Laboratory Diagnosis Of Cancer

LABORATORY DIAGNOSIS OF CANCER Dr RAMASWAMY A S ASSISTANT PROFESSOR DEPARTMENT OF PATHOLOGY PESIMSR KUPPAM

OBJECTIVES • HOW TO APPROACH A CASE OF CANCER? • MODALITIES AVAILABLE IN DIAGNOSING CANCER • CONCEPT OF TUMOR MARKERS

• BEFORE ANY FORM OF EVALUATION CLINICAL HISTORY AND EXAMINATION IS A MUST

• WHATEVER SAMPLE WHICH IS SENT FOR DIAGNOSIS SHOULD BE ADEQUATE, REPRESENTATIVE AND PROPERLY PRESERVED • WHENEVER POSSIBLE EXCISIONAL BIOPSY, OTHERWISE INCISIONAL BIOPSY

PRESERVATION • Formalin fixative – for routine hematoxylin and eosin staining of tissue sections • Bouin’s fixative for testicular biopsy • Glutaraldehyde for electron microscopy • Refrigeration – for hormone, receptor or other molecular analysis • Frozen section – for determining the nature of the lesion and the margin

METHODS OF EVALUATION 1. Cytologic methods 2. Histologic methods 3. Special tests i. Immunohistochemistry ii. Molecular diagnosis iii. flow cytometry iv. Tumor markers

CYTOLOGIC METHODS • EXFOLIATIVE CYTOLOGY • ASPIRATION CYTOLOGY

CYTOLOGY • Commonest example quoted for the early detection and diagnosis of cancer is the PAPANICOLAOU SMEAR ( Pap smear ) examination for carcinoma cervix.

TUMOR MARKERS • They are biochemical indicators for the presence of a tumor • They cannot be construed as primary diagnostic modalities for cancer • They only support a diagnosis of cancer • They also help in determining the response of a cancer to therapy • Useful in detecting relapse during follow up period

CLASSES OF TUMOR MARKERS 1. 2. 3. 4. 5. 6.

HORMONES ONCOFETAL ANTIGENS ISO ENZYMES SPECIFIC PROTEINS MUCINS AND GLYCOPROTEINS NEW MOLECULAR MARKERS

HORMONES HORMONES

ASSOCIATED CANCER

HCG

Trophoblastic tumors, non seminomatous germ cell tumors

Calcitonin

Medullary carcinoma of thyroid

Catecholamine and metabolites

Pheochromocytoma and related tumors

Ectopic hormones

Paraneoplastic syndromes of many cancers

ONCO FETAL ANTIGENS • These are the antigens which are normally expressed during embryonic life • These get re expressed in many diseased states including malignancy • They are not specific for any cancer • The two main onco fetal antigens are 1. α fetal protein 2. Carcino embryonic antigen

α FETAL PROTEIN • It is a glyco protein • Normally synthesised in early fetal life by - Yolk sac - Fetal liver - Fetal gastro intestinal tract

1. 2. 3. 4.

Non neoplastic conditions in which α feto protein is elevated Cirrhosis Toxic liver injury Hepatitis Pregnancy especially with fetal distress or death

Neoplastic conditions in which α feto protein is elevated 1. Hepato cellular carcinoma 2. Germ cell tumor of testis Less commonly elevated in 4. Carcinoma colon 5. Carcinoma lung 6. Carcinoma pancreas

• Markedly elevated AFP levels in the plasma is an useful indicator of hepato cellular carcinoma and germ cell tumor of testis • AFP levels rapidly decline after surgical resection of these tumors • Serial post therapy levels of AFP in these patients indicate a sensitive index of response to therapy and recurrence

CARCINO EMBRYONIC ANTIGEN (CEA) • It is a complex glycoprotein • Normally synthesised in the embryonic tissue of - gut - pancreas - liver

Non neoplastic conditions in which CEA is elevated 1. 2. 3. 4. 5.

Alcoholic cirrhosis Hepatitis Ulcerative colitis Crohn disease Smokers

Neoplastic conditions in which CEA is elevated 1. 2. 3. 4.

Colorectal carcinoma – 60-90% Pancreatic carcinoma – 50-80% Gastric carcinoma – 25-50% Breast carcinoma – 25-50%

• CEA lacks the sensitivity and specificity required for the detection of early cancers • Pre operative CEA levels corelate with the tumor burden • In patients with CEA positive colon cancers, the presence of elevated CEA levels 6 weeks after surgery indicates residual disease • Rising CEA levels indicates recurrence • Serum CEA is also useful in monitoring metastatic breast cancer

SPECIFIC PROTEINS PROTEIN

CANCER

Immunoglobulins

Multiple myeloma and gammopathies Carcinoma prostate

Prostate specific antigen (PSA) and Prostate specific membrane antigen (PSMA)

