categories include: Nondiagnostic/Unsatisfactory, Benign, Atypia of Undetermined Significance/Follicular Lesion of Undetermined Significance, Follicular Neoplasm/Suspicious for Follicular Neoplasm, Suspicious for Malignancy, and Malignant. Each category has specific criteria for cellularity, architecture, and nuclear features to provide consistent terminology for thyroid FNA interpretation and patient management.
Molecular profiling of breast cancer can classify tumor types, identify appropriate therapeutic targets, determine prognosis, and predict treatment response. Techniques include immunohistochemistry, fluorescence in situ hybridization, reverse transcription PCR, microarrays, and next generation sequencing to analyze protein expression, gene copy number, mutations, and gene expression levels. Breast cancers are classified into intrinsic subtypes including luminal A/B, HER2-enriched, basal-like, and claudin-low based on distinct gene expression patterns that predict clinical behavior and response to therapy.
The document provides an outline and overview of a presentation on cytopathology of the breast. It discusses the normal breast anatomy and cells seen on fine needle aspiration (FNA). It covers patient workup, techniques for FNA, and considerations for interpreting results. Inflammatory conditions, benign and malignant breast tumors are addressed. The accuracy and limitations of FNA are summarized. Reporting categories for breast FNA results are also outlined.
The document discusses cell blocks, which are used in cytopathology to provide tissue samples from fluid specimens for histological examination. Cell blocks allow for maintaining tissue architecture, performing ancillary tests, and archiving samples. Various cell block preparation methods are described. Cell blocks provide diagnostic advantages over smears for certain tumor types and body fluids. While cell blocks increase diagnostic accuracy, some methods can result in low cellularity or inadequate samples for ancillary testing. Overall, the document provides an overview of the utility and methods of cell block preparation in cytopathology.
This document discusses patterns of fine needle aspiration cytology (FNAC) findings in benign and malignant breast lesions. It provides classifications of breast lesions, descriptions of cytology findings for specific lesions such as fibroadenomas, papillomas, cysts, fat necrosis, and various types of breast carcinomas. It notes that while FNAC is useful for evaluating breast lesions, its ability to distinguish between some proliferative benign lesions and low-grade carcinomas is limited. Histopathological examination is often needed for definitive diagnosis.
The Paris System for Reporting Urinary CytologyRawa Muhsin
The Paris System for Reporting Urinary Cytology provides standardized diagnostic categories for urine cytology specimens. It divides results into negative for high-grade urothelial carcinoma, positive for high-grade urothelial carcinoma, atypical urothelial cells, and suspicious for high-grade urothelial carcinoma based on the number and features of abnormal cells seen. The system aims to determine whether high-grade urothelial carcinoma is present or not, as this has important implications for patient management and prognosis. Risk of malignancy increases from negative to atypical to suspicious to positive categories.
The document discusses the development and benefits of the Milan System for Reporting Salivary Gland Cytopathology. It aims to standardize terminology for salivary gland FNA reports which previously lacked uniformity. The system categorizes specimens as non-diagnostic, non-neoplastic, atypia of undetermined significance, neoplastic (benign or uncertain malignant potential), suspicious for malignancy, or malignant. It is intended to improve communication between pathologists and clinicians, enhance patient care, and facilitate research by allowing standardized data collection across institutions. While validation is ongoing, the system provides a practical framework for uniform reporting of salivary gland cytology.
Cell blocks provide tissue fragments from FNA specimens that are processed into paraffin blocks. This allows examination of histological structure and use of ancillary tests like immunohistochemistry. Cell blocks increase diagnostic sensitivity and specificity compared to cytology alone. They require minimal effort and preserve tissue for second opinions without losing the original smears. The document discusses FNAC and cell block techniques, materials used, advantages like increased cellularity and diagnostic yield, and importance of clinical information for optimal diagnosis.
The bethesda system for reporting thyroid cytopathology dhanya89
The Bethesda System for Reporting Thyroid Cytopathology document outlines the standardized categories for reporting thyroid fine needle aspiration biopsy results. The categories include: Nondiagnostic/Unsatisfactory, Benign, Atypia of Undetermined Significance/Follicular Lesion of Undetermined Significance, Follicular Neoplasm/Suspicious for Follicular Neoplasm, Suspicious for Malignancy, and Malignant. Each category has specific criteria for cellularity, architecture, and nuclear features to provide consistent terminology for thyroid FNA interpretation and patient management.
Molecular profiling of breast cancer can classify tumor types, identify appropriate therapeutic targets, determine prognosis, and predict treatment response. Techniques include immunohistochemistry, fluorescence in situ hybridization, reverse transcription PCR, microarrays, and next generation sequencing to analyze protein expression, gene copy number, mutations, and gene expression levels. Breast cancers are classified into intrinsic subtypes including luminal A/B, HER2-enriched, basal-like, and claudin-low based on distinct gene expression patterns that predict clinical behavior and response to therapy.
The document provides an outline and overview of a presentation on cytopathology of the breast. It discusses the normal breast anatomy and cells seen on fine needle aspiration (FNA). It covers patient workup, techniques for FNA, and considerations for interpreting results. Inflammatory conditions, benign and malignant breast tumors are addressed. The accuracy and limitations of FNA are summarized. Reporting categories for breast FNA results are also outlined.
The document discusses cell blocks, which are used in cytopathology to provide tissue samples from fluid specimens for histological examination. Cell blocks allow for maintaining tissue architecture, performing ancillary tests, and archiving samples. Various cell block preparation methods are described. Cell blocks provide diagnostic advantages over smears for certain tumor types and body fluids. While cell blocks increase diagnostic accuracy, some methods can result in low cellularity or inadequate samples for ancillary testing. Overall, the document provides an overview of the utility and methods of cell block preparation in cytopathology.
The document discusses the history, utility, and methods of preparing cell blocks from fine needle aspiration cytology samples. Cell blocks allow examination of histological structure and use of ancillary tests. Key methods include the fixed sedimentation method using a 1:1 ratio of 100% alcohol and 40% formalin, the plasma thrombin method using equal parts plasma and thrombin, and the bacterial agar method using 3% agar. Cell blocks provide increased diagnostic sensitivity and specificity compared to cytology alone through examination of tissue architecture and ability to perform special stains and molecular testing.
This document discusses cytology of the urinary tract. It describes three methods for collecting urine specimens: voided urine, catheter specimens, and bladder washings. Voided urine is the simplest but cells may degrade over time. Catheter specimens avoid contamination but can damage cells. Bladder washings provide high cellularity and preservation through saline irrigation. Specimens can be prepared via several methods including cytocentrifugation and direct smears. Normal urinary tract cytology shows a range of superficial and deep urothelial cell morphologies depending on collection method and location in the tract.
Immunohistochemistry in diagnosis of soft tissue tumours seminarPannaga Kumar
This document discusses immunohistochemistry in the diagnosis of soft tissue tumors. It begins by introducing soft tissue and the classification of soft tissue tumors. It then discusses various ancillary techniques used, focusing on immunohistochemistry. It provides details on common markers used to identify muscle, neural, melanocytic, endothelial and other types of differentiation. It discusses the applications and diagnostic utility of various markers for different tumor types. In summary, the document is a comprehensive overview of immunohistochemistry techniques and markers useful in the diagnosis and classification of soft tissue tumors.
The document summarizes the International Academy of Cytology (IAC) System for classifying breast malignancy based on fine-needle aspiration cytology (FNAC) results. The system was developed at a meeting in Yokohama in 2016. It aims to standardize breast cytology reporting to improve diagnosis and patient management. The system categorizes FNAC results as insufficient, benign, atypical, suspicious of malignancy, or malignant, with associated risks of malignancy. Cytological features and management recommendations are provided for each category. The goal is to link cytology reports to optimal breast care.
