Lecture 10: Diagnosis of cancer Learning objectives: This lecture provides an understanding of various approaches in the diagnosis of malignancy Learning outcomes At the end of this lecture student will be able to Discuss methods in the diagnosis of malignancy including cytology, histopathology,frozen sections, biochemical, molecular and tumour markers Relate the important information provided by the pathologic diagnosis in the clinical setting

Various approaches in the diagnosis of malignancy CLINICAL FEATURES Clinical data are invaluable for optimal pathologic diagnosis B. DIAGNOSTIC IMAGING X RAY - PA/Lateral, Double contrast, Barium studies Ultrasound, Mammography CT, MRI

Various approaches in the diagnosis of malignancy C. LABORATORY DIAGNOSIS Histological Diagnosis Cytological Diagnosis Tumor markers Examination of blood & bone marrow Molecular techniques DNA probe analyses DNA flow cytometry

GENERAL signs & symptoms : LOW, LOA, FATIGUE, PALLOR GIT Dysphagia, epigastric mass, vomiting, mass per abdomen, intestinal obstruction, mass per rectum, change in bowel habit, malaena Lung Hemoptysis Kidney / urinary bladder Painless Hematuria Pancreas / ampulla Progressive persistence obstructive jaundice Examples : Clinical Examination

Imaging techniques  Plain films x-rays  Computed tomography (CT scan)  Magnetic resonance imaging (MRI)  Mammography (breast lesion)  Ultrasonography (US) detect the presence location of mass lesions FNAB of deep seated lesion aid staging & determination of therapy

LABORATORY DIAGNOSIS OF CANCER Histological Diagnosis Diagnostic interpretation of tissue samples, Most important method of tumour diagnosis Provides definitive diagnosis (Gold standard)

LABORATORY DIAGNOSIS OF CANCER laboratory evaluation of a lesion can be only as good as the specimen made available for examination It must be adequate, representative, and properly preserved

LABORATORY DIAGNOSIS OF CANCER Type of tissue samples in histopathological examinations Excision biopsy Incision biopsy Punch biopsy Fine needle aspiration biopsy Endoscopic biopsy frozen sections

Preparation for hitopathological examination The tissue is then prepared for viewing under a microscope using either chemical fixation or frozen section Chemical fixation Tissue processing & Embedding Sectioning of tissues into very thin (2 - 7 micrometer) sections using a microtome Staining of the Processed Histology Slides Routine used stain in histopathology is a combination of hematoxylin and eosin (often abbreviated H&E)

Histopathology

Frozen-section The tissue is frozen and sliced thinly using a microtome mounted in a below-freezing refrigeration device called the cryostat The principal use of the frozen section procedure is the examination of tissue while surgery is taking place (for rapid microscopic analysis of a specimen) in determining the nature of a mass lesion or in evaluating the margins of an excised cancer to ascertain that the entire neoplasm has been removed (resection margin clearance)

Cryostat

Cytologic Examination Diagnostic interpretation of the morphology and other characteristics of cells; commonly used in cancer screening and diagnosis cancer cells have lowered cohesiveness and exhibit a range of morphologic changes encompassed by the term anaplasia shed cells can be evaluated for the features of anaplasia indicative of their origin from a tumor

Cytologic Examination Fine-needle aspiration cytology The procedure involves aspirating cells and attendant fluid with a small-bore needle is used most commonly for the assessment of readily palpable lesions in sites such as the breast, thyroid, and lymph nodes Modern imaging techniques permit extension of the method to lesions in deep-seated structures, such as pelvic lymph nodes and pancreas

Cytologic Examination Exfoliative cytology Cytologic (Pap) smears is widely used to screen for carcinoma of the cervix also used endometrial carcinoma, bronchogenic carcinoma, bladder and prostatic tumors, and gastric carcinomas; for the identification of tumor cells in abdominal, pleural, joint, and cerebrospinal fluid

Cytopathology

An abnormal cervicovaginal smear shows numerous malignant cells that have pleomorphic, hyperchromatic nuclei; interspersed are some normal polymorphonuclear leukocytes

Immunohistochemistry identification of cell products or surface markers by using specific antibodies

