LABORATORY DIAGNOSIS OF CANCER
Q. Write short note on laboratory diagnosis of cancer.
Confirmation of lesion as neoplastic usually requires cytological and/or
histopathological examination of the suspected organ or tissue. Different
laboratory methods available for the diagnosis of malignant tumors are:
Morphological Methods
Histopathological Examination
Histopathological diagnosis is based on the microscopic features of
neoplasm and by this method of examination, accurate diagnosis can be
made in majority of cases.
v Clinical data: It should be provided for accurate pathologic diagnosis
e.g.,
n Radiation causes changes in the skin or mucosa that mimic changes
seen in cancer.
n Sections taken from the site of a healing fracture can mimic an
osteosarcoma.
v Adequate and representative area of the specimen should be sent.
v Proper fixation.
Frozen Section
Q. Write short essay/note on frozen section and its
uses.
In this method, tissue is hardened rapidly by freezing (hence termed
frozen section) and sections are cut by special instrument called freezing
microtome or cryostat (microtome in a refrigerated chamber). Its uses are:
v Rapid diagnosis: Frozen section is used for quick histologic
diagnosis (within minutes) and useful for determining the nature of
a tumor (benign or malignant) lesion, especially when the patient is
still on the operation table. It will help to determine whether a
lesion is a neoplasm and, if so, whether it is benign or malignant..
v Evaluation of the margins of an excised cancer to know whether
excision of the neoplasm is complete. If resection margins are
inadequate, additional tissue can be removed immediately, without
the need for a subsequent operation.
v Demonstration of fat mainly in non-neoplastic lesions.
v To know the lymph node (sentinel) status in carcinoma. Knowing
the extent of regional tumor metastases may be useful in deciding
the further surgery.
v To determine whether additional workup is necessary for a
particular tissue specimen while it is still fresh. For example, if the
metastatic tumor found in a lymph node is recognized as a poorly
differentiated carcinoma, special fixation and electron microscopy
may be needed for proper diagnosis.
Various Techniques for Tissue Sampling
v Needle biopsy: It is called as cutting-needle or core- needle or drill
biopsy. Using cutting needle, a core of tissue1– 2 mm wide and 2 cm long
is obtained for histologic examinations or special studies that permit
evaluation of architectural structure. Tissue obtained is small and
interpretation may be difficult.
v Endoscopy biopsy: It is performed through endoscopy. Usually
performed for lesions in gastrointestinal, respiratory, urinary and genital
tracts.
v Incision biopsy: In this representative tissue sample is obtained by
incising the lesions. Incisional biopsy (along with fine-needle aspiration)
is often the method of choice for inoperable lesions, or too large for
excision, or when excision causes functional or cosmetic impairment.
v Excision biopsy: In this entire abnormal lesion is surgically removed. It
provides generous amounts of tissue for diagnosis and may be surgical
therapy for some tumors (e.g., small- to medium-sized breast cancers.
Pitfalls in biopsy interpretation: These include inadequate tissue sampling
and artifacts due to procedure itself (e.g., thermal damage caused by an
electrocautery or laser).
Cytological Examination
It is performed on many tissues and usually done for identifying neoplastic
cells.
Methods of Obtaining Cells
Exfoliative cytology: It is the study of spontaneously exfoliated (shed)
cells from the lining of an organ into a body cavity. Discussed in detail on
pages 279-81.
Fine-needle Aspiration Cytology
It involves aspiration of cells and attendant fluid with a small-bore
needle. The smears are prepared and stained, followed by microscopic
examination of cells. It is widely used, simple and quick procedure (For
details refer pages 281-3).
Liquid-based cytology (thin prep): This is a special technique for
preparation of samples that provides uniform monolayered dispersion of
cells on smears.
Cytological Characteristics of Cancer Cells
Cancer cells have decreased cohesiveness and show cellular features of
anaplasia. Cytologically, differentiation can be made between normal,
dysplastic, carcinoma in situ and malignant cells.
Disadvantages of Cytological Examination
v Diagnosis is based on the features of individual cells or a clump of
cells, without the supporting evidence of loss of orientation.
v The invasion which is diagnostic of malignant tumor under histology
cannot be assessed by cytology.
Histochemistry and Cytochemistry
These are stains, which identify the chemical nature of cell contents or their
products. H&E staining cannot demonstrate certain specific
substances/constituents of cells. This requires some special stains. Common
histochemical and cytochemical stains useful in diagnosis of tumors are
listed in Table 7.13.
Immunohistochemistry
TABLE 7.13: Common histochemical and cytochemical stains useful in diagnosis of tumors.
Chemical substance Name of the stain
Q. Write short note on immunohistochemistry and its
role in the diagnosis of tumors.