This document provides guidance on grossing (examining) different types of breast specimens in pathology. It describes how to properly collect, orient, measure, sample and submit lumpectomy, mastectomy, microdochectomy and lymph node specimens. Key steps include fixation in formalin, inking, slicing, measuring margins, evaluating tumors and lymph nodes, and submitting representative sections for histology. The goal is to thoroughly examine specimens and obtain high quality samples for accurate diagnosis.
The document provides guidance on grossing Whipple's specimens. It discusses that a Whipple's specimen contains the pancreatic head, duodenum, gallbladder and portions of the stomach and small intestine. Key steps in grossing include inking surfaces, slicing the pancreatic head, examining the ampulla and margins, and submitting sections of the tumor, ducts, margins and lymph nodes for histology. Performing a thorough gross examination is important for accurately assessing resection status and staging the tumor.
Cytologic assessment of bronchopulmonary lesionsAseem Jain
This document provides an overview of cytologic assessment of bronchopulmonary lesions. It discusses the normal histology of the respiratory system and various cytologic sampling techniques used to evaluate the lungs, such as sputum samples, bronchial brushings, washings and lavage. The cytology of normal respiratory cells and endogenous material is described. A variety of benign pulmonary conditions and infectious processes are outlined. Specific lung diseases like tuberculosis, sarcoidosis and nocardiosis are discussed through their characteristic cytologic findings.
The document discusses several pediatric neoplasms that appear as small round blue cell tumors due to their primitive histological features. These include neuroblastoma, Wilms tumor, rhabdomyosarcoma, Ewing's sarcoma, medulloblastoma, retinoblastoma, and lymphoma. For each tumor, the document outlines characteristics such as common age of diagnosis, clinical features, histopathological appearance under the microscope, immunohistochemistry profiles, genetics where relevant, and important prognostic factors. Differential diagnosis of these small round blue cell tumors in children is provided for accurate diagnosis and treatment.
This document summarizes urine cytology and various urinary markers for detecting bladder cancer. Urine cytology is the gold standard but has low sensitivity of 40-62%. Newer urinary markers like BTA, ImmunoCyt, NMP-22, and UroVysion have higher sensitivities than cytology of 50-86%, but lower specificities and can be affected by benign conditions. No single marker currently has a sensitivity of 90%, which patients indicate is needed to replace cystoscopy. Therefore, the best approach is a combination of cystoscopy and urinary markers for non-muscle-invasive bladder cancer surveillance.
The document discusses effusion cytology. It begins by describing the anatomy of serous cavities and membranes that line them, producing serous fluid. Any excess fluid is an effusion, indicating a pathological process. Effusions can be classified as hydrostatic, infectious, inflammatory, or malignant. Samples are collected and prepared as smears for staining.
Normal components in effusions include mesothelial cells, histiocytes, lymphocytes, and other inflammatory cells. Reactive mesothelial cells can appear atypical but maintain a uniform appearance. Malignant effusions result from direct extension or metastasis of cancers. Identifying malignant cells involves comparing size, shape and number to determine the primary tumor type and origin. The most
This document discusses liquid biopsy, a non-invasive technique to detect tumor biomarkers shed into body fluids like blood. It can identify circulating tumor cells, circulating tumor DNA, exosomes, and newly, tumor educated platelets. Liquid biopsy offers advantages over tissue biopsy like being less invasive, allowing repeated sampling over time. While sensitivity remains a limitation, liquid biopsy shows promise for early cancer detection, monitoring treatment response and tumor evolution, and assessing tumor heterogeneity. The document reviews technologies to analyze various biomarkers and potential clinical applications of liquid biopsy.
This document discusses the role of immunohistochemistry (IHC) in diagnosing soft tissue tumours. It begins by defining soft tissue and the WHO classification of soft tissue tumours. IHC is an important ancillary technique that can be used to identify discrete tissue components using antigen-antibody binding. The document outlines the IHC protocol and discusses various markers that can help diagnose different types of soft tissue tumours, including markers for fibroblastic, adipocytic, vascular, neural, osseous and cartilaginous tumours. Specific markers and the tumours they are useful for identifying are provided. The document emphasizes that IHC should be used along with other techniques as markers sometimes show cross-reactivity.
Synovial biopsy provides tissue that can be used to better understand the pathophysiological mechanisms of arthritis through techniques like immunohistochemistry, electron microscopy, and molecular biology. It is not normally required for routine diagnosis but can help evaluate new treatment approaches. There are different types of synovial biopsies including needle biopsy, arthroscopic biopsy, and open surgical biopsy. Needle biopsy is the most common technique and samples are obtained from joints like the knee using a 14-gauge needle. Synovial biopsy helps diagnose conditions like infectious arthritis, autoimmune diseases like rheumatoid arthritis, and crystal-induced arthritides.
Bethesda system for reporting thyroid cytologyariva zhagan
The document discusses the Bethesda System for Reporting Thyroid Cytopathology (BSRTC), which provides a standardized classification system for thyroid fine needle aspiration (FNA) results. The BSRTC aims to improve communication between clinicians by establishing uniform diagnostic terminology. It categorizes FNA results as non-diagnostic, benign, atypia of undetermined significance/follicular lesion of undetermined significance, follicular neoplasm/suspicious for follicular neoplasm, suspicious for malignancy, or malignant. The document outlines the criteria for each category and risk of malignancy. It notes recent enhancements in the 2017 version of BSRTC, including recalculated risk of malignancy and the
Small round cell tumors are a group of highly aggressive cancers composed of small, undifferentiated cells. The diagnostic approach involves clinical findings, imaging, pathology, and molecular genetics testing. Key small round cell tumors in pediatric patients include Ewing sarcoma, neuroblastoma, nephroblastoma, rhabdomyosarcoma, medulloblastoma, retinoblastoma, and lymphoblastic lymphoma. Immunohistochemistry and genetic testing are used to determine the specific tumor type to help guide treatment.
this PPT is all about case base approach to kidney tumors. clinical approach and their radiological findings. indication and contra-indications of Kidney FNAC of Kidney lesions.
This presentation in mainly focused of understanding of automation and its utility in cytopathology. It will be very usefull for postgraduate in pathology, cytopathologist and cytotechnicians.
This document provides background information on microsatellite instability (MSI). It discusses how microsatellites are short repetitive sequences prone to mutations during DNA replication due to slipped strand mispairing or unequal crossing over. MSI occurs when mutations inactivate DNA mismatch repair genes, leading to length alterations in microsatellite regions. This causes microsatellite instability, which is seen in certain cancers like colorectal cancer and can be assessed through testing tumor DNA for instability in microsatellite markers. Immunohistochemistry for mismatch repair proteins or direct testing for MSI can help identify tumors associated with defective mismatch repair and microsatellite instability.
MULTIPROBE FLUORESENCE IN SITU HYBRIDISATION For Detection Of.pptxDrmustafa Ali
Urinary cytology is useful for detecting high-grade urothelial carcinoma but has low sensitivity for low-grade carcinoma. Fluorescence in situ hybridization (FISH) aids in detecting chromosomal abnormalities associated with urothelial carcinoma like gains on chromosomes 3, 7 and 17 and deletions on chromosome 9p21. FISH analysis of urine samples provides higher sensitivity and specificity than cytology alone, especially for low-grade carcinoma and carcinoma of the upper urinary tract. It can help manage cases with atypical cytology and monitor patients after BCG treatment when cytology results are difficult to interpret.