Role of immunohistochemistry in the diagnosis & management of malignant neoplasms Categorization of undifferentiated malignant tumors Determination of site of origin of metastatic tumors Detection of molecules that have prognostic or therapeutic significance

Categorization of undifferentiated malignant tumors Tumors which are often quite difficult to distinguish on the basis of routine hematoxylin and eosin (H&E)–stained tissue sections ( for example :certain anaplastic carcinomas, lymphomas, melanomas, and sarcomas may look quite similar)

Categorization of undifferentiated malignant tumors By using antibodies specific to intermediate filaments Solid tumor cells often contain intermediate filaments characteristic of their cell of origin For example the presence of cytokeratins detected by immunohistochemistry, points to an epithelial origin (carcinoma) desmin is specific for neoplasms of muscle cell origin

Anti-cytokeratin immunoperoxidase stain of a tumor of epithelial origin

Determination of site of origin of metastatic tumors In cases in which the origin of the tumor is obscure immunohistochemical detection of tissue- specific or organ-specific antigens in a biopsy specimen of the metastatic deposit can lead to the identification of the tumor source For example, prostate-specific antigen (PSA) and thyroglobulin are markers of carcinomas of the prostate and thyroid, respectively

Detection of molecules that have prognostic or therapeutic significance Immunohistochemical detection of hormone receptors (estrogen/progesterone) in breast cancer cells is of prognostic and therapeutic value because these cancers are susceptible to anti- estrogen therapy

Protein products of oncogenes such as ERBB2 in breast cancers can also be detected by immunostaining Breast cancers with overexpression of ERBB2 protein generally have a poor prognosis Herceptin: anti-HER-2/neu

Flow Cytometry Flow cytometry can rapidly and quantitatively measure several individual cell characteristics, such as membrane antigens and the DNA content of tumor cells Flow cytometry has also proved useful in the identification and classification of tumors arising from T and B lymphocytes and from mononuclear-phagocytic cells

Clinical chemistry measurement and interpretation of substances in blood, other body fluids and tissues

Tumor Markers Biochemical indicators of tumor contribute to the detection of cancer useful in determining the - effectiveness of therapy - appearance of a recurrence

Tumor marker These markers may be Surface Antigens Glycoproteins Tumor-associated Enzymes & Hormones

Clinically useful Tumor Markers 1. ONCOFETAL ANTIGENS a.Alpha - Foeto - Protein (AFP) Abnormal elevation seen in HCC b. Carcino-embryonic antigen ( CEA ) Elevated in CA of the colon, Breast & pancreas

Clinically useful Tumor Markers 2. HORMONES Beta HCG - increased in trophoblastic tumors, choriocarcinoma Calcitonin - in medullary CA of thyroid 3. ISOENZYMES Placental alkaline phosphatase - seminoma Neuron-specific enolase -cancer of lung, neuroblastoma

Clinically useful Tumor Markers 4. SPECIFIC PROTEINS Immunoglobulins - multiple myeloma Prostate-specific antigen (PSA) - Prostate cancer 5. MUCINS AND OTHER GLYCOPROTEINS CA ovarian cancer CA Colon & pancreatic cancer CA Breast cancer

NEW MOLECULAR MARKERS p53, APC, RAS mutants in blood and body fluids (serum, stool, sputum and urine) Colon cancer, Pancreatic cancer,lung cancer, bladder cancer

Molecular Diagnosis  Diagnosis of malignant neoplasm  Prognosis of malignant neoplasm  Detection of minimal residual disease  Diagnosis of hereditary predisposition to cancer

Molecular Techniques Several techniques are used in DNA analysis Molecular cytogenetic techniques Hybridization-based methods PCR - based Methods

Cytogenetics Analysis of chromosomal and genetic abnormalities analyze the chromosome constitution of an individual is known as a karyotype Detection of translocations in hematopoietic neoplasms (leukemias and lymphomas)

The study of chromosomes by usual procedure of producing a chromosome spread is to arrest mitosis in dividing cells in metaphase by the use of mitotic spindle inhibitors (e.g., colcemid) and then to stain the chromosomes

Preparation of a karyotype.

