Molecular diagnostic techniques play an important role in elucidating the mechanisms of disease by identifying molecular and pathway alterations. These techniques rely on labeled probes and antibodies and include karyotyping, amplification, blotting, in situ hybridization, microarrays, and next generation sequencing. Molecular assays are used for diagnostic, predictive, carrier, prenatal, and pharmacogenomic testing. The structure of DNA and variations that can lead to disease are also discussed.

1. ROLES AND LIMITATION OF
MOLECULAR DIAGNOSTIC
TECHNIQUES
Presenter: Dr. Sakshi
Dubey
Moderator: Dr.
Manjunath GV

2. CONTENTS
Introduction
Structure of genome
Implicating molecular pathology in various
systemic diseases
Molecular diagnostic techniques
Recent advances in molecular pathology

3. INTRODUCTION
Molecular pathology can be broadly defined as the testing of nucleic acid
within a clinical context.
The purpose of molecular pathology is to elucidate the mechanisms of disease
by identifying molecular and pathway alterations
The applications of molecular diagnostics span a range of human disorders,
including hereditary, neoplastic, and infectious diseases.

4. Many of the techniques of molecular pathology rely on the use of labeled
antibodies and nucleic acid probes and are either slide-or fluid-based.
These are;
Karyotyping
Amplification techniques
Blotting methods
In situ hybridization (ISH)
 DNA Microarray
Next generation sequency

5. Molecular-based assays are used for specific purposes:
• Establishing the basis of an existing disorder (diagnostic testing)
• Determining the presence of a genetic condition when there are no obvious
symptoms (predictive testing)
• Carrier testing
• Assessing a fetus for abnormalities (prenatal testing)
• Detecting cancer-causing gene mutations
• Selecting pharmacotherapy

6. STRUCTURE OF DNA
Deoxyribonucleic acid, DNA, is a
double-stranded molecule
consisting of two antiparallel
polymers composed of
ribonucleosides linked together
by phosphodiester bonds.
Weak hydrogen bonding between
complementary bases allows for
easy denaturing and
reassociation of double-stranded
DNA.

7. Each chromosome is made up of
DNA tightly coiled many times
around proteins called histones that
support its structure.
A chromatid is one of the two
identical halves of a chromosome
that has been replicated in
preparation for cell division
representing one molecule of DNA.
The two “sister” chromatids are
joined at a constricted region of the
chromosome called the centromere.

8. THE HUMAN GENOME
In 1953, James D. Watson and
Francis H. C. Crick discovered the
double helical structure of DNA.
The genomes of any two people are
more than 99% similar; therefore the
small fraction of the genome that
varies among humans is very
important.
Variations in DNA can occur in the
form of genetic mutations in which a
base is missing or changed result in
an aberrant protein and can lead to
disease.

9. The natural history of a disease describes the expected course of disease,
including chronicity, functional impairment, and survival.
Not all patients with a given disease will naturally follow the same disease
course, so differences in patient outcome do not necessarily correspond to
incorrect diagnosis.
Variables that independently correlate with clinical outcome differences are
called independent prognostic variables, and are assessed routinely in an
effort to predict the natural history of the disease in the patient.
Understanding molecular
pathogenesis in disease causation..

10. Molecular pathogenesis describes the molecular alterations that occur in
response to;
Environmental insults
Exogenous exposures
Genetic predispositions, and other contributing factors to produce
pathology.
By developing a complete understanding of molecular pathogenesis,
1. The pathways that contribute to disease outcome can be elucidated.
2. Involvement of specific genes, proteins, and pathways in the causation of
specific diseases facilitate the development of targeted therapies for
particular diseases.