This document discusses various methods used in pathologic diagnosis of tumors, including histological examination of biopsy samples, cytological methods like exfoliative cytology and fine needle aspiration cytology, special staining techniques, immunohistochemistry, electron microscopy, tumor markers, and modern techniques like flow cytometry, in situ hybridization, and molecular diagnostic methods. The key information provided is an overview of the major diagnostic tools and techniques used in pathological analysis of tumors.
The document discusses the history, utility, and methods of preparing cell blocks from fine needle aspiration cytology samples. Cell blocks allow examination of histological structure and use of ancillary tests. Key methods include the fixed sedimentation method using a 1:1 ratio of 100% alcohol and 40% formalin, the plasma thrombin method using equal parts plasma and thrombin, and the bacterial agar method using 3% agar. Cell blocks provide increased diagnostic sensitivity and specificity compared to cytology alone through examination of tissue architecture and ability to perform special stains and molecular testing.
This document discusses cytology of the urinary tract. It describes three methods for collecting urine specimens: voided urine, catheter specimens, and bladder washings. Voided urine is the simplest but cells may degrade over time. Catheter specimens avoid contamination but can damage cells. Bladder washings provide high cellularity and preservation through saline irrigation. Specimens can be prepared via several methods including cytocentrifugation and direct smears. Normal urinary tract cytology shows a range of superficial and deep urothelial cell morphologies depending on collection method and location in the tract.
Immunohistochemistry in diagnosis of soft tissue tumours seminarPannaga Kumar
This document discusses immunohistochemistry in the diagnosis of soft tissue tumors. It begins by introducing soft tissue and the classification of soft tissue tumors. It then discusses various ancillary techniques used, focusing on immunohistochemistry. It provides details on common markers used to identify muscle, neural, melanocytic, endothelial and other types of differentiation. It discusses the applications and diagnostic utility of various markers for different tumor types. In summary, the document is a comprehensive overview of immunohistochemistry techniques and markers useful in the diagnosis and classification of soft tissue tumors.
The document summarizes the International Academy of Cytology (IAC) System for classifying breast malignancy based on fine-needle aspiration cytology (FNAC) results. The system was developed at a meeting in Yokohama in 2016. It aims to standardize breast cytology reporting to improve diagnosis and patient management. The system categorizes FNAC results as insufficient, benign, atypical, suspicious of malignancy, or malignant, with associated risks of malignancy. Cytological features and management recommendations are provided for each category. The goal is to link cytology reports to optimal breast care.
This document provides guidance on grossing (examining) different types of breast specimens in pathology. It describes how to properly collect, orient, measure, sample and submit lumpectomy, mastectomy, microdochectomy and lymph node specimens. Key steps include fixation in formalin, inking, slicing, measuring margins, evaluating tumors and lymph nodes, and submitting representative sections for histology. The goal is to thoroughly examine specimens and obtain high quality samples for accurate diagnosis.
The document provides guidance on grossing Whipple's specimens. It discusses that a Whipple's specimen contains the pancreatic head, duodenum, gallbladder and portions of the stomach and small intestine. Key steps in grossing include inking surfaces, slicing the pancreatic head, examining the ampulla and margins, and submitting sections of the tumor, ducts, margins and lymph nodes for histology. Performing a thorough gross examination is important for accurately assessing resection status and staging the tumor.
Cytologic assessment of bronchopulmonary lesionsAseem Jain
This document provides an overview of cytologic assessment of bronchopulmonary lesions. It discusses the normal histology of the respiratory system and various cytologic sampling techniques used to evaluate the lungs, such as sputum samples, bronchial brushings, washings and lavage. The cytology of normal respiratory cells and endogenous material is described. A variety of benign pulmonary conditions and infectious processes are outlined. Specific lung diseases like tuberculosis, sarcoidosis and nocardiosis are discussed through their characteristic cytologic findings.
The document discusses several pediatric neoplasms that appear as small round blue cell tumors due to their primitive histological features. These include neuroblastoma, Wilms tumor, rhabdomyosarcoma, Ewing's sarcoma, medulloblastoma, retinoblastoma, and lymphoma. For each tumor, the document outlines characteristics such as common age of diagnosis, clinical features, histopathological appearance under the microscope, immunohistochemistry profiles, genetics where relevant, and important prognostic factors. Differential diagnosis of these small round blue cell tumors in children is provided for accurate diagnosis and treatment.
This document summarizes urine cytology and various urinary markers for detecting bladder cancer. Urine cytology is the gold standard but has low sensitivity of 40-62%. Newer urinary markers like BTA, ImmunoCyt, NMP-22, and UroVysion have higher sensitivities than cytology of 50-86%, but lower specificities and can be affected by benign conditions. No single marker currently has a sensitivity of 90%, which patients indicate is needed to replace cystoscopy. Therefore, the best approach is a combination of cystoscopy and urinary markers for non-muscle-invasive bladder cancer surveillance.
The document discusses effusion cytology. It begins by describing the anatomy of serous cavities and membranes that line them, producing serous fluid. Any excess fluid is an effusion, indicating a pathological process. Effusions can be classified as hydrostatic, infectious, inflammatory, or malignant. Samples are collected and prepared as smears for staining.
Normal components in effusions include mesothelial cells, histiocytes, lymphocytes, and other inflammatory cells. Reactive mesothelial cells can appear atypical but maintain a uniform appearance. Malignant effusions result from direct extension or metastasis of cancers. Identifying malignant cells involves comparing size, shape and number to determine the primary tumor type and origin. The most
This document discusses liquid biopsy, a non-invasive technique to detect tumor biomarkers shed into body fluids like blood. It can identify circulating tumor cells, circulating tumor DNA, exosomes, and newly, tumor educated platelets. Liquid biopsy offers advantages over tissue biopsy like being less invasive, allowing repeated sampling over time. While sensitivity remains a limitation, liquid biopsy shows promise for early cancer detection, monitoring treatment response and tumor evolution, and assessing tumor heterogeneity. The document reviews technologies to analyze various biomarkers and potential clinical applications of liquid biopsy.
This document discusses the role of immunohistochemistry (IHC) in diagnosing soft tissue tumours. It begins by defining soft tissue and the WHO classification of soft tissue tumours. IHC is an important ancillary technique that can be used to identify discrete tissue components using antigen-antibody binding. The document outlines the IHC protocol and discusses various markers that can help diagnose different types of soft tissue tumours, including markers for fibroblastic, adipocytic, vascular, neural, osseous and cartilaginous tumours. Specific markers and the tumours they are useful for identifying are provided. The document emphasizes that IHC should be used along with other techniques as markers sometimes show cross-reactivity.
Synovial biopsy provides tissue that can be used to better understand the pathophysiological mechanisms of arthritis through techniques like immunohistochemistry, electron microscopy, and molecular biology. It is not normally required for routine diagnosis but can help evaluate new treatment approaches. There are different types of synovial biopsies including needle biopsy, arthroscopic biopsy, and open surgical biopsy. Needle biopsy is the most common technique and samples are obtained from joints like the knee using a 14-gauge needle. Synovial biopsy helps diagnose conditions like infectious arthritis, autoimmune diseases like rheumatoid arthritis, and crystal-induced arthritides.