It is an immunological method of identifying the antigenic component in the
cell or one of its components by using specific antibodies. It is widely used
in the diagnosis or management of malignant neoplasms.
Uses of Immunohistochemistry
v To categorize undifferentiated cancers: Many malignant tumors of
diverse origin resemble each other and are difficult to distinguish on
routine hematoxylin and eosin (H&E) sections.
n Example: Few anaplastic carcinomas, lymphomas, melanomas and
sarcomas may look almost similar. They should be accurately
diagnosed because of their different modes of treatment and
prognosis.
t In poorly differentiated carcinoma intermediate filaments (e.g.,
cytokeratins) shows positivity (Table 7.14).
t Malignant melanomas when unpigmented (amelanotic
melanoma) appear similar to other poorly differentiated
carcinomas. They express HMB-45 and S-100 protein, but
negative for cytokeratins.
t Desmin is found in neoplasms of muscle cell origin.
v To determine the origin of poorly differentiated metastatic tumors: It
may be determined by using tissue-specific or organ-specific antigens
(Table 7.15).
v For prognosis or to select the mode of treatment:
n Identification of hormone (estrogen/progesterone) receptors in
breast cancer cells is of prognostic and therapeutic value. These
cancers respond well to antiestrogen therapy and have a better
prognosis.
n Breast cancers with ERBB2 protein (HER2/neu) positivity have a
poor prognosis.
Immunohistochemical Markers
Q. List tumors in which immunohistochemistry has
diagnostic role.
Apart from the various immunochemical markers mentioned above, other
markers useful are as follows:
v Neuroendocrine tumors show positivity for cytokeratins like carcinomas,
but they can be identified by their contents, namely:
n Chromogranins (proteins found in neurosecretory granules)
n Neuron-specific enolase (NSE)
n Synaptophysin.
v Soft tissue sarcomas: They show intermediate filament positivity
n Vimentin
n Desmin is positive in smooth or striated muscle fibers
n Muscle-specific actin marker for muscle tissue.
v Neurofilament proteins: Marker for tumors of neurons,
neuroblastomas and ganglioneuroma.
v Neuron-specific enolase in neuroblastomas.
v Glial fibrillary acidic protein (GFAP), also intermediate filament
expressed in glial cell neoplasms.
v Malignant lymphomas: Generally positive for leukocyte common
antigen (LCA, CD45). Markers for lymphomas and leukemias are called
cluster designations (CDs) and useful to differentiate T and B
lymphocytes, monocytes, and granulocytes and the mature and immature
variants of these cells.
v Vascular tumors derived from endothelial cells, include benign
hemangiomas, and malignant hemangiosarcomas and are positive for
factor VIII-related antigen or certain lectins.
v Proliferating cells: Cells in cell cycle show positivity for Ki-67 and
proliferating cell nuclear antigen (PCNA).
Electron Microscopy
It helps in the diagnosis of poorly differentiated/undifferentiated cancers,
which cannot identify the origin by light microscopy, e.g., carcinomas show
desmosomes and specialized junctional complexes, structures which are not
seen in sarcomas or lymphomas.
Flow Cytometry
Q. Write short note on modern techniques in tumor
diagnosis.
It quantitatively measures various individual cell characteristics, such as
membrane antigens and the DNA content of tumor cells. Flow cytometry is
useful for identification and classification of tumors of T and B
lymphocytes (leukemias and lymphomas) and mononuclear-phagocytic
cells.
Circulating Tumor Cells
Detection, quantification, and characterization of rare solid tumors cells
(e.g., carcinoma, melanoma) circulating in the blood is emerging as a
diagnostic modality though presently in research stage. Few latest devices
detect three-dimensional flow cells coated with antibodies specific for tumor
cells of interest (e.g., carcinoma cells) in the blood. It will be useful for early
diagnosis, to assess the risk of metastasis and assess the response of tumor
cells to therapy.
Tumor Markers
Q. Write short note on tumor markers.
Q. List tumor markers giving one example for each.
TABLE 7.16: Common tumor markers.
Tumor marker Associated tumors
Tumor markers are products of malignant tumors that can be detected in
the cells themselves or in blood and body fluids.
Usefulness
v Detection of cancer, e.g., PSA is the most common and useful tumor
markers used to screen prostatic adenocarcinoma. High levels of PSA
are found in the blood of prostatic carcinoma patients but it also may be
elevated in benign prostatic hyperplasia.
v Determine the effectiveness of therapy.
v Detection of recurrence.
Types of markers (Table 7.16): These may be tumor-associated hormones,
oncofetal antigens, specific proteins, mucin and glycoproteins, enzymes and
molecular markers.