11. MOLECULAR PATHOLOGY OF HUMAN
DISEASE
NON- NEOPLASTIC CONDITIONS

12. DOWN’S SYNDROME
Karyotypes:
oTrisomy 21 type: 47,XX, +21
oTranslocation type:
46,XX,der(14;21)(q10;q10),+21
oMosaic type: 46,XX/47,XX, +21
Prenatal screening by the help of
polymorphic genetic marker or by
NGS can be done

13. TUBERCULOSIS
Diagnosis of TB has entered an era of
molecular detection that provides
faster and more cost-effective methods
for diagnosis.
There are various methods of detection
of drug resistance cases by molecular
techniques
Multiplex –PCR
Direct DNA sequencing analysis
Acid-fast bacilli

15. HCV INFECTION
The natural history of HCV infection
varies from patient to patient.
Through improved understanding
of the biology of the HCV virus and
its life cycle in the infected host,
Effective and sensitive diagnostic
tests like Real time RTPCR have
been developed
determination of hepatitis C
genotype, as a key tool, is essential

17. GLYCOGEN STORAGE DISEASES
•HEPATIC TYPE
Von Gierke disease (type I)
Point mutation
MYOPATHIC TYPE
•McArdle disease (type V)
• Point mutation
•MISCELLANEOUS TYPE
•Pompe disease (type II)
• Point mutation Lysosomal acid
alpha-glucosidase
Muscle
phosphorylase
Glucose-6-
phosphatase

18. SYSTEMIC LUPUS ERYTHEMATOSUS
SNP
Homozygous deficiency of
early complement
component cascade (C1q,
C2, or C4)
susceptibility to the
development of SLE

19. CHRONIC GRANULOMATOUS
DISEASE (CGD)
Genetic defects in the five
subunit enzyme NADPH oxidase
phagocytes are unable to produce
reactive oxygen species to kill
ingested pathogens
recurrent, indolent infections
caused by catalase positive
organisms including S. aureus and
Aspergillus spp.

20. CONGENITAL PRIMARY ADRENAL
INSUFFICIENCY
Etiologically
Adrenal Hypoplasia Congenita
(AHC)
• X-linked disorder caused by
inactivating mutations of NR0B1
(DAX1)
• defective development of the
adrenal glands
Congenital Adrenal Hyperplasia
(CAH)
• Inactivating mutations of
CYP21A2 and CYP11B1
• Inborn errors in adrenal steroid
biosynthesis

21. POLYCYSTIC KIDNEY DISEASE
(PCKD)
Renal cysts of tubular epithelial cell
origin are a hallmark of ADPKD and
ARPKD.
Causative genetic loci are:
 PKD1 and PKD2, which code for
the polycystins, and
 PKHD1, which codes for fibrocystin
Due to the size and complexity of
these genes, diagnosis is still made
predominantly using radiologic
findings and family history.

22. MOLECULAR BASIS OF HEMOSTATIC
DISEASES
The cascade of events that occur immediately after injury to the blood
vessels and preceding wound healing : vasoconstriction primary
hemostasis secondary hemostasis

23. Defect in primary
hemostasis
i. Von Willebrand
disease
ii. Platelet
dysfunction
(adhesion or
aggregation
defect)
Deficient thrombin
generation
i. Deficiencies of factor
VIII or IX
(hemophilia A and
B)
ii. Deficiencies of
factors in the final
common pathway of
thrombin generation
Defect in fibrin
polymerization
i. Deficiencies or
abnormalities of
fibrinogen
ii. Deficiency of factor
XIII (required for
cross linking fibrin)

24. MOLECULAR BASIS OF
THROMBOTIC DISEASES
Have a genetic
basis

25. PRIMARY CAUSES:
Deficiencies of protein C or
protein S
Prothrombin
G20210A mutation
Antithrombin III deficiency
Factor V Leiden
mutation causing
activated protein
C resistance

26. EMPHYSEMA
Smoking
excess of elastase production
centrilobular pattern of
emphysema
panacinar pattern of
emphysema
Mutant SERPINA1
gene

27. MOLECULAR PATHOLOGY IN NEOPLASTIC
CONDITIONS

28. SCHEMATIC REPRESENTATION
Initiating mutation ( LOF mutation / GOF mutation ) in
a single precursor cell
Clonal expansion GENETICALLY
IDENTICAL
Genetically heterogenous cancer Constituting various
subclones
Additional DRIVER
MUTATIONS
Additional PASSENGER
MUTATIONS

29. MOLECULAR BASIS OF MYELOID
AND LYMPHOID MALIGNANCIES
All types of differentiated blood cells originate from hematopoietic stem cells.
Growth factor receptor signaling and expression of specific hematopoietic
transcription factors regulate hematopoietic cell differentiation.
Mutations that cause hyperactive growth factor receptor signaling often
contribute to myeloid or lymphoid cancers.
Mutational inactivation of transcription factors that promote differentiation
often contributes to myeloid or lymphoid cancer.
Cancer-causing mutations can be spontaneous or inherited.