Bethesda system for reporting thyroid cytologyariva zhagan
The document discusses the Bethesda System for Reporting Thyroid Cytopathology (BSRTC), which provides a standardized classification system for thyroid fine needle aspiration (FNA) results. The BSRTC aims to improve communication between clinicians by establishing uniform diagnostic terminology. It categorizes FNA results as non-diagnostic, benign, atypia of undetermined significance/follicular lesion of undetermined significance, follicular neoplasm/suspicious for follicular neoplasm, suspicious for malignancy, or malignant. The document outlines the criteria for each category and risk of malignancy. It notes recent enhancements in the 2017 version of BSRTC, including recalculated risk of malignancy and the
Small round cell tumors are a group of highly aggressive cancers composed of small, undifferentiated cells. The diagnostic approach involves clinical findings, imaging, pathology, and molecular genetics testing. Key small round cell tumors in pediatric patients include Ewing sarcoma, neuroblastoma, nephroblastoma, rhabdomyosarcoma, medulloblastoma, retinoblastoma, and lymphoblastic lymphoma. Immunohistochemistry and genetic testing are used to determine the specific tumor type to help guide treatment.
this PPT is all about case base approach to kidney tumors. clinical approach and their radiological findings. indication and contra-indications of Kidney FNAC of Kidney lesions.
This presentation in mainly focused of understanding of automation and its utility in cytopathology. It will be very usefull for postgraduate in pathology, cytopathologist and cytotechnicians.
This document provides background information on microsatellite instability (MSI). It discusses how microsatellites are short repetitive sequences prone to mutations during DNA replication due to slipped strand mispairing or unequal crossing over. MSI occurs when mutations inactivate DNA mismatch repair genes, leading to length alterations in microsatellite regions. This causes microsatellite instability, which is seen in certain cancers like colorectal cancer and can be assessed through testing tumor DNA for instability in microsatellite markers. Immunohistochemistry for mismatch repair proteins or direct testing for MSI can help identify tumors associated with defective mismatch repair and microsatellite instability.
MULTIPROBE FLUORESENCE IN SITU HYBRIDISATION For Detection Of.pptxDrmustafa Ali
Urinary cytology is useful for detecting high-grade urothelial carcinoma but has low sensitivity for low-grade carcinoma. Fluorescence in situ hybridization (FISH) aids in detecting chromosomal abnormalities associated with urothelial carcinoma like gains on chromosomes 3, 7 and 17 and deletions on chromosome 9p21. FISH analysis of urine samples provides higher sensitivity and specificity than cytology alone, especially for low-grade carcinoma and carcinoma of the upper urinary tract. It can help manage cases with atypical cytology and monitor patients after BCG treatment when cytology results are difficult to interpret.
This document discusses various methods used in pathologic diagnosis of tumors, including histological examination of biopsy samples, cytological methods like exfoliative cytology and fine needle aspiration cytology, special staining techniques, immunohistochemistry, electron microscopy, tumor markers, and modern techniques like flow cytometry, in situ hybridization, and molecular diagnostic methods. The key information provided is an overview of the major diagnostic tools and techniques used in pathological analysis of tumors.
The Evolution of In Situ Genetic Technologyasclepiuspdfs
In situ genetic technology was historically developed and mainly focused on detection purpose, allowing specific nucleic acid sequences to be visualized in morphologically preserved tissue sections. With the synergy of genetics and immunohistochemistry, in situ detection can correlate microscopic topological information with gene activity at the transcriptional or post-transcriptional levels in specific tissues. Furthermore, its resolution allows spatial distribution of nucleic acid products to be revealed in a heterogeneous cell population. The newest member to the franchise of in situ genetic technology is a direct-on-specimen enrichment methodology specifically for cell-free DNA liquid biopsy. Contrary to in situ detection, this in-well in situ innovation tackles the very first sample preparation step to reduce material loss, thereby improving overall sensitivity. Genomic nucleic acids purified from specimens have been proven to be time consuming and suffered from damages and losses; the evolution of in situ genetic technology offers a powerful tool for precision functional genomics, enabling cross-check between in vitro and in vivo findings. It further opens the door to ultimate genetic engineering in situ.
This document discusses lung cancer diagnoses made using minimally invasive techniques on small samples obtained non-surgically. It outlines various procedures like fine needle aspiration that can acquire adequate tissue for molecular testing to guide targeted therapy. Key points include:
- Molecular drivers like ALK, ROS1, RET, and EGFR mutations are increasingly detected to select effective targeted therapies with fewer side effects than chemotherapy.
- Immunohistochemistry, fluorescence in situ hybridization, and next generation sequencing can identify these mutations using small samples from minimally invasive procedures.
- A single fine needle aspiration can provide a diagnosis, tumor subtype, and theranostic molecular information to personalize treatment.
Karyotyping is the process by which photographs of chromosomes are taken in order to determine the chromosome complement of an individual, including the number of chromosomes and any abnormalities.
The term is also used for the complete set of chromosomes in a species or in an individual organism and for a test that detects this complement or measures the number.
This document provides an overview of flow cytometry, including its history, components, principles, and applications. Flow cytometry involves passing cells in suspension through a laser beam to measure physical properties like size and granularity, as well as cell markers detected by fluorescent antibodies. This allows identification of cell types, lineages, and abnormalities. The document discusses sample preparation, common specimens analyzed, immunophenotyping using multiple fluorochromes, and applications like DNA content analysis, erythrocyte analysis, and reticulocyte counting.
This document discusses various cytogenetic techniques used to study chromosomes, including their structure and abnormalities. It describes karyotyping to analyze all chromosomes for changes, as well as techniques like fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), spectral karyotyping (SKY), and multicolor FISH (M-FISH) that use fluorescent probes to detect DNA sequences on chromosomes. It also discusses banding techniques like G-banding and Q-banding that identify chromosomes based on dark and light bands corresponding to GC-rich and AT-rich regions. These techniques are used to diagnose chromosomal abnormalities and further cytogenetic research.
fish- Fluorescence in situ hybridization gaurav raja
FISH is a technique that uses fluorescent probes that bind to complementary DNA sequences on chromosomes to detect chromosomal abnormalities. It allows detection of structural changes like deletions and duplications not visible under a microscope. FISH gained recognition supporting genome mapping efforts. It involves labeling probes, denaturing probe and target DNA, and allowing hybridization. Results are then viewed under a fluorescence microscope. FISH has applications in detecting abnormalities, identifying marker chromosomes, and monitoring therapy. It provides advantages over traditional cytogenetics like rapid analysis of non-dividing cells.
This document provides an overview of Papanicolaou testing and cervicovaginal smear cytology. It discusses the history and development of the Pap test, including the contributions of Dr. Papanicolaou. It also describes normal cervical cytology findings, abnormalities that may be seen, the Bethesda System for reporting, and advantages of liquid-based cytology over conventional smears. The goal of the document is to provide lectures on Pap testing, cytology methodology, interpretation, and screening.
Flow cytometry can be used for a variety of applications including medical research, diagnostics, and basic science. It allows for precise quantification of multiple antigens on individual cells through fluorescent labeling and detection. Key uses of flow cytometry include cell counting, sorting, analysis of characteristics and function, detection of microorganisms, biomarker analysis, and protein engineering detection. It is a routine technique in research, clinical practice, and clinical trials.
This study used SNP microarray analysis to reanalyze 50 blastocysts that had previously been diagnosed as aneuploid by FISH at the cleavage stage. 58% of blastocysts were found to be euploid in all sections analyzed despite an aneuploid FISH result. Aneuploid blastocysts showed no evidence of preferential segregation of abnormalities to the trophectoderm. Additionally, mechanisms of self-correction like chromosome extrusion or duplication were not observed. These findings support the conclusion that cleavage-stage FISH has poor predictive value for aneuploidy in morphologically normal blastocysts.
This document discusses cell blocks, liquid based cytology, and the Bethesda system for reporting cervical cytology. It defines cell blocks and describes their advantages like allowing histological examination and ancillary tests. It outlines the advantages and disadvantages of liquid based cytology compared to conventional pap smears. Finally, it explains the Bethesda system for standardized reporting of cervical cytology results, including categories for negative, epithelial abnormalities, organisms, and other non-neoplastic findings.
