Surgical Pathology: Special Study

SURGICAL PATHOLOGY
Special Studies in Surgical Pathology
Dr.Tesoro
Direct method-primary antibody only

INTRODUCTION
Haematoxylin and eosin staining (H&E)
o Is like a clinicians history and physical
examinations
o Basic examinations to e performed on every
patient or specimen forming the cornerstone of
diagnosis
Special studies
o Familiarity is important as the initial processing Indirect method primary and secondary antibodies
may limit the types of studies that can be
performed
SPECIAL STUDIES:
Histochemistry studies
o Are suitable for formalin fixed tissues
o These are used to highlight special structures
o Acid Fuschin orange G (AFOG) / Modified
Massons trichrome - evaluation of renal
biopsies
o Alcian blue- identify mucosubstances
o Acid Fast stains (Fite-Faraco) identific AFB
o Bodians stains Neural tumors Enzyme linkage indirect method
o Condo Red- Amyloid
Immunoperoxidase studies
o Immunohistochemical studies
o Detect antigens in sections with antibodies
o Use: gain evidence for or agianst diagnostic
possibilities
o Panels: used as interpretation
immunohistochemical profile
o Introduction
o Used for the following:
Classification of tumours
Identification of in-situ vs invasive
lesions
Prognostic factors Multiple Immunofluorescence
Predictive factors to guide specific
treatments
Identification of extracellular material
Identification of infectious agents
o Factors affecting immunogenecity:
Type of fixative: 10% buffered formalin
Length of fixation time: 6-24 hours
Prior decalcification : HCl vs EDTA
Tempearture
Length of time since the glass slide was
cut
Antigen retrieval procedure
Type of antibody
Incubation time, temperature, dilution
of antibody
Methods of signal amplification

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 Surgical Pathology: Special Study
Pleural Effusions
CASE: TUMOUR in Liver of 65 year old male
o Males: Lung primary
o Females: Breast primary
Cancer of Unknown Primary Site (CUPS) o Lymphomas in both sexes
o Heterogenous group of cancers o Sites of Involvement
o Presence of metastatic disease
o No identifiable primary tumor at presentation Malignant ascites
o Males:
CUPS comprise 5-15% of all diagnosed cancers GI (colon, rectum or stomach)
o Recent improvements in imaging have reduced o Females:
CUPS to 3-7% Ovaries, endometrium, cervix

Identification of prognostically favorable disease is Peritoneal carcinomatosis of non-gynecological origin

important o Stomach, colon, pancreas

o Directed treatment may provide substantial Lymph node metastases

benefit, including prolonged survival o Anatomic site of lymph node involvement
Leukemias / Lymphomas o Morphologic clues
Germ Cell Tumors (GCT) Adenocarcinoma, squamous cell
Small Cell Lung Carcinoma (SCLC) carcinoma, undifferentiated carcinoma
Carcinomas of breast, ovary,
endometrium, adrenal gland, thyroid Axillary Adenocarcinoma in Females
and prostate o Ipsilateral breast
Sites of Involvement
Metastatic neck adenocarcinoma
Liver o Male: Lung
o One of the largest repositories of metastatic o Female: Breast
malignancy o GI and prostate adenocarcinoma show a
o Males predilection for the left side of the neck
Colorectal, Lung, Prostate
o Females Undifferentiated carcinomas of the head and neck
Colorectal, Breast, Lung o Majority are squamous mucosal derivation
o Other GI malignancies, Malignant Melanoma o Sites of Involvement
Lung Squamous cell carcinomas of the upper and mid-cervical
o Also a major repository of metastatic lymph nodes
carcinomas, especially adenocarcinoma o Endoscopic investigation of oro-nasopharyngeal
o Distinction of lung primary versus metastasis and upper esophageal tract with biopsy of
may be difficult suspicious lesions
Scant biopsy materials from FNAB o CT scan, PET scan can help determine primary
Most frequent lung primary is also site
adenocarcinoma
Brain Metastatic squamous cell carcinoma of lower cervical
o Distinguishing glial tumors from metastatic lymph nodes
adenocarcinoma or poorly differentiated o Lung
carcinoma is straightforward o Esophagus
However, determining source of
metastasis may be problematic Squamous cell carcinoma of inguinal nodes
o Common: Lung, breast, kidney, thyroid, GI o Anogenital area primary
o Rare: ovary, prostate, pancreacticobiliary o Diagnostic Algorithm

