LP 6 TesteMoleculareGeneticeDermatologie

Disciplina de Dermatologie, Universitatea de Medicină Victor Babeş Timişoara
METODE DE DIAGNOSTIC MOLECULAR SI

GENETIC IN DERMATOLOGIE
1
The Central Dogma
• The central dogma (due to
Francis Crick in 1958) states
that information flows are all
unidirectional:
“The central dogma states that
once `information' has
passed into protein it cannot
get out again.”
Phenotype
Genotype
Genome Selection
DNA RNA Protein
Evolution
Transcription Translation
• Oncogenes: BAX, BCL2L1, CASP8, CDK4, ELK1, ETS1,
HGF, JAK2, JUNB, JUND, KIT, KITLG, MCL1, MET, MOS,
MYB, NFKBIA, NRAS, PIK3CA, PML, PRKCA, RAF1,
RARA, REL, ROS1, RUNX1, SRC, STAT3, ZHX2.
• Tumor Suppressor Genes: ATM, BRCA1, BRCA2,

CDH1, CDKN2B, CDKN3, E2F1, FHIT, FOXD3, HIC1,
IGF2R, MEN1, MGMT, MLH1, NF1, NF2, RASSF1,
RUNX3, S100A4, SERPINB5, SMAD4, STK11, TP73,
TSC1, VHL, WT1, WWOX, XRCC1.
• Oncogenic & Tumor Suppressor Properties: BCR, EGF,

ERBB2, ESR1, FOS, HRAS, JUN, KRAS, MDM2, MYC,
MYCN, NFKB1, PIK3C2A, RB1, RET, SH3PXD2A,
TGFB1, TNF, TP53.
• Transcription Factors: ABL1, BRCA1, BRCA2, CDKN2A,

CTNNB1, E2F1, ELK1, ESR1, ETS1, FOS, FOXD3, HIC1,
JUN, JUNB, JUND, MDM2, MEN1, MYB, MYC, MYCN,
NF1, NFKB1, PML, RARA, RB1, REL, RUNX1, RUNX3,
SMAD4, STAT3, TGFB1, TNF, TP53, TP73, TSC1, VHL,
WT1, ZHX2.
• Epithelial-to-Mesenchymal Transition: BRCA2,

CDKN2B, CTNNB1, ERBB2, HGF, JAK2, KIT, MCL1, NF1,
RUNX3, S100A4, SMAD4, TGFB1, VHL.
• Angiogenesis: AKT1, CTNNB1, EGF, ERBB2, NF1, PML,

RUNX1, TGFB1.
• Apoptosis: BAX, BCL2, BCL2L1, BRCA1, CASP8, E2F1,

MCL1, MGMT, TNF, VHL.
• Cell Adhesion: APC, CDH1, CDKN2A, CTNNB1, KITLG,

NF1, NF2, TGFB1.
• Cell Cycle: ATM, BRCA1, BRCA2, CCND1, CDK4,

CDKN1A, CDKN2A, CDKN2B, CDKN3, E2F1, HGF,
MEN1, STK11, TP53.
• Chemotaxis, Cell Migration & Motility: HRAS, JAK2,