MOLECULAR AND CYTOGENETIC ANALYSIS -BMLS GENERAL &HBT-1.pptxAmosiRichard
Molecular and cytogenetic analysis are essential techniques for diagnosing and managing hematological disorders. Key methods include DNA extraction, PCR, FISH, and next generation sequencing. Clinical applications involve investigating diseases like sickle cell anemia, thalassemias, leukemias, lymphomas, and coagulation disorders. Molecular analysis allows identification of genetic mutations and translocations that underlie these conditions and guides treatment decisions. While providing critical diagnostic information, these techniques also have limitations like risk of infection and interference from therapies.
Dr Dinah Parums. The Role of the Pathologist in Targeted Therapy and Personal...Dinah Parums
This document discusses the role of pathologists in targeted cancer therapy and personalized medicine. It outlines the key components of a pathology capability including tissue acquisition, processing, staining, and molecular analysis techniques like immunohistochemistry and in situ hybridization. The document emphasizes how pathologists can help validate drug targets and biomarkers through localization of proteins and nucleic acids in tissues. It also discusses challenges like antibody characterization and validation, and the importance of standardizing immunohistochemistry methods for clinical trials and companion diagnostics. Overall, the document illustrates how pathology informs targeted cancer treatment by identifying biomarkers that can predict a patient's response to specific therapies.
This document summarizes a case study of a 41-year old female with adenocarcinoma of the lung. Biopsy of a lung lesion showed well-differentiated adenocarcinoma. Immunohistochemistry, molecular testing, and fluorescence in situ hybridization were performed. Molecular testing found no mutations in EGFR, KRAS, or BRAF genes. FISH analysis detected an ALK gene rearrangement in 55.5% of cells tested. The patient was referred for molecular profiling to determine potential targeted therapies.
This document discusses immunohistochemistry (IHC), which combines immunology, histology, and chemistry to identify tissue antigens using antibodies. IHC allows identification of cell types and origins. The document outlines the principles and history of IHC, the IHC process, common markers used to identify tissues like epithelial, mesenchymal and hematopoietic tissues. Examples of IHC use in identifying lung cancer subtypes, gastrointestinal cancers, breast cancer subtypes, and lymphoma subtypes are provided. In summary, the document provides a comprehensive overview of the field of immunohistochemistry.
Pap Smear: A Bird's Eye View from a CytopathologistDr. Shubhi Saxena
The document provides information about Pap smear tests, including:
1. A Pap smear is a screening test to detect precancerous and cancerous lesions of the cervix in order to treat them and reduce cervical cancer rates. New guidelines recommend screening starting at age 21 or within 3 years of becoming sexually active.
2. Screening intervals are yearly until age 30, then every 3 years. Screening can end after age 70, or after hysterectomy with 3 normal previous Pap smears.
3. Limitations include low sensitivity and potential for false negatives due to sampling, fixation, or interpretation errors. Proper sampling of the transformation zone is important for detection of early cancers.
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This document provides guidelines for laboratory diagnosis of Chlamydia trachomatis infections in East European countries. It discusses sample collection and transport, diagnostic methods including culture, antigen detection, nucleic acid amplification tests, and considerations for implementing and interpreting these tests. Commercial nucleic acid amplification tests are highlighted but caution is given around using non-approved tests.
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O estabelecimento da amamentação e o manejo das principais intercorrências é contemplada.
Recomendamos muito.
Vamos discutir essas recomendações no nosso curso de pós-graduação em Aleitamento no Instituto Ciclos.
Esta publicação só está disponível em inglês até o momento.
Prof. Marcus Renato de Carvalho
www.agostodourado.com
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Overall life span (LS) was 1671.7±1721.6 days and cumulative 5YS reached 62.4%, 10 years – 50.4%, 20 years – 44.6%. 94 LCP lived more than 5 years without cancer (LS=2958.6±1723.6 days), 22 – more than 10 years (LS=5571±1841.8 days). 67 LCP died because of LC (LS=471.9±344 days). AT significantly improved 5YS (68% vs. 53.7%) (P=0.028 by log-rank test). Cox modeling displayed that 5YS of LCP significantly depended on: N0-N12, T3-4, blood cell circuit, cell ratio factors (ratio between cancer cells-CC and blood cells subpopulations), LC cell dynamics, recalcification time, heparin tolerance, prothrombin index, protein, AT, procedure type (P=0.000-0.031). Neural networks, genetic algorithm selection and bootstrap simulation revealed relationships between 5YS and N0-12 (rank=1), thrombocytes/CC (rank=2), segmented neutrophils/CC (3), eosinophils/CC (4), erythrocytes/CC (5), healthy cells/CC (6), lymphocytes/CC (7), stick neutrophils/CC (8), leucocytes/CC (9), monocytes/CC (10). Correct prediction of 5YS was 100% by neural networks computing (error=0.000; area under ROC curve=1.0).
3. Molecular cytopathology is defined as the application of
molecular studies to any type of cytology specimen, whether
gynecological, exfoliative, or fine-needle aspiration cytology.
• Molecular genetic analyses have been increasingly performed on
cytological specimens to facilitate management of cancer
patients.
• Advances in minimally invasive interventional procedures have
resulted in an increasing reliance on small biopsies along with
exfoliative and fine-needle aspiration (FNA) cytology specimens
not only for diagnostic purposes
• but also for ancillary molecular testing to guide the increasingly
“personalized” management of patients with cancer.
4. Recognition of tumor heterogeneity at the molecular level
are in increasing demand for disease like thyroid neoplasms,
lung CAs, Urothelial carcinoma etc.
Rapid development of new high-throughput & multiplex
molecular testing modalities holds the promise to address
the issue of simultaneously testing for a multitude of
different genomic abnormalities using cytology samples.
5. Technical Feasibility
Cytologic specimen contains less amount of target
cells
However, cytological specimens, especially those
obtained through fine needle aspiration, are often
more suitable for molecular assays due to the high
quality nucleic acids by non-formalin fixation and
less fragmented genome.
6. • Considering clinical utility as initial step, following
factors should be considered before conducting
validation testing of a molecular assay.
1. Types of genetic alteration, such as amplification,
mutation, indels, and gene fusion
2. Clinical sensitivity and specificity
3. Accuracy, precision and detection of low limit
4. simplicity, associated with shorter turn-around time
and lower cost
5. Availability of tissue type, such as all smears , cell
blocks or fresh cytological specimen (Either FNA or
Exfoliative)
6. Clinical volume and cost effective issue.
10. Commonly used molecular assays
Florescence in situ hybridization (FISH)
NucleicAcid Hybridization
Polymer chain reaction (PCR),
Reverse transcriptional PCR (RT-PCR)
Next generation sequencing (NGS)
DNA/RNA microarray
Sequenom’s MassARRAY system
Conventional (Sanger) DNA sequencing
11. • Fluorescence in situ hybridization (FISH) is a technique that is well
suited for detecting many types of genomic abnormalities in
cytology specimens.
• FISH involves hybridization of fluorescently labeled nucleic acid
sequences (probes) to complementary nucleic acid sequences
(targets) which are typically in the form of metaphase
chromosomes obtained from cultured, dividing cells or
decondensed chromosomes within interphase nuclei of
nondividing cells.
• FISH allows visualization of the physical location of the probe(s)
to their target(s) and can be used to interrogate specific areas of
the genome.
12. a) Enumeration Probe
– used to detect genomic gains and losses.