Skeletal
DIAGNOSTIC ALGORITHM
o Metastatic lesions from:
Lung Carcinomas form 90-95% of CUPS
Breast o Virtually all are positive for cytokeratins (CK)
Kidney More specific subcategorization
Urogenital tract o Site-specific cytokeratins
o Imaging studies particularly useful in elucidating o Antibodies against various cellular products
primary tumor

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 Surgical Pathology: Special Study
Diagnostic Algorithm: STEP 1 Diagnostic Algorithm: STEP 2

Determine line of differentiation Use site-specific cytokeratins

o First-line panel of stains o CK7 and CK20 as a screening panel
Pancytokeratin (CK) epithelial o High molecular weight (HMW) keratins to
Leukocyte common antigen (LCA) identify basal and myoepithelial cells, squamous
lymphoid epithelial origin and mesotheliomas
S-100 melanocytic Ex. 34E12 in carcinoma-in-situ in
Vimentin* mesenchymal breast and prostate
*Vimentin may also be expressed by o CK5/6 is a good indicator of squamous and
poorly differentiated carcinomas transitional cell differentiation
Lymphoid Line of Differentiation Ex. Mesothelial versus
o If LCA (+) and CK (-): adenocarcinoma in lung
Subclassify the lymphoma Ex. CK5 in basal-like carcinoma of the
pan-B (CD20, CD79a) breast
markers
pan-T (CD3) markers CK7 and CK 20 Expression in Carcinomas
o If LCA () and pan-B and pan-T are negative, with
lymphoid morphology:
Consider Myeloid neoplasm
Myeloperoxidase (MPO),
CD117
Melanocytic Line of Differentiation
o If S-100 (+) and CK (-):
Confirm if melanoma using additional
markers:
HMB-45
Melan-A
o S-100 is very sensitive but not specific for
melanoma
Also expressed by some carcinomas
and sarcomas (liposarcoma,
chondrosarcoma, neural tumors) Diagnostic Algorithm: STEP 3

Mesenchymal Line of Differentiation

Carcinomas with Frequent Vimentin Coexpression [CK (+),
o If Vimentin (+), LCA (-), S-100 (-):
Vimentin (+)]
Sarcomas
o Vimentin stains virtually all spindle cell
Synovial sarcoma may stain
neoplasms
with epithelial markers in
o Stains a particular subset of carcinomas
gland-like component
o Vimentin is an internal quality measure for
o Small round blue cell morphology:
antigen assessment in any tissue
Ewings sarcoma, desmoplastic small
o But not useful for body cavity effusion
round cell tumor (DSRCT),
specimens
rhabdomyosarcoma
Cytokeratin/Vimentin Coexpression Patterns
o Spindle and epithelioid morphology:
Synovial sarcoma, clear cell sarcoma,
angiosarcoma
o Pure epithelioid morphology:
Epitheloid sarcoma
Epithelial Line of Differentiation
o If CK (+), LCA (-), S-100 (-):
Carcinoma
70% adenocarcinomas
15-20% poorly differentiated
carcinomas
5% squamous cell
carcinomas
5% neuroendocrine
carcinomas