30. Acute myeloid leukemia with defining genetic
abnormalities may be diagnosed with <20% blasts.
A novel scalable model is introduced for
myeloid neoplasms with germline
predisposition with or without preexisting
thrombocytopenia or organ dysfunction
For acute leukemia of mixed or ambiguous
lineage, genetic abnormalities have been
defined.

31. ACUTE PROMYELOCYTIC
LEUKEMIA
It is a distinct subtype of AML that is cytogenetically characterized by a balanced
reciprocal translocation between chromosomes 15 and 17 [t(15;17)(q21;q21)],
which results in a gene fusion forms PML –RARa protein.
This disease is associated with a severe bleeding tendency and a fatal course of
only weeks in affected individuals.
With the introduction of all-trans retinoic acid (ATRA), the complete remission
rate increased from 75-80% ( with cytotoxic chemotherapy) to 90–95% and five-
year disease free survival improved to 74%.

32. NON- HODJKIN’S LYMPHOMA
Characteristic cytogenetic abnormalities are found
The translocation detection is done with help of conventional
cytogenetics or FISH or PCR
Subtypes of Lymphoma Cytogenetic abnormality
Follicular lymphoma t(14;18)(q32;q21)
Mantle cell lymphoma t(11;14)(q13;q32)
Burkitt’s lymphoma t(8;14)(q24;q32)
Anaplastic large cell
lymphoma
t(2;5)(p23;q35)

33. IN LUNG CANCER
Genetics of lung cancer:
• Oncogenes involved in the pathogenesis of lung cancer include MYC, K-RAS,
Cyclin D1, BCL2, and ERBB family genes such as EGFR and HER2/neu.
• TSG abnormalities involving TP53, RB and p16INK4a.
Targeted therapeutic agents in use and under investigation for treatment of lung
cancers include;
EGFR pathway inhibitors, VEGF/VEGFR pathway inhibitors, Ras/Raf/MEK
pathway inhibitors, PI3K/Akt/PTEN pathway inhibitors, tumor suppressor gene
therapies, others.

34. GASTROINTESTINAL
MALIGNANCIES

35. GASTRIC CARCINOMA
It is the fourth most frequent
cancer worldwide and the
second most common cause
of death from cancer.

36. THE PATHOGENESIS OF GASTRIC
CANCER IS MULTIFACTORIAL
Other environmental
factors:
-Dietary factors
-Smoking
Chronic gastritis related to H.
Pylori
Host genetic
susceptibility
• CDH1 – Hereditary
diffuse gastric cancer
syndrome
• STK11 – Peutz- jeghars
syndrome
• SMAD4 – Juvenile
polyposis
• MLH1 and MSH2 – Lynch
syndrome
• APC – Familial
adenomatous polyposis
• TP53- Li- Fraumeni
syndrome

37. COLORECTAL CANCER
Most colorectal cancers occur sporadically.
The molecular pathways of colon cancer development include a
stepwise acquisition of mutations, epigenetic changes, and alterations
of gene expression, resulting in uncontrolled cell division, and
manifestation of invasive neoplastic behavior.
The major molecular pathways of colorectal cancer development
include:
(1) The chromosomal instability pathway (CIN)
(2) The microsatellite instability pathway (MSI), or
(3) The CpG island methylator pathway (CIMP).

38. CHROMOSOMAL INSTABILITY
PATHWAY

39. MICROSATELLITE INSTABILITY
PATHWAY

40. Several hereditary colon cancer syndromes have been characterized;
Lynch syndrome (HNPCC)
 Autosomal dominant inheritance of germline mutations in one of the DNA
mismatch repair genes (most commonly in the MLH1 and MSH2),
leading to defects in the corresponding DNA mismatch repair.
 Early onset CRC (accounting for 4–6% of CRC) and tumors in other
organs (including endometrium, ovary, urothelium, stomach, brain, and
sebaceous glands).
Familial adenomatous polyposis (FAP)
 Autosomal dominant inheritance of germline mutations in the APC
gene on chromosome 5q
 Presence of numerous (sometimes >1000) adenomatous polyps
distributed throughout the colon and rectum.