– Centromere probes,which are composed of alpha satellite DNA
used for detecting whole-chromosome aneuploidies
– locus−/gene-specific (unique sequence) probes are
commonly used used to detect loss or gain/amplification
of a region of interest.
b) Break-apart probes
– are optimal for assessing rearrangements of a gene that
could have more than one translocation partner, such as
MLL (KMT2A)
c) Fusion probes
are most frequently employed when fusions involve
consistent gene partners, such as BCR/ABL1
13. (a) Enumeration probe set consisting of a locus-specific probe (red) and a
centromere-specific probe (green) on the same chromosome.
14. (b) Dual fusion probe strategy consisting of two differentially
labeled, locus-specific probes that span the genes involved in a
fusion event.
(c) Break-apart probe strategy consisting of two differentially
labeled probes that flank the breakpoint (dashed lines) of one
of the gene partners involved in a fusion.
15. A wide range of cytologic preparations can be used for
FISH,
•touch preparations,
•unstained cytological smears,
•archival stained cytology slides,
•Cytospin preparations,
•liquid-based cytological preparations,
•formalin or alcohol-fixed paraffin-embedded cell
blocks
16. Preparation type Advantages Disadvantages
FFPE cell
block
1. Allows for
correlation with
adjacent H&E- or
IHC-stained section
2. Preservation of
morphology and
partial tissue
architecture
3. Cell blocks made
from aspirates may
be enriched for
tumor cells
1. Probe signal loss due
to nuclear truncation
from sectioning
2. Overlapping nuclei
can cause difficulty
scoring signals
3. May have
autofluorescent
background that
obscures signals
4. May have
inadequate tumor
cellularity
17. Preparation type Advantages Disadvantages
Cytology
smear
1. No nuclear
truncation artifact
2. Previously stained
slides can be used
3. Only technique
that allows rapid
on-site
determination of
specimen
adequacy at time
of procedure
4. Smears made from
aspirates may be
enriched in tumor
cells
1. Nuclear distortion
due to crushing
during slide
preparation
2. Overlapping nuclei
can cause difficulty
scoring signals
3. May have
autofluorescent
background that
obscures signals
18. Preparation type Advantages Disadvantages
Liquid-based
cytology
1. No nuclear
truncation artifact
2. Thin, monolayer
preparation
minimizing nuclear
overlap
3. Less background
than other
preparations
4. Less hands-on time
than many other
preparations
5. Cells are
concentrated in a
smaller area
1. More expensive than
other cytologic
preparations
2. Special
instrumentation
needed for slide
preparation
3. May have
inadequate
cellularity
19. Preparation type Advantages Disadvantages
Liquid-based
cytology
1. No nuclear
truncation artifact
2. Thin, monolayer
preparation
minimizing nuclear
overlap
3. Less background
than other
preparations
4. Less hands-on time
than many other
preparations
5. Cells are
concentrated in a
smaller area
1. More expensive than
other cytologic
preparations
2. Special
instrumentation
needed for slide
preparation
3. May have
inadequate
cellularity
23. • Traditionally UCC is detected and monitored
by the combination of cystoscopy and urine
cytology tests
• Cystoscopy is an expensive and invasive
procedure and often may miss a flat lesion,
whereas urinary cytology, though noninvasive
has very low sensitivity (between 20 and 50
%) for low-grade papillary tumors.
• Adjunct molecular markers with high accuracy
for the detection of both low and high grades
of urothelial carcinoma is necessary.
24. • Two commercially available cellular-based
tests –
– more widely used UroVysion or fluorescence in
situ hybridization (FISH) test and
– ImmunoCyt or uCyt test
25. • Cytogenetically, urothelial cell carcinoma,
especially high grade UCC is an aneuploid
cancer, and contains multiple copies of
chromosomes
• Centromere enumeration probes for
chromosome 3, 7, and 17 label the
centromere of each respective chromosome.
• Presence of more than two signals within a
cell would indicate an abnormal DNA content
and increase the suspicion for malignancy.
26. DAPI stain (4′6-diamidino-2-phenylindole) is used to
stain the nucleus blue under fluorescence microscopy
Benign urothelial
cells will show a
homogeneous
staining pattern,
Malignant cells
show large nuclei
and a clumped,
heterogeneous
chromatin pattern
(reflects an aneuploid cell with dark, coarse
chromatin distribution and nuclear irregularity)
27. • Centromere enumeration probes (CEP)
directed toward the chromocenter of
chromosomes 3 (red), 7 (green), and 17
(aqua) reflect the number of copies of
chromosomes.
• Screening for large (presence of 25 urothelial cells
may be accepted in most cases) abnormal cells on
DAPI, then examining them with each filter
• When one cell with an abnormal number of
probes is detected, a search for at least 4 or
more abnormal cells should be performed
28. Once four cells are found, the
case may be signed out as “Positive
for aneusomy”.
These patients are at increased
risk for cancer, even when the
cytology is negative.
An abnormal cell shows more
than 2 signals in 2 or more
probes.
True 9p21 (gold) loss occurs in
clusters of urothelial cells which
may represent low grade papillary
lesions of the bladder.
30. The use of cytological smears for FISH testing as an
advantage over the use of FFPE section in that
tumor cells on smears are mostly monolayered,
It facilitates enumerating all the HER2 signals
within an entire nucleus without a truncating
artifact.
When collecting a cytology specimen that is highly
suspicious for breast cancer, collecting a sample in
formalin or saving air-dried direct smears for HER2 ISH
testing should be considered.
31. HER2 FISH can be
performed by either
a single-probe assay
or dual-probe assay.
HER2 is considered
amplified when the
assay shows ≥ 6.0
HER2 signals/cell
using a single-probe
system.
By dual-probe testing, it is positive if there is a
HER2/CEP17 ratio of ≥ 2.0 with an average HER2 copy
number of ≥ 4.0
33. • As with any technology, FISH has its limitations,
and there are many examples of cases in which
FISH results are negative
• False-negative FISH results can be due to a variety
of variables, such as failure to score tumor nuclei
or atypical rearrangements.
• Complex rearrangements, or submicroscopic
insertions of part of a gene into another gene,
have been documented to result in fusions that
yield false-negative FISH results.
• Aberrant molecular results can also be
encountered even if known fusion partners with
unusual breakpoints are involved
35. The traditional PCR assay involves 3 main steps –
1. DNA denaturation,
2. annealing, and
3. extension,
with the use of primers (sequence of nucleotides complementary
to the target DNA)
A forward and reverse primer flank the designated area
containing the desired sequence of DNA to be amplified
Multiple copies (amplicons) of the target DNA (known sequence to
verify the presence of mutations) are generated.
The amplified DNA can then be sequenced for the verification of
any mutations.
36. Cytology samples provide high-quality DNA, sufficient for a wide
array of DNA-based sequencing assays, including next-generation
sequencing (NGS)
This novel high-throughput technology represents an evolution
of conventional DNA sequencing methodologies, such as Sanger
sequencing and pyrosequencing
Sanger sequencing has long been the gold standard for the
identification of point mutations, deletions, and small insertions.
But with the advancement of NGS it is now becoming obsolete
nowadays.
37. Direct sequencing is a simple technique that uses chemically
modifi ed nucleotides labeled with distinct fluorescent dyes
a chemically modified nucleotide (dideoxynucleotide)
terminates the extension of the DNA strand at the point
of incorporation.
This results in a mixture of DNA fragments of varying
lengths. Each dideoxynucleotide, (A, T, C, or G) is labeled
with a different fluorescent dye (dye terminator).
The newly synthesized and labeled DNA fragments are
sequentially separated by size through capillary gel
electrophoresis.