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 Surgical Pathology: Special Study
TTF-1
Diagnostic Algorithm: STEP 4 o Nuclear staining in all carcinomas of thyroid
origin (except anaplastic type)
o Nuclear staining in vast majority of lung
Use supplemental epithelial markers
carcinomas
o Carcinoembryonic Antigen (CEA)
Adenocarcinoma (66%), large cell
o Epithelial Membrane Antigen (EMA)
neuroendocrine and small cell (99%)
o BER-EP4
o Small cell carcinomas of other sites (GIT, bladder,
Best marker to distinguish
cervix, prostate) frequently TTF-1(+)
adenocarcinomas from mesotheliomas
o Merkel cell carcinomas are negative for TTF-1
Almost never expressed in squamous
Thyroglobulin
cell carcinomas
o Positive in almost all thyroid carcinomas
CEA Staining in Adenocarcinoma
Reduced staining in poorly
differentiated carcinoma
Complete absence in anaplastic types
o Highly specific for thyroid carcinomas
Rare positivity in some leukemic blast
cells
Calretinin and WT-1
o Calretinin and Wilms tumor-1 (WT-1) most
sensitive mesothelial markers with high
specificity
o Calretinin may be (+) in up to 8% of
adenocarcinomas
o Ovarian serous carcinomas strongly WT-1(+)
EMA Staining in Carcinomas and Nonepithelial Tissues
Majority of endometrial serous
carcinomas negative
o Mucinous breast carcinomas may be WT-1(+)
Less intense staining vs serous ovarian
GCDFP-15 and Mammaglobin
o Gross cystic disease fluid protein (GCDFP-15)
>90% specificity but lower sensitivity for breast
carcinoma
o Mammaglobin has high sensitivity (~60%) but
lower specificity for breast carcinoma
o Mammaglobin expression in endometrioid
carcinomas may be useful diagnostically
Hormone Receptors (ER and PR)
o Diffuse strong expression indicates breast or
gynecologic primary
o Weak/moderate expression should be judged
more carefully
Clinical presentation
Results of other IHCs in panel
Diagnostic Algorithm: STEP 5 CDX-2
o CDX-2 is highly sensitive and specific for
intestinal differentiation
Use cell-specific products o Positive in urinary bladder (urachal origin)
o Most metastases can be identified with the adenocarcinoma, colorectal carcinomas
initial panel Uterine carcinomas CDX-2(+) in 30% of
o IHCs to cell-specific products have a high cases
specificity, allowing fine focus in searching for Endometrioid adenocarcinomas of
tumor ovary and uterus CDX-2(+) in up to
Neuroendocrine Differentiation 25% of cases
o Typical cytokeratin profile is CK7(+) Colloid lung carcinomas, ovarian
o Use of chromogranin and synaptophysin in a mucinous carcinomas
panel complement each other HepPar-1
o Subsets of peripheral neuroendocrine o Specific marker of hepatocellular differentiation
carcinomas may be TTF-1(+) Characteristic cytoplasmic granular
o Subsets of neuroendocrine carcinomas of the GI staining
tract may be CDX-2(+)