ISO ENZYMES ISOENZYME

CANCER

Prostatic acid phosphatase

Prostate cancer

Neuron specific enolase

Small cell cancer lung, neuroblastoma

MUCINS AND OTHER GLYCOPROTEINS MUCINS

CANCER

CA-125

Ovarian cancer

CA 19-9

Colon cancer, pancreatic cancer

CA 15-3

Breast cancer

NEW MOLECULAR MARKERS NEW MOLECULAR MARKERS

CANCERS

P53, APC, RAS mutations in stool and serum

Colon cancer

P53 and RAS mutations in stool and serum

Pancreatic cancer

P53 and RAS mutations in sputum Lung cancer and serum P53 mutations in urine

Bladder cancer

IMMUNOHISTOCHEMISTRY • This is the special branch of pathology where antibodies against cellular antigens are used in identification of cellular products or surface markers • The components are visualized using chromogens which stain up when the antigen antibody reaction is completed. • Depending on the location and the staining intensity the results are

UTILITY OF IHC IN NEOPLASMS 1. Categorisation of undifferentiated malignant tumors 2. Categorisation of leukemias and lymphomas 3. Determination of site of origin of metastatic tumors 4. Detection of molecules that have prognostic or therapeutic significance

CATEGORISATION OF UNDIFFERENTIATED MALIGNANT TUMORS

• To differentiate anaplastic carcinoma from malignant lymphomas, melanomas and sarcomas – antibodies to intermediate filaments. Eg., cytokeratin – epithelial origin desmin – muscle cell origin

CATEGORISATION OF LEUKEMIA AND LYMPHOMA • IHC used in conjuction with immunofluorescence • Separation of myeloid from lymphoid neoplasms • Separation of T, B cells and mono nuclear phagocytic neoplasms • Prognostication of leukemias and lymphomas

DETERMINATION OF SITE OF ORIGIN OF METASTATIC TUMORS

• When origin of a metastatic tumor is obscure on morphological grounds then IHC is helpful by the utilisation of tissue specific or organ specific antigens. • eg., PSA – prostate thyroglobulin - thyroid

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DETECTION OF MOLECULES OF THERAPEUTIC OR PROGNOSTIC IMPORTANCE Most useful parameter in certain tumors. Eg., breast cancer – ER / PR receptor status. In general receptor positive tumors are responsive to anti estrogen therapy and have a better prognosis. Product of oncogenes like ERB B2 if it is overexpressed in ca breast then it has got poor prognosis

Cytokeratin positive gastric adenocarcinoma

Sarcoma positive for vimentin

Bcl-2 positivity in lymphoma

C – erb – B2 positivity in breast cancer

MOLECULAR DIAGNOSIS • • • •

Diagnosis of malignant neoplasms Prognosis of malignant neoplasms Detection of minimal residual disease Diagnosis of hereditary pre disposition to cancer • DNA micro array analysis and proteomics

DETECTION OF MALIGNANT NEOPLASMS • Not the primary modality of cancer diagnosis • Useful in differentiating monoclonal from polyclonal proliferations off cells like T / B cells • Specific translocations which cause neoplasms can be identified by techniques like routine cytogenetic analysis or FISH or PCR . Eg., detection of BCR – ABL transcripts in CML • It is also helpful in the differential diagnosis of morphologically similar neoplasms. Eg., ewing sarcoma has all the small round blue cell tumors in its differential diagnosis. It can be diagnosed by demonstration of t

PROGNOSIS OF MALIGNANT NEOPLASMS • Certain genetic alterations have a bearing on the tumor prognosis • Detection of these helps in the prognostication • Eg., amplification of N – MYC and deletion of 1p = poor prognosis in neuroblastoma. • t(15;17) in AML M3 carries good prognosis

DETECTION OF MIMINAL RESIDUAL DISEASE • After the treatment of patients with leukemia or lymphoma the presence of minimal residual disease can be monitored by PCR based amplification of specific tumor genetic sequences

DETECTION OF HEREDITARY PREDISPOSITION TO CANCERS • Detection of germ line mutations in tumor suppressor genes helps in early detection of cancers or warns the persons of increased risk of developing neoplasm • eg,., RET gene analysis in multiple endocrine neoplasia (MEN syndrome)

FLOW CYTOMETRY • This procedure can rapidly and quantitatively measure several individual cell characteristics like - Membrane antigens - DNA content - Cell surface antigens this information can be used both diagnostically and prognostically.

Flow cytometry - aneuploidy

NEWER TECHNIQUES 1. 2. 3. 4. 5. 6.

Spectral karyotyping Comparative genomic hybridisation DNA micro array analysis Proteomics Tissue arrays Electron microscopy

Tissue microarray

Spectral karyotyping of a tumor

Electron microscopy of adenocarcinoma

Summary • Clinical history a must in evaluation of tumor • Morphological approach adopted first in any cancer both gross and microscopic • Hematoxylin and eosin stained tissue sections first line of investigation in all solid cancers • Additional newer techniques should be used judiciously

REFERENCES • GENERAL PATHOLOGY – WALTER & ISRAEL • PATHOLOGIC BASIS OF DISEASE – ROBBINS AND COTRAN • TEXT BOOK OF SURGERY – BAILEY AND LOVE • HARRISON PRINCIPLES OF INTERNAL MEDICINE