41. In liver pathologies like;
Hepatitis, fibrosis, and other forms of chronic hepatic injuries
There is presence of progenitors or transiently amplifying cells
Predisposes the liver to neoplastic transformation
The cancer stem cells as a source of a subset of HCC is
often suggested.

42. HEPATOCELLULAR CARCINOMA
Chronic liver insults
Cirrhosis
Presence of regenerating nodules in the liver
Low- or high- grade dysplastic nodules
Hepatocellular carcinoma
• Aberrations in
many receptor
tyrosine kinases
and
• Other pathways
such as the Wnt/b-
catenin signaling
Agents targeting these pathways
are at various stages of
development for chemoprevention
and chemotherapy.

43. GYNAECOLOGICAL
MALIGNANCIES

44. CERVICAL CARCINOGENESIS
High-risk HPVs contribute to the genesis of almost all human cervical
carcinomas and have also been associated with a number of other
anogenital malignancies including vulval, anal, and penile carcinomas.
High-risk HPV E6 and E7 oncoproteins
These proteins and/or the processes that they regulate should
provide targets for intervention.
Initiation
Progressio
n
Maintenance of the
transformed
phenotype of cervical
cancer cells

46. ENDOMETRIAL TUMORS
Uterine cancer is the most common gynecologic cancer in the
United States.
It can be broadly divided into Type I and Type II categories,
based on
Risk factors such as unopposed estrogen use, polycystic ovarian
syndrome
Natural history, and
Molecular features.

47. TYPE I Normal
endometrium
Atypical endometrial
hyperplasia
Low grade Endometrioid
carcinoma
High- grade endometrioid
carcinoma
PTEN mutation
hMLH1
methylation
hMSH6 mutation
PTEN mutation
K-RAS mutation
ẞ-catenin mutation
TP53
mutation

48. TYPE II Atrophic
endometrium
Endometrial intraepithelial
carcinoma
Serous/ Clear cell
carcinoma
Genotoxic
stress
TP53 mutation
TP53 mutation
Loss of p16
HER 2/neu
amplification

49. MALIGNANT OVARIAN TUMORS..
The majority of malignant ovarian tumors in adult women are epithelial
ovarian cancer, which can be classified into serous, mucinous,
endometrioid, clear cell, transitional, squamous, mixed, and
undifferentiated. They all have different pathogenetic pathways.
BRCA1 and BRCA2 are the key genes involved in the development of
familial ovarian cancer.

50. Based on genetic analysis, it has been hypothesized that low-grade and
high-grade serous ovarian cancers are developed through a two-tier system.
Immunohistochemical stains for p53, p16, and Ki-67 for distinction of low-
from high-grade tumors
Type II pathway
a proportion appear to originate from
intraepithelial carcinoma in the
fallopian tube.
high-grade serous carcinomas have
high-grade nuclei and numerous mitotic
figures.
Type 1 pathway
adenofibromas or borderline tumors
Low-grade serous carcinomas exhibit
low-grade nuclei with infrequent
mitotic figures.
KRAS, BRAF, or ERBB2
TP53

51. MOLECULAR BASIS OF PROSTRATE
CANCER
Prostatic adenocarcinomas arise from precursor lesions termed proliferative
inflammatory atrophy (PIA) and prostatic intraepithelial neoplasia (PIN).
PIA lesions are characterized by:
 Epithelial damage,
 Regeneration, and
 Inflammatory cell infiltration
That appear in response to a variety of procarcinogenic stresses.

52. Heredity contributes significantly to the risk of prostate cancer
development
Inherited prostate cancer susceptibility genes/loci include;
RNASEL gene and MSR1 gene.
Somatic epigenetic alterations, like hypermethylation and
transcriptional silencing of several key genes, are the most abundant
and earliest genome abnormalities, evident in PIA and PIN as well as in
prostate cancer.