MET, NF1, NF2, PRKCA, SERPINB5, STAT3.
• DNA Damage & Repair: ABL1, APC, ATM, BRCA1,

BRCA2, CDKN1A, MEN1, MGMT, MLH1, PML, TP53,
TP73, XRCC1.
Immunohistochemistry (IHC)
Cytoplasmic marker
Nuclear markers Membranous Marker
Immunohistochemistry
• Immunohistochemistry or IHC refers to the process
of localizing antigens (e.g. proteins) in cells of a
tissue section exploiting the principle of antibodies
binding specifically to antigens in biological
tissues.
• Immunohistochemical staining is widely used in the
diagnosis of abnormal cells such as those found in
cancerous tumors.
• Specific molecular markers are characteristic of
particular cellular events such as proliferation or
cell death (apoptosis).
• IHC is also widely used in basic research to
understand the distribution and localization of
biomarkers and differentially expressed proteins in
different parts of a biological tissue.
• Visualising an antibody-antigen interaction can be
accomplished in a number of ways. In the most
common instance, an antibody is conjugated to an
enzyme, such as peroxidase, that can catalyse a
colour-producing reaction.
Surface Markers - CD
• CD markers, an abbreviation for human cluster of differentiation markers, are a classification
system for monoclonal antibodies against cell surface molecules on leukocytes and antigens
from other cells.
• Currently, more than 400 CD markers have been identified, although not all of them are of
diagnostic value.
• Immunophenotyping can be used on paraffin-embedded samples, frozen sections, or with flow
cytometry.
• When faced with a possible cutaneous lympho-proliferative disorder, the dermatopathologist
evaluates the overall histological architectural pattern of the biopsy.
• Interpretation of CD marker staining on fixed tissue samples should be based on the cellular
distribution of staining (i.e., membranous, cytoplasmic, nuclear).
• Negative and positive controls are also used in the staining process to allow for comparison, to
confirm the specificity and sensitivity of the staining process, and to assist in determining the
affinity of a particular stain.
• The first step in the immunophenotypic evaluation is determination if the dominant population of
cells are B-cells, T-cells, or neither.
• Three markers are typically used for this initial classification: CD20, CD3, and CD45.
– T-cell processes are typically CD3+, CD20-, CD45+.
– B-cell processes are typically CD3-, CD20+, and CD45+.
• CD markers are specific for a particular cell type or origin, but there can be overlap.
• CD markers serve as an imperfect attempt to identify and classify some neoplastic cells. It is
probably more accurate and practical to state that the pattern of CD marker expression is
strongly suggestive of a certain cell type or lineage, but may not be definitive
Flow cytometric immunophenotyping
• Some antibodies do not work with sections

cut from paraffin-embedded samples or with
frozen sections and necessitate flow
cytometry. However, flow cytometry requires
that cells being immunophenotyped be
individually suspended in liquid, an easy
task for circulating cells in peripheral blood
samples, but more complicated when
dealing with skin samples.
• It allows simultaneous multiparametric
analysis of the physical and/or chemical
characteristics of up to thousands of
particles per second.
• Flow cytometry uses the principles of light
scattering , light excitation, and emission of
fluorochrome molecules to generate specific
multi-parameter data from particles and cells
in the size range of 0.5um to 40um diameter.
• In the flow cytometric evaluation of mature B-cell lymphoid neoplasms, it is useful to consider 4 broad groups as determined by their expression of CD5 and CD10.
• For each group, additional flow cytometric data in combination with the morphology can narrow down the diagnostic.
• Among mature lymphoid neoplasms with a T-cell phenotype, expression of CD4 and CD8 can be used to formulate a list of diagnostic
possibilities and determine what additional information is required for further classification
Clonality Diagnosis
T-Cell Receptor and Immunoglobulin Gene
Rearrangements in Diagnosing Skin Disease
• The most important advance in the molecular immunological features of lymphomas has been the
recognition that each normal T and B cell bears a unique antigen receptor on its cell surface that
serves as a specific marker for that cell and all of its clonal progeny.
• If the cell should undergo malignant transformation, then this same structure becomes a tumor-
specific marker, as well.
– For B cells, this marker is the immunoglobulin (Ig) molecule.
– For T cells, it is the T-cell receptor (TCR).
B-Cell Receptor
• B cell development occurs through several stages, each stage representing
a change in the genome content at the antibody loci.
• An antibody is composed of two identical light (L) and two identical heavy
(H) chains, and the genes specifying them are found in the 'V' (Variable)
region and the 'C' (Constant) region.
• In the heavy-chain 'V' region there are three segments; V, D and J, which
recombine randomly, in a process called VDJ recombination, to produce a
unique variable domain in the immunoglobulin of each individual B cell.
• Similar rearrangements occur for light-chain 'V' region except there are only
two segments involved.
• The B-cell receptor is a
transmembrane receptor protein
located on the outer surface of B-
cells.
• When a B-cell is activated by its
first encounter with an antigen that
binds to its receptor (its "cognate
antigen"), the cell proliferates and
differentiates to generate a
population of antibody-secreting
plasma B cells and memory B cells.
Stage Heavy chain Light chain