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o Other HepPar-1(+) tumors
Signet ring cell carcinoma of the
stomach, hepatoid differentiation in
carcinomas from other sites (ex. Ovary)
PSA
o Prostate-specific Antigen (PSA) is highly specific
for prostate tissue
Immunostaining of tumor cells
decreases as Gleason grade increases
o Patchy staining in some salivary gland ductal
carcinomas, up to 1/3 of breast carcinomas and
sweat gland tumors, periurethral glands, cystitis
glandularis of bladder, urachal remnants, some
anal glands
Renal Cell Carcinoma Markers
o Common renal clear cell carcinoma profile: CK7(-
), CK20(-), EMA(+), CD10(+), Renal cell carcinoma
(RCC)(+), Vimentin(+)
None are specific; a panel approach is
preferred
o Other CD10(+) carcinomas:
Transitional cell, prostate carcinoma
o Other RCC(+) carcinomas:
Breast, colon, adrenocortical, prostate
CD5
o Found in T-lymphocytes and mantle zone
lymphocytes
o Most thymic carcinomas are CD5(+)
o Spindle cell thymic carcinomas CD5(-)
o Lung carcinomas are largely CD5(-)
Germ Cell Tumor Markers
Summary
o IHC is a cost-effective tool in diagnosing CUPS
Performed in most hospitals
Can be automated
Rapid TAT
o No IHC stain is entirely site-specific
IHC panels give statistical power to
morphologic diagnosis
IMMUNOFLOURESCENCE
Immunofluorescence (IF) or cell imaging techniques rely
on the use of antibodies to label a specific target antigen
with a fluorescent dye (also called fluorophores or
fluorochromes) such as fluorescein isothiocyanate (FITC).
Antibodies that are chemically conjugated to fluorophores
are commonly used in IF.
Surgical Pathology: Special Study
ELECTRON MICROSCOPY
Method
o It uses a type of microscope that uses a beam
of electrons to create an image of the specimen.
o It is capable of much higher magnifications and
has a greater resolving power than a
light microscope, allowing it to see much smaller
objects in finer detail.
Indications
o Diagnostic renal biopsies
o Difficult to classify tumors
o Adenocarcinoma vs mesothelioma
o Bullous skin lesions
o Ciliary dysmorphology
o Endomyocardial biopsies
o Prion diseases
MOLECULAR GENETIC PATHOLOGY
The subspecialty of Molecular Genetic Pathology employs
testing of nucleic acids (DNA and RNA) for disease
diagnosis
A variety of tests for the presence of mutations in DNA,
chromosomal translocations, and altered levels of RNA
(gene expression) are used in four general areas of
disease:
o Hereditary diseases (e.g., cystic fibrosis)
o Hematologic malignancies (e.g., leukemia)
o Solid tumors (e.g., lung cancer)
o Infectious diseases (e.g., HIV/AIDS)
Hereditary diseases
o identification of inherited diseases
o Identification of genes conferring susceptibility
to diseases (BRCA)
o Infectious diseases
o Detection of organisms
o Identification of specific organisms
o Quantitation of viral infections
Hematologic malignancies
o Hematolymphoid proliferations with difficult
diagnosis due to frequent and characteristic
rearrangements
Solid tumors
o Identification of specific genetic alterations
associated with tumors
o Identification of gene mutations associated with
susceptibility to treatments detection of minimal
residual disease after treatment
CYTOGENETICS
Cytogenetics is the study of the structure of normal and
abnormal chromosomes
o chromosome numbers and structure
o learning and describing the relationships
between chromosome structure and phenotype
o seeking out the causes of chromosomal
abnormalities
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POLYMERASE CHAIN REACTION
PCR (Polymerase Chain Reaction) is to make a huge
number of copies of a gene. This is necessary to have
enough starting template for sequencing.
The cycling reactions : There are three major steps in a
PCR, which are repeated for 30 or 40 cycles. This is done
on an automated cycler, which can heat and cool the tubes
with the reaction mixture in a very short time.
o Denaturation at 94C
o Annealing at 54C
o Extension at 72C
The cycling reactions
o Denaturation at 94C
the double strand melts open to single
stranded DNA, all enzymatic reactions
stop (for example : the extension from
a previous cycle).
o Annealing at 54C
The primers are jiggling around, caused
by the Brownian motion. Ionic bonds
are constantly formed and broken
between the single stranded primer
and the single stranded template.
The more stable bonds last a little bit
longer (primers that fit exactly) and on
that little piece of double stranded
DNA (template and primer), the
polymerase can attach and starts
copying the template. Once there are a
few bases built in, the ionic bond is so
strong between the template and the
primer, that it does not break
anymore.
o Extension at 72C
This is the ideal working temperature
for the polymerase
The bases (complementary to the
template) are coupled to the primer on
the 3' side (the polymerase adds
dNTP's from 5' to 3', reading the
template from 3' to 5' side, bases are
added complementary to the
template)
FLOURESCENCE IN-SITU HYBRIDIZATION
This technique is used for the detection of target
molecules with a system of coupled antibodies and
fluorochromes.
The detection of nucleotidic sequences on a combed DNA
molecule is performed indirectly, by first hybridizing the
seeked nucleotidic sequences (the probes) with the
combed DNA (also called the matrix DNA or target)
If the probes are synthesized with incorporated
fluorescent molecules or antigenic sites which can be
recognized with fluorescent antibodies, the direct
visualization of the relative position of the probes is
possible.
Surgical Pathology: Special Study
ANALYTIC CYTOLOGY (FLOWCYTOMETRY)
Flowcytometry
o analyze populations of thousands of
disaggregated cells as they pass pass through a
fluid stream by means of one or more optical or
electronic sensors.
o measures multivariate physical, chemical and/or
biological characteristics of individual cells
o Applications
RBC analysis
Differential WBC Counts
Reticulocyte count
Platelet analysis
Bone marrow analysis
Applications of Flow Cytometry
Oncology
o DNA ploidy analysis DNA/RNA content
o Oncogene analysis
o Nuclear antigens (proliferation associated)
o Tumor markers (estrogen/progesterone
receptors
o Cell cycle analysis
o Rate of DNA synthesis in a tumor
Immunology
o Transplant applications
Pre-transplant
donor or reactive antibody
detection
HLA cross matching
Post-transplant
detection, diagnosis and
follow-up of transplant
rejection
o Immunophenotyping
Immunodeficiency diseases
o Cellular functional assays
Lymphocyte subset analysis
Mixed lymphocyte cultures
Microbiology
o Identification of bacteria using DNA stains
o Virus identification
o Identification of intracellular parasites
Endocrinology
o Sperm analysis by DNA content
o X:Y sperm sorting
Genetics
o Chromosome analysis
Blood Bank
o Red cell aging studies
What Can a Flow Cytometer Tell Us About a Cell?
Its relative size (Forward ScatterFSC)
Its relative granularity or internal complexity (Side
ScatterSSC)
Its relative fluorescence intensity
(FL1, FL2, FL3, FL4, and FL5)
Applications of Flow Cytometry
Used predominantly for measurement of
o cell surface antigens