53. MOLECULAR CLASSIFICATION OF
BREAST CANCER
Major molecular subtypes of breast cancer are:

54. LUMINAL A
•Gene expression: Expression of Low molecular weight CKs,
High expression of hormonal receptors.
•Good Prognosis
•Responsive to endocrine therapy & variable response to
chemotherapy

55. LUMINAL B
•Gene expression: Expression of low molecular weight
cytokeratins, moderate-low expression of hormone
receptors.
•Prognosis not as good as Luminal A
•Responsive to endocrine therapy not as good as Luminal A
and variable response to chemotherapy (better than luminal
A)

56. HER2/NEU
•Gene expression: High expression of Her2/neu, low
expression of ER. Diffuse TP53 mutation
•Usually unfavourable prognosis
•Response to Trastuzumab (Herceptin) and response to
chemotherapy with anthracyclins.

57. BASAL LIKE
•Gene expression: High expression of basal ( high molecular
weight) cytokeratins, low expression of ER and HER 2/neu.
•Usually worse prognosis
•No response to Endocrine therapy or Trastuzumab and
sensitive to Platinum based chemotherapy and PARP
inhibitors.

58. MOLECULAR BASIS OF CNS
TUMORS
Gliomas are primary CNS neoplasms that have genetic defects associated
with cell cycle control and proliferation, apoptosis, cell motility, and invasion.
These tumors are generally diffusely infiltrative and cannot be cured by
resection.
Specific genetic abnormalities are recognized to drive both the histologic
appearance and behavior of gliomas.

59. ASTROCYTOMAS
Grade I or pilocytic astrocytoma: KIAA1549-BRAF gene fusion/duplication
Grade II or diffuse astrocytoma: IDH1 and IDH2 mutation
Grade III or anaplastic astrocytomas: IDH1 and IDH2 mutation
Grade IV or Glioblastomas:
o Primary Glioblastoma: IDH- WT, gains of Chr.7, loss of Chr.10, TERT
promotor mutations, EGFR gene amplification
o Secondary Glioblastoma: IDH1 and IDH2 mutation

60. OLIGODENDROGLIOMAS
Low-grade oligodendrogliomas: IDH mutation with codeletion of 1p
and 19q
Anaplastic oligodendrogliomas: IDH mutation, codeletion of 1p and
19q, deletion of CDKN2A TSG and TERT- promotor mutation

61. PRACTICE OF MOLECULAR
PATHOLOGY

62. MOLECULAR DIAGNOSTIC
TECHNIQUES
The techniques used for molecular diagnosis involves
manipulation, analysis of DNA and RNA.
These techniques have utility in every area of
diagnostic pathology
Neoplastic disorders
Infectious diseases
Inherited conditions and
Identity determination.

63. CYTOGENETIC STUDY- KARYOTYPING
Cytogenetics involve study of chromosomes
Both the number and structure of chromosomes are
analyzed..

65. STEPS OF
KARYOTYPING

66. G-banding/chromomosome
morphology

67. IDENTIFYING CHROMOSOMES

68. ANEUPLOIDY

69. Changes in chromosome structure

70. APPLICATIONS

71. LIMITATIONS

72. OTHER TECHNIQUES..

73. AMPLIFICATION
TECHNIQUES

74. POLYMERASE CHAIN REACTION
(PCR)
Developed by Kary Mullis
in 1984
Test tube method
Amplifies a selected DNA
segment.

75. PCR
CYCL
E

78. APPLICATIONS OF PCR
In recent years, the role of PCR in diagnostics has
increased.
The use of PCR in clinical settings can be broadly
divided into 3 categories:
To amplify human genes to check for mutations
To amplify microbial genes in a sample
To amplify human genes from a limited samples to
complete a DNA profile of an individual.

79. LIMITATIONS
1. PCR cannot be used to amplify unknown targets.
Prior information about the target sequence is
necessary to design the primers.
2. DNA polymerases are prone to error
3. DNA Polymerases are sensitivity to inhibitors
4. PCR is very sensitive to contamination.