Normal male karyotype with G banding 2n = 46

Molecular techniques in Diagnosis of malignancy fluorescence in situ hybridization (FISH) uses DNA probes that recognize sequences specific to particular chromosomal regions detection of numeric abnormalities of chromosomes demonstration of subtle microdeletions complex translocations not detectable by routine karyotyping analysis of gene amplification (e.g., HER2/NEU in breast cancer or N-MYC amplification in neuroblastomas)

Fluorescence in Situ Hybridization Uses fluorescent labelled DNA probes that recognize chromosome-specific sequences to bind to its complementary sequence on the chromosome and thus labels the specific chromosome, which can then be visualized under a fluorescent microscope

Fluorescence in Situ Hybridization FISH can be used to demonstrate SUBTLE MICRODELETIONS COMPLEX TRANSLOCATIONS AND TELOMERE ALTERATIONS that are not readily detectable by routine karyotyping

Fluorescence in Situ Hybridization

F.I.S.H. greatly enhances G-banding FISH is POWERFULLY more sensitive, accurate, and specific, than G-banding

Fluorescence in situ hybridization (FISH) Interphase nuclei of a childhood hepatic cancer (hepatoblastoma) stained with a fluorescent DNA probe that hybridizes to chromosome 20. Under ultraviolet light, each nucleus reveals three bright yellow fluorescent dots, representing three copies of chromosome 20. Normal diploid cells (not shown) have two fluorescent dots TRIPLE CHROMOSOME #20

Chromosome painting with a library of chromosome 22- specific DNA probes. The presence of three fluorescent chromosomes indicates that the patient has trisomy 22 Fluorescence in situ hybridization (FISH)

SPECTRAL KARYOTYPING Chromosome painting has limited ability to visualize all 46 human chromosomes simultaneously This hurdle has been overcome by the introduction of spectral karyotyping

SPECTRAL KARYOTYPING By using fluorochromes and appropriate computer- generated signals, the entire human genome can be visualized detect all types of chromosomal rearrangements in tumor cells It can also detect the origin of unidentified chromosomes, called marker chromosomes, seen in many hematopoietic malignancies

SPECTRAL KARYOTYPING

Comparative Genomic Hybridization This technique enabled the detection of regions of allele loss and gene amplification Tumor or 'test' DNA was labeled with a green paint, and control normal DNA with a red paint Tumor-specific alterations in gene copy number can be determined by comparative genomic hybridization

PCR-based methods Detection of microsatellite repeats Mutation detection by allele-specific PCR

Polymerase Chain Reaction (PCR) exponential amplification of DNA PCR analysis, which involves exponential amplification of DNA used in the molecular diagnosis of human diseases using appropriate DNA polymerases and thermal cycling

Molecular techniques in Diagnosis of malignancy Polymerase chain reaction (PCR) PCR-based detection of T-cell receptor or immunoglobulin genes allows distinction between monoclonal (neoplastic) and polyclonal (reactive) proliferations to detect translocations characteristic of Ewing sarcoma and several leukemias and lymphomas

Molecular techniques in Diagnosis of malignancy PCR-based detection of BCR-ABL transcripts provides the molecular diagnosis of chronic myeloid leukemia

Prognosis of malignant neoplasm Detection of certain gene asscited with poor prognosis by PCR or FISH eg : amplification of the N-MYC gen in neuroblastoma amplification of HER-2/NEU in breast cancer

Detection of minimal residual disease detection of minimal residual disease after treatment For example, detection of BCR-ABL transcripts by PCR gives a measure of residual disease, in patients treated for chronic myeloid leukemia

Diagnosis of hereditary predisposition to cancer Germ-line mutation of several tumor suppressor genes, such as BRCA1, increases a patient's risk of developing certain types of cancer detection of these mutated alleles allow the patient and physician to devise an aggressive screening protocol, as well as to consider prophylactic surgery allows genetic counselling of relatives at risk

Molecular Profiling of Tumors One of the most exciting advances in the molecular analysis of tumors has been made possible by DNA-microarray analysis

simultaneous measurements of the expression levels of several thousand gene The principle - gene chip technology