Progenitor (or pre-pro) B cells germline germline
Early Pro (or pre-pre)-B cells undergoes D-J rearrangement germline
Late Pro (or pre-pre)-B cells undergoes V-DJ rearrangement germline
Large Pre-B cells is VDJ rearranged germline
Small Pre-B cells is VDJ rearranged undergoes V-J rearrangement
Immature B cells is VDJ rearranged VJ rearranged
Mature B cells is VDJ rearranged VJ rearranged
T-Cell Receptor
• The TCR, which is anchored in the cell membrane,
consists of two halves which form a pair (or dimer) of
protein chains. The halves are called the alpha (α) and
beta (β) fragments (in γ/δ T cells, the halves are gamma
(γ) and delta (δ) fragments).
• Each fragment is divided in turn into a constant (C) and
variable (V) region. The constant region has an end which
is anchored in the cell membrane.
• The variable region faces outward and binds to the HLA
molecule and the antigen it presents. On the α chain, the
variable region is called Vα and the constant region is
called Cα; on the β chain they are called Vβ and Cβ
respectively.
• Processes for TCR formation are similar to those

described for B cell antigen receptors
• The TCR alpha chain is generated by VJ recombination,

whereas the beta chain is generated by V(D)J
recombination (both involve a somewhat random joining of
gene segments to generate the complete TCR chain).
• Similarly, generation of the TCR gamma chain involves VJ
recombination, whereas generation of the TCR delta chain
occurs by V(D)J recombination.
Clonality
• Mycosis fungoides can arise from a background of chronic inflammation via the gradual selection
of one dominant T-cell clone that becomes increasingly malignant over time, probably as a result
of sequential somatic mutations.
• Cutaneous patches containing superficial T-cell infiltrates with deletion of certain antigens and the
presence of dominant clonality are often diagnosed as mycosis fungoides, even when the
histopathological features are not fully diagnostic.
• Clinically nodular skin lesions composed of atypical lymphoid infiltrates that exhibit abnormal
patterns of antigen expression and contain molecular evidence of dominant clonality are usually
regarded as lymphomas, even when this diagnosis cannot be made on morphological grounds
alone.
• Thus, the principle has emerged that cutaneous lymphomas do not necessarily arise de novo but
can instead develop gradually from different types of chronic inflammatory processes.
Clonality
• Once a diagnosis of lymphoma has been established, TCR or IgH gene rearrangement assays
can also be used to determine the disease stage of patients and to monitor their response to
therapy.
• Occult involvement of lymph nodes by mycosis fungoides is prognostically relevant.
• Because of their enhanced sensitivity relative to routine histological testing, molecular assays
can more accurately define remission and detect early relapse.
• Patients who stop treatment when representative skin biopsy specimens are nonspecific
histologically but still positive by molecular analysis tend to relapse rapidly.
Polymerase Chain Reaction (PCR)
and
Single Nucleotide Polymorphism
What Is the Human Genome?
Human Cell
Nucleus
Chromosomes
DNA and Chromosome Structure
Chemical
DNA molecule bases
(chromosome)
A
G
C
The Genome Contains Genes
Gene 1 Coding region Protein 1
Noncoding region
Gene 2 Coding region Protein 2
Noncoding region
Variation in the Human Genome
Person 1 Person 2
= Variations in DNA
What Is Variation in the Genome?
Common Sequence
Variations
Polymorphism
Deletions
Insertions
Chromosome
Translocations
Variations Causing No Changes
= Variations in DNA that cause no changes

Variations Causing Harmless Changes
= Variations in DNA that cause harmless changes