84. APPLICATIONS
•It is used for gene mapping.
•To analyze the genetic patterns which appear in a
person’s DNA.

87. APPLICATION
•It serves as a standard for the study of gene
expression at the level of mRNA.
•Detection of mRNA transcript size.

90. APPLICATIONS
•It is the confirmatory HIV test
•Definitive test for Bovine spongiform encephalopathy
(BSE)

91. LIMITATION
Southern blotting - requirement for large amounts of input
DNA
Nothern blotting - only one gene is analyzed at a time, with
an important amount of RNA and reagents required
Western blotting - it can only be carried out if a primary
antibody against the protein of interest is available

92. HYBRIDIZATION
TECHNIQUES

95. (FISH)

96. Whole chromosome paint
Telomere
Locus
Centromer
e

102. APPLICATIONS OF FISH

103. LIMITATIONS
•The design, preparation, and labeling of these probes is still
very labor-intensive.
•The experimental implementation of FISH is time-
consuming and requires experienced personnel.
•Even if probes are chemically synthesized, depending on
the nucleic acid length and fluorescent labels, they can be
expensive.

104. CYTOGENOMIC ARRAY
TECHNOLOGY
• Global genomic survey in which genomic abnormalitites
can be detected without prior knowledge
•Platforms:
CGH arrays
SNP arrays Routinely used in labs

105. WORKFLOW

106. APPLICATIONS
•To uncover copy number abnormalities in paediatric
patients.
•To diagnose disorders caused by uniparental disomy.
LIMITATIONS
•Reliance upon existing knowledge about the genome
sequence.
•Limited dynamic range of detection owing to both
background and saturation signals

107. NEXT GENERATION SEQUENCING
Principles:
1. Spacial separation
2. Local amplification
3. Parallel sequencing

109. APPLICATIONS
•to sequence the whole genome rapidly.
•To sequence the target regions deeply.
•Novel pathogens identification.
•Study the rare somatic variants, tumor subclones, and more
by sequencing cancer samples.
•used for ‘gene therapy’ by identifying the isolated genes and
providing the correct copy of that defective gene.

110. LIMITATIONS
•It requires different infrastructures such as computer
capacity and storage and staff for analyzing and interpreting
the subsequent data.
•Trained personnels are required to process this raw,
sequenced data.

119. RECENT
ADVANCES

120. BREAST CARCINOMA
NGS can simultaneously detect alterations of multiple genes
involved in breast cancers.
Clinically actionable genomic changes include:
ERBB2 (HER2) amplification (prevalence~15%),
BRCA1/BRCA2 mutations (5%–10%)
Poly-ADP-ribose polymerase (PARP) inhibitors are FDA approved targeted
therapy for BRCA1/BRCA2 associated breast cancer.,
PIK3CA mutations (30%–40%)
API3K (phosphoinositide 3-kinase) inhibitor, alpelisib, is FDA approved for
advanced breast cancers that are PIK3CA altered, HER2 (-) and hormone
receptor (+).

121. MULTIGENOMIC PROGNOSTIC PROFILING Is
being used to predict the risk of breast cancer
recurrence:
1. Oncotype DX® (Genomic Health Inc.):
21 gene RTPCR assay
For predicting the likelihood of recurrence and chemotherapy benefit in
hormone receptor (+) patients on endocrine therapy.
2. MammaPrint (Agendia, Inc.):
70 gene expression profile by microarray for predicting the risk of distant
metastasis.
Patients with high clinical risk but low genomic risk based on MammaPrint
might not require chemotherapy.

122. 3. EndoPredict® (Myriad Genetics):
11 gene RNA expression profile
For predicting the risk of recurrence in hormone receptor (+) / HER2 (-)
patients who are node (+) or (-) on endocrine therapy.
4. Prosigna® (Veracyte):
50 gene RTPCR assay
For assessing the risk of recurrence in 10 years for hormone receptor (+)
early stage breast cancer and identifying the subset of patients who may not
benefit from chemotherapy.