38. The fluorescence is detected by an automated sequence
analyzer, and the order of nucleotides (base calling) in the
target DNA is visualized as a sequence electropherogram
In case a mutation is present, two different overlapping
peaks will be seen (from the wild-type and mutant cells).
Similarly, a deletion will be seen as a “truncation” of the
peak signals.
39. Sequencing
methods
Sanger
sequencing
NGS
Yield (MB/Run) 0.06 MB 600GB–1.8TB
Cost ($$$/MB) $1500 $0.04–$0.007
Speed (per human
genome)
13 years 2–3 days
Amount of DNA
required
500–5000 ng 10–1000 ng
Sequencing sensitivity >20 % mutation rate >1 % mutation rate
Multiplexing capability Single Multiple
40. • Data suggest that NGS can be reliably applied on cytology specimens
with high sensitivity, specificity and reproducibility
• Some of the NGS systems are –
1. Roche 454 – first commercially successful next generation
system; uses pyrosequencing technology.
2. AB SOLiD (Sequencing by Oligo Ligation Detection) System –
adopts the technology of two-base sequencing based on ligation
sequencing.
3. Illumina GA/HiSeq System –adopts the technology of
sequencing by synthesis (SBS). HiSeq 2000 uses two lasers and
four filters to detect four types of nucleotide (A,T, G, and C)
4. Compact PGM (Personal Genome Machine)
Sequencers – uses semiconductor sequencing technology.
PGM is the first commercial sequencing machine that
does not require fluorescence and camera scanning, resulting in
higher speed, lower cost, and smaller instrument size
41. • PCR based assays are the methods of choice for procurement
of mutations in
– EGFR in lung cancer
– BRAF in melanomas and papillary thyroid carcinomas
– KRAS in colon cancer.
• All sorts of cytological samples, including fresh cell
suspensions, smears (stained and unstained), cytospins and
FFPE cell blocks can be used to extract DNA for PCR based
assays.
42. Potential Applications of NGS in Cytology
• Recent improvements of FNA procedures and technological
advancement in making DNA library with the small amount of
DNA have made NGS technology applicable to cytology
specimens in clinical setting
• In Lung Carcinoma :
– The majority of NSCLC are diagnosed at an advanced stage, and missed
the best surgery time.
– Therefore, the diagnosis and therapeutic decision for lung cancer
heavily rely on minimally invasive procedures, like cytology samples
43. • Thyroid
– In approximately 25 % of thyroid nodules, the diagnosis
cannot be established and consequently classified as
indeterminate (Bethesda Cat III) by FNA cytology, hampering
clinical management of these patients
– Because some molecular markers (BRAF, NRAS, KRAS, and
PTEN) are highly specific in thyroid cancer, NGS offers the
potential to improve the accuracy of cancer diagnosis and
prognosis in thyroid nodules.
• Pancreatic Cancer
– The combination of cytological evaluation and tumor marker
mutation analysis (oncogene KRAS and tumor suppressor genes
CDKN2A/p16, SAMD4, and TP53), especially for inconclusive
cases, can potentially enhance the diagnostic power.
44. Various alternatives to classic PCR followed by direct sequencing have been
used for cytological samples
1. Real time-PCR –
uses oligonucleotide primers that bind specifically to flank regions
of the most common mutations.
2. High-resolution melting analysis (HRMA) –
A rapid and cost-effective method that relies on the combination of
real time PCR and evaluation of DNA melting curves
3. Restriction fragment length analysis(RFLP)
uses mutation-specific restriction endonucleases, and amplification is
only possible in the mutated sites.
4. MASS-Array spectrometry
uses multiplex mutation analysis with preset commercial panels, such
as the Oncocarta Panel, have been successfully used in FNA
specimens, with reliable results.
48. Recommendations for using HPV testing for
cervical cancer prevention
Age-based recommendation
<20 or >65 years of age: no screening
21–29 years of age: cytology alone every 3 years with an option of
reflex HPV testing for women withASCUS
30–65 years of age: HPV co-screening along with Pap test every
5 years
• In conjunction with Pap cytology
– ASCUS (21 years and older)
– Pap/HPV+ co-testing results: HPV16/18 genotyping
– LSIL in postmenopausal women
– Post-colposcopy management of women withAGC orASC-H
– Post-colposcopy management of women 21 years or older with
– ASCUS or LSIL
– Post treatment surveillance
49. Primary HPV screening
25–65 years of age: primary HPV screening every 5 years, if
tested negative
HPV16/18 genotyping test, colposcopy referral if positive
Non-HPV16/18 positive (12 hrHPV genotypes)
Pap cytology triage
50. • To date, a total of seven commercially available HPV testing
assays under five commercial brand names were approved by
US FDA for cervical cancer screening –
1. Hybrid Capture 2 (HC2, Qiagen,Valencia, CA)
2. Cervista HPV HR and Cervista HPV16/18
3. Cobas HPV assay
4. Aptima HPV andAptima HPV16 18/45 assays
5. BD Onclarity HPV assay
51. All the assays use liquid based preparations
To date, the FDA approved most of HPV testing assays
specifically in ThinPrep Pap cytology
In 2016 and 2018, FDA approved Cobas HPV assay and BD
Onclarity HPV assay for SurePath Pap specimen, respectively.
Although bothThinPrep and SurePath Pap tests are FDA-
approved for cervical cancer screening, SurePath Pap cytology
showed a significantly lower unsatisfactory rate thanThinPrep
for Pap cytology testing.
52. Hybrid
Capture 2
Cervista
HPV
Aptima
HPV
Cobas
HPV
BD
Onclarity
HPV
PCR-based No No Yes Yes Yes
Amplification Signal Signal E6, E7
RNA
E6, E7
DNA
E6, E7
DNA
HPV
detection
13 types 14 types 14 types 14 types 14 types
HPV
genotyping
No HPV16,
18
HPV16, 18,
45
HPV16, 18 HPV16,18,
45
Internal
controls
No Yes Yes Yes Yes
Equivocal
Zone
Yes No No No No
Company Qiagen Hologic Hologic Roche Becton,
Dickinson
53. What Is the Role of Molecular
Testing?
Lung Cancer
54. • Lung cancer is the leading cause of cancer-related
death worldwide.
• diagnosis and therapeutic decision for lung cancer
heavily rely on minimally invasive procedures, either
small biopsies or cytology samples.
• Lung cancer has the most available targeted therapies.
• The targeted genes include EGFR, BRAF, KRAS, ALK, and
ROS1 . Many more potential targets, such as PIK3CA,
FGFR1, and DDR2 , are in clinical trials. Therefore, the
number of predictive biomarkers for novel targeted
drugs entering into clinical practice is expected to
rapidly increase.
55. Types of Specimens
FNA
In past 5 years, EBUS-TBNA has become the most popular
approach for clinical staging and obtaining cytological materials
for diagnosis and molecular testing
Recent studies have demonstrated that cell block preparations
from FNA and other specimens, such as BAL and pleural
effusions, are superb for molecular testing.
Transthoracic FNA
EBUS-TBNA
•BAL
•Sputum
•Pleural Effusion
•Exhaled breath
condensate (EBC)
56. • Major Concerns Extracting nuclear material
– Contamination from
• blood cells,
• Inflammatory cells,
• plasma
• stromal cells
• This problem can be overcome by
– Isolating tumor cells by laser capture microdissection
(LCM)
– Microdissection of tumor tissue using microscope and
analyses potential biomarkers from selective areas of the
tumor
57. A NIH pathology group showed that as a few as 50 tumor
cells microdissected from cell block can be used for the
detection of EGFR and K-RAS mutational testings.