Molecular Diagnosis
Molecular diagnosis can be done by different techniques such as FISH
technique and PCR (polymerase chain reaction) analysis.
a. Diagnosis of Cancer
v Monoclonal (malignant) versus polyclonal (benign): To differentiate
benign (polyclonal) proliferations of T- or B-cells from malignant
(monoclonal) proliferations.
v Chromosomal alterations: Many hematopoietic neoplasms (leukemias
and lymphomas) and few solid tumors (e.g., Ewing sarcoma) are
characterized by particular translocations that can be detected by FISH
technique or by PCR analysis.b. Prognosis of Cancer
Certain genetic alterations are of prognostic value. They can be detected by
routine cytogenetics and also by FISH or PCR assays. Example of poor
prognostic feature is amplification of the N-MYC gene and deletions of 1p in
neuroblastoma and amplification of HER-2/Neu in breast cancer.
c. Detection of Minimal Residual Disease
PCR can detect minimal residual disease or the onset of relapse in patients
who are treated for leukemia or lymphoma, e.g., detection of BCR-ABL
transcripts in treated patients with CML.
d. Detection of Hereditary Predisposition to Cancer
Germline mutations in many tumor suppressor genes are associated with
increased risk for specific cancers. This will help in prophylactic surgery,
and counseling of relatives at risk, e.g., BRCA1, BRCA2 and the RET protooncogene.
e. For Therapeutic Decision
It is useful in target therapy.
Molecular Profiling of Tumors
Traditional Cancer Typing
Traditionally cancer is diagnosed and classified according to the
morphological (histopathology/cytopathology) appearance of the cells and
its surrounding tissue. It has limitations such as (i) it relies on a subjective
review of the tissue which is dependent on the knowledge and experience of
a pathologist, and therefore may not be reproducible, (ii) limited ability to
determine the individual recurrence risk of cancer, (iii) insufficient to reflect
the complicated underlying molecular events that drive the neoplastic
process and (iv) histopathology reports lack or offer very little information
regarding the potential drug treatment regime to which a cancer will
respond. Traditional pathology reports helps to determine treatment that
leads to better outcomes. However, tumors with identical pathology may
have different origins and respond differently to treatment.
Newer Cancer Diagnostics
Molecular profiling of tumors is the identification and documentation of
the structure of a specific DNA, RNA, or protein molecule. Till recently,
molecular studies of tumors consisted of analyzing individual genes.
However, the revolutionary technologies which have achieved completion of
the human genome sequence and more reliable methods of gene expression
analysis have revolutionized cancer diagnosis and treatment. Present
methods like DNA microarray technology can measure the expression single
gene to all genes in the genome instead of only one gene at a time. By
providing a molecular portrait of an individual cancer, this technology will
help the clinicians to determine the origin of the cancer, its potential for
metastasis, its specific drug responsiveness, and the probability of its
recurrence. Classification of cancer typing by molecular profile overcomes
the limitations of traditional cancer typing. A molecular profile determines
the level of gene expression within the cancer by hybridizing the cellular
RNA with known genes. However, RNA is prone to degradation and is a
more difficult analyte to work with than DNA in clinical practice.
DNA Microarray
Currently, the most common method for large-scale analysis of RNA
expression is based on DNA microarrays. A DNA microarray (also known
as DNA chip or biochip) is a collection of microscopic DNA spots attached
to a solid surface. This provides information on thousands of genes
simultaneously. Once the gene expression pattern is determined, this
information is compared to the expression profiles of cancers with known
outcomes using a predetermined algorithm.
DNA Sequencing
It is the process of determining the precise order of nucleotides within a
DNA molecule and is technically simpler than RNA sequencing. Nextgeneration sequencing refers to non-Sanger-based high-throughput DNA
sequencing technologies. Millions or billions of DNA strands can be
sequenced in parallel, yielding substantially more throughput and
minimizing the need for the fragment-cloning methods that are often used in
“Next-generation [NextGen] sequencing” (refer page 250) which can be
readily performed on virtually any tissue specimen. By nextGen sequencing,
the process of whole-genome of individual tumors can be completed in as
little as a few weeks. The cancer genome sequencing can identify new
mutations in various cancers; describe the full, extensive collection of
genetic lesions in individual cancers; and a presence of genetic heterogeneity
in individual cancers from area to area. It is particularly useful to tumors,
such as lung carcinomas, that are genetically diverse and require a
“personalized” approach for successful targeted therapy. However,
histopathological examination of tumors provides information about
important characteristics of cancers, such as anaplasia, invasiveness, and
tumor heterogeneity. These cannot be obtained from DNA sequences. Thus,
the most accurate diagnosis and assessment of prognosis in cancer can be
arrived by a combination of morphologic and molecular techniques (i.e.,
molecular profiling of tumors by RNA expression profiling, DNA
sequencing, and DNA copy number arrays). In points