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 Surgical Pathology: Special Study
o intracellular antigens
o and cellular nucleic acid (DNA & RNA) contents Antibodies in Immunophenotyping of Leukemias and
Qualitative and quantitative changes in these parameters Lymphomas
have been used to define normal and abnormal cellular
differentiation and function. Blasts
o Anti-terminal deoxynucleotidyl transferase (TdT)
Basic Components of Flow Cytometry T- and B-Lymphoblasts
Less in Myeloid blasts
o HLA-DR
Myeloid blasts and B-Lymphoblasts
o CD34
Myeloid
o CD13, CD33
o Anti-myeloperoxidase
Monocytic
o CD11b, CD14
Megakaryocyte
o CD41, CD61
Erythroid
o Anti-glycophorin
B-Lymphoid
o CD10, CD19, CD20, CD22
o Surface immunoglobulins
T-Lymphoid
o CD3, CD5, CD7

IMMUNOTYPING OF ACUTE LEUKEMIAS

5 major groups identified
o ALL (3 subgroups)
Precursor B-ALL
Light Scattering Burkitt B-ALL
Precursor T-ALL
o AML
Provide correlation with the FAB
subtype
Diagnosis of AML-M0 and AML-M7
o Acute Leukemias of Ambiguous Lineage

Normal Bone Marrow Immunophenotype

Lysed Whole Blood

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 Surgical Pathology: Special Study
Acute Myeloid Leukemia
PERIPHERAL BLOOD STEM CELL TRANSPLANTATION
Effective means of reconstituting patients hematopoietic
cells following high-dose chemotherapy
o Progenitor cell mobilization is accomplished by
use of G-CSF with peak reached in 5-10 days and
harvest is initiated at that time
o Use of CD34 monoclonal Ab enables detection of
stem cells, and is combined with other
antibodies to distinguish true progenitor cells

DIANOSIS AND MANAGEMENT OF AIDS

Confirmation of Diagnosis
o Uses immunoreactive microbeads to p24, p31,
p41 and gp120
o More sensitive and specific than ELISA or
Western Blot
CD4 & CD8 Lymphocyte counts
o Serial counts give good correlation with different
stages and onset of complications

AML S/P Chemotherapy TRANSPLANT APPLICATIONS

Detection of donor reactive antibodies in the serum of
potential recipients
o Hyperacute rejection is observed in allograft
recipients who have preformed antibodies
against their donors
o A FCM based cross-match is more sensitive than
traditional complement dependent technique
Evaluation of T lymphocyte subsets in recipients
o Expression of IL-2R on CD8 cells is good predictor
for acute rejection
Immune monitoring of transplant recipients receiving
OKT3 for acute rejection

Summary
Histopathologic morphology is still the basis of surgical
pathology
o It may be dangerous to base any distinction in
tumor pathology primarily on the basis of the
pattern of immunoreactivity of a given marker,
DNA ANALYSIS OF TUMORS no matter how specific it is purported to be.
DNA content/Ploidy analysis determines whether a tumor Juan Rosai, MD
has abnormal amount of DNA
o Using fluorescent dyes that bind to cellular DNA,
the specimens fluorescent intensity is directly
related to amount of DNA present
Cell cycle analysis determines the percentage of cells in
each phase of mitosis
Has prognostic and diagnostic significance depending on
tumor type
Common tumors for which DNA analysis is utilized:
o Breast
o Prostate
o Bladder
o Lymphoid

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