Recent studies/data from the Cleveland Clinic Foundation
have demonstrated that leftover cytolyte from the Thinprep
preparation maybe an alternative choice for both
conventional and next-generation sequencing based
mutational assays.
In a study Ion PGM sequencing technology was applied for
targeted gene mutations analysis for 38 lung adenocarcinomas
cytology specimens
– Of the 38 specimens, 36 cases were successfully sequenced (95
%). 24/36 cases identified at least one mutations
60. Thyroid cancer typically occurs in thyroid nodules. FNA
followed by cytological examination is an accurate and cost
effective diagnostic method for evaluating thyroid nodules.
This commonly used approach allows detecting cancer or
establishing a diagnosis of a benign nodule in most cases.
However, in approximately 25 % of nodules, the diagnosis
cannot be established and classified as Cat III/IV/V by FNA
cytology, hampering clinical management of these patients.
Because some molecular markers are highly specific in
thyroid cancer, NGS offers the potential to improve the
accuracy of cancer diagnosis & prognosis in thyroid nodules.
61. What Is the Role of Molecular
Testing inThyroid Cytology?
62.
63. • Ancillary molecular testing has emerged as a promising tool to
improve risk stratification among thyroid nodules placed in these
low-risk indeterminate categories ofThe Bethesda System for
ReportingThyroid Cytopathology (TBSRTC)
• Molecular testing has dual aims in this context
(1) to identify biologically benign nodules that can be
followed clinically rather than surgically and
(2), for nodules that warrant resection, to help guide the
extent of initial surgery (lobectomy versus total
thyroidectomy)
64. DNA, microRNA, mRNA, and proteins have all been
investigated as analytes for ancillary testing on thyroid cytology
specimens
The four molecular tests that are currently offered by
commercial laboratories for cytologically indeterminate thyroid
FNAs are all nucleic acid-based tests –
Afirma Gene Expression Classifier
RosettaGX Reveal
ThyGenX/ThyraMIR
ThyroSeq
65. Afirma
GEC
RosettaGX
Reveal
ThyGenX/
ThyraMIR
ThyroSeq
Testing
approach
Expression
profiles of
142mRNAs by
DNA
microarray
Expression
profiles
of 24
microRNAs by
qRT-PCR
ThyGenX:
Hotspot
mutations in 5
genes and 3
gene fusions by
targeted NGS
ThyraMIR:
Expression
profiles of 10
microRNAs by
qRT-PCR
Hotspot
mutations in
14 genes and
42 gene fusions
by targeted
NGS
Substrate for
molecular
testing
Fresh cells
collected into
nucleic acid
preservative
Fixed cells on
routine
cytology slides
(direct smear
or LBC)
Fresh cells
collected
into nucleic
acid
preservative
Fresh cells
collected into
nucleic acid
preservative
67. BCR-ABL trans. for CML , germline mut in the BRCA1/2 for HBOC.
Rule out tests are much less common (CEA, CA-125, PSA, HPVDNA)
In thyroid mutation of BRAF ,RAS and rearrangement of RET/PTC
and PAX8/PPAR-γ are most common.
Analysis of those abnormatlities are considered as rule-in tests for
thyroid.
NGS likeThyGenX,ThyroSeq are used for this.
BRAFV600E mutation and RET-PTC1/3 gene fusions are associated with
near-100% risk for PTC.
In contrast, RAS mut & PAX8-PPARG fusions have been identified in a broad
spectrum of benign, premalignant, and malignant neoplasms (FA, FC,
NIFTP, encapsulated Fptc ) – may be best considered markers of
neoplasia rather than malignancy per se.
68. • An alternative to the rule in approach of mutation panels for the
evaluation is rule out approach of mRNA expression analysis
exemplified by theAfirma Gene Expression Classifier (GEC).
• Unlike the mutation panels, the GEC utilizes an approach that is
designed to look for the presence of benign mRNA expression
patterns in cytologically indeterminate nodules rather than the
absence of specific mutations.
• Has an advantage over mutation analysis in identifying gene
signatures that reflect whole patterns of pathway activation
resulting from both upstream mutations and environmental
factors rather than alterations in a small number of genes
69. At present, molecular testing is meant to complement and not
replace clinical judgment, sonographic assessment, and visual
cytopathology interpretation
AmericanThyroidAssociation (ATA) Recommendation
For patients with a preference for surgical excision, a
molecular test with high specificity and PPV (AFIRMA
GEC) was recommended
For patients with a preference of conservative management,
a molecular test with high sensitivity and NPV (Four panel
mutation test) was recommended
72. Salivary gland tumors (SGTs) are heterogeneous nature with
more than 40 different types of neoplasms described in the
currentWHO classification scheme
Therefore, in some cases a specific diagnosis may not be
rendered based on morphology alone,
Several authors have demonstrated that the use of ancillary
techniques (including molecular testing) can overcome the
morphological limitations and refine the diagnostic practice of
salivary gland cytology
FISH and PCR based assays can be done using freshly prepared
FNA smears, CYTOSPIN preparations, cell blocks
74. a, b, low- and
high-power, air-
dried smears
stained with Diff-
Quik® stain.
The alcohol-fixed
on-site smears
stained with
Papanicolaou stain
highlight the
delicate cytoplasm
and nuclear
pleomorphism
(low and high
power, d, e).
A case of fine-needle aspiration
diagnosed as secretory carcinoma
(previously known as mammary
analogue secretorycarcinoma)
75. The break-apart fluorescence
in situ hybridization to
evaluate for disruption of
ETV6 gene shows ETV6
rearrangement (c, f low and
high power)
76. The integration of molecular diagnostic assays in cytopathology has
added a genomic dimension to the world of diagnostic
cytopathology.
The variety and versatility of cytology specimen preparations
provide multiple options for performingmolecular assays.
Novel applications of cytology specimens for molecular diagnostic
assays have redefined and expanded the role of cytopathology in
patient care
Laboratories must evolve with the changing landscapes of
molecular medicine, embrace new technological advancements,
and optimize these methods into routine cytopathology practice.
78. • HPVTesting for Head and Neck Cancers
– HPV-positive OPSqCC have a better prognosis and outcome
than conventional head and neck SqCC.
– p16 IHC is a commonly used surrogate marker for hrHPV
detection
– However criteria for detecting hrHPV using p16 in cytology
specimens is not well defined
– CAP guidelines recommend hrHPV testing on all FNA
specimens of known or suspected OPSqCC when hrHPV
status has not previously been established and for metastatic
SqCC of unknown primary
– RNA / DNA in situ Hybridizations , HPV PCR, liquid-phase
test like Cervista assay can be used
80. • Molecular Diagnostics in Pancreatic and Biliary Cytology (PBC)
– Molecular testing has limited diagnostic utility in PBC but can be
useful in certain settings, particularly for the diagnosis of pancreatic
cyst.
– KRAS/GNAS mutations highly sensitive and specific for the detection
of neoplastic mucinous cysts
– TP53 mutation,loss of SMAD4/DPC4,or loss of p16 detected in pancreatic
cyst fluid supports a high-risk cyst.
– Addition of FISH (UroVysion probe set, cholangiocarcinoma-
/PDAC-specific FISH probe set) and/or NGS to cytology may
improve the diagnostic sensitivity of PBC
82. QUESTIONS
• In UroVysion when all probes show 4 signals,
shall we consider it as positive for
malignancy?
83. • These cells may represent malignancy;
however,
– they may also represent a dividing urothelial cell,
which may be 2N.
– Tetrasomic cells are found more frequently in the
upper urothelial tract and should be interpreted
with caution.
– Once 4 abnormal cells are found, some
laboratories may count 100 consecutive urothelial
cells and provide a percentage of abnormal cells.