123. RENAL CELL CARCINOMA
Clear cell RCC
Recurrent VHL alterations and 3p deletion
BAP1 or SETD2 mutated RCCs behave aggressively
Papillary RCC
Type 1 Chromosome 7 or 17 polysomy and trisomy
Type 2 CDKN2A silencing, SETD2 mutations and other alterations.
Hybrid oncocytic chromophobe tumors (HOCT) are associated with Birt-Hogg-
Dubé syndrome (BHD) or renal oncocytosis while others are sporadic. BHD
can be confirmed by germline mutations in the FLCN tumor suppressor
gene.

124. Renal cell neoplasms with TFE3, TFEB and MITF rearrangements are now
grouped together as MiT family translocation RCC.
Molecular profiling has helped to define new and emerging RCC variants, e.g.,
 Eosinophilic solid and cystic RCC associated with tuberous sclerosis (TSC1 or
TSC2 gene alterations)
 RCC with TSC/MTOR mutations
 TCEB1 mutated RCC
 RCC with TFEB/6p21/VEGFA amplification
 ALK rearranged RCC

125. BRAIN TUMORS
Primary glioblastoma (GBM)
10q loss of heterozygosity (LOH) (70%),
EGFR amplification (36%),
CDKN2A deletion (31%) and
PTEN mutations (25%)
MGMT promoter hypermethylation (30%–40% of primary GBM) predicts
improved response to alkylating agents and a lower risk of tumor recurrence.
Associated with improved survival, IDH1 or IDH2 mutations are common in;
Grade II and III astrocytoma
Oligodendroglioma and Secondary GBM
Denote aggressive
disease

126. Co-deletion of 1p / 19q (detectable by
FISH / PCR / microarray) is
characteristic of oligodendroglioma
predicting response to conventional
therapy.
BRAF fusion, most commonly with
KIAA1549, is observed in 59%–90%
of pilocytic astrocytomas.
Co-deletion of 1p (B) and 19q (C) by dual color
FISH. Target (orange) to control (green) signal
ratio of ≤ 0.8 indicates loss of the target allele.

127. IMMUNE CHECKPOINT INHIBITOR
BIOMARKER
Programmed cell death ligand 1 (PDL1) immunohistochemistry (IHC) uses
various scoring systems and cutoffs for particular antibody clones to quantify
PDL1 expression as a predictive biomarker for specific FDA
approved ICI.

128. IN NSCLC…
Non small cell lung carcinoma (NSCLC)
PDL1 pharmDX 22C3 immunostain
Pembrolizumab immunotherapy.
The PDL1 SP142 clone (given by either the % of tumor area occupied
by PDL1 expressing tumor infiltrating immune cells of any intensity or
the % of PDL1 expressing tumor cells of any intensity)
-is an FDA approved complementary diagnostic to predict response of
metastatic NSCLC to atezolizumab.
+

129. PDL1 SCORING SYSTEMS
The tumor proportion score (TPS) is calculated by dividing the
total number of PDL1 positive tumor cells by the total number of
tumor cells (≥ 1% positive cutoff).
The combined positive score (CPS) is based on dividing the
number of PDL1 positive cells (tumor cells, lymphocytes,
macrophages) by the number of tumor cells to predict
pembrolizumab efficacy in
Metastatic gastric and gastroesophageal junctional carcinoma,
Bladder cancer and
Cervical cancer

130. ICI efficacy
Recently, Foundation Medicine’s FoundationOne® CDx, which
includes MSI and TMB assessment, was approved as a companion
diagnostic for pembrolizumab with a cutoff of ≥ 10 mutations per
megabase, regardless of solid tumor type.
Deficient mismatch repair
(dMMR) / high
microsatellite instability
(MSI-H)
High tumor mutational
burden (TMB)

131. REFERENCES
•Anderson: Diagnostic molecular pathology. Pg-199-122,2009
•U. Satyanarayana, U. Chakrapani, Biochemistry, Arunabha Sen
Baaks Pvt. Ltd,2010
•George J.N, Rana D.S, and Peter A.D. Diagnostic molecular
pathology: current techniques and clinical applications. 2003
Oct; 16: 379-283
•William B. Coleman, Gregory J Tsongalis Essential concepts in
molecular pathology.
•Dr. Tsang, Dr. Chen. Whats new in molecular pathology