Variations Causing Latent Changes
= Variations in DNA that cause latent effects

Many years later Many years later
SNPs Are the Most Common
Type of Variation
At least 1 percent
Most of the population of the population
G to C
Common Variant
sequence sequence
SNP
site
Why Are SNPs Significant?
Person 1 Person 2
Gene A Gene B
SNP marks Gene A SNP may cause Gene B

to make altered protein
= SNP variations in DNA
Amino Acids
20 Different Amino Acids
Amino group
Carboxyl group
Lysine side chain Lysine
Graphic Representation
of an Amino Acid
Basic Structure
of an Amino Acid
Genes to Proteins I
A T
U A
G C
C G
G C
U A
U A
A T
U A
A T
C G
G C
U A
A T
A T
DNA mRNA
Genes to Proteins II
Genes to Proteins III
Methionine
Arginine
Ribosome
Threonine
tRNA
Tyrosine
mRNA
A U G C G U U A U A C G U A A
Methionine Tyrosine Stop

Codons:
AUG=Methionine=Start
Arginine Threonine CGU=Arginine
UAU=Tyrosine
ACG=Threonine
UAA=Stop
Protein Folding and Function
Amino acid chain grows
and folds
into a 3-D structure.

SNPs in Coding Regions –
No Changes in Protein
GAC GAG
DNA SNP C to G
mRNA
CUG CUC
RNA Codon
CUG to CUC
CUG CUC
Leucine Protein Leucine

Leucine to Leucine
No change in shape
Subtle, Harmless Changes in Protein
CTA CTC
DNA SNP A to C
mRNA
GAU GAG
RNA Codon
GAU to GAG
GAU GAG
Aspartic acid Protein Glutamic acid

Aspartic acid
to Glutamic acid
Slight change in shape

Harmful Changes in Protein – Mutations
C T A DNA SNP T to A C A A
mRNA
GAU GUU
RNA Codon
GAU to GUU
GAU GUU
Aspartic acid Protein Valine

Aspartic acid
to Valine
Change in shape
PCR Requirements
• Magnesium chloride: .5
2.5mM
• Buffer: pH 8.38.8
• dNTPs: 20200µM
• Primers: 0.10.5µM
• DNA Polymerase: 12.5
units
• Target DNA:  1 µg
Gel electrophoresis
Heterozygous = having two

different alleles for a single
trait.
– Wild type
– Mutant
Homozygous = having identical

alleles for a single trait.
Subtle Changes in Proteins
That Only Switch on Under Certain Conditions
Smoking
Switched-on
genes
Pattern A Pattern B
Many years later Many years later
= SNPs causing latent effects

SNP Profiles and Response to
Drug Therapy
Breast Cancer Patients
Individual SNP Profiles Are Sorted
Responds to Standard Drug Treatment Does Not Respond to Standard Drug Treatment
SNP profile A SNP profile B
SNP profile E SNP profile C
SNP profile D
Gene SNP
TNFa Chromosome: 6; Location: 6p21.3 rs2228088, rs3179060, rs35131721, rs4645843, rs1800620, rs1800618, rs11574936, ………
IL-1a Chromosome: 2; Location: 2q14 rs3783588, rs55910084, rs1801715, rs3783581, rs17562, rs17561, rs61538608, rs20540, rs3783531,
………
IL-2 Chromosome: 4; Location: 4q26-q27 rs1051753, rs2069763, rs3087209, ………
IL-4 Chromosome: 5; Location: 5q31.1 rs4986964, rs56279116, rs55743996, rs35648164, rs71645915, ………
IL-6 Chromosome: 7; Location: 7p21 rs34280821, rs2069830, rs11544633, rs56383910, rs34012176, rs71708959,
rs2069860, rs13306435, rs34709428, rs2069849, ………
IL-8 Chromosome: 4; Location: 4q13-q21 rs1803205, rs71745371, ………
IL-12 Chromosome: 5; Location: 5q31.1-q33.1 rs34012639, rs55780930, rs2230052, rs56272177, rs35990253, rs55691228, rs56043315, rs1042154,
rs1042155, ………
IL-13 Chromosome: 5; Location: 5q31 rs55733734, rs56035208, rs34255686, rs34654684, rs20541, rs56258826, ………
IL-17 Chromosome: 6; Location: 6p12 rs17880588, rs17878530, ………
IL-22 Chromosome: 12; Location: 12q15 rs2227507, ………
IL-23 Chromosome: 12; Location: 12q13.3 rs61937689, rs11465746, rs11171806, rs71772333, ………
VEGF Chromosome: 6; Location: 6p12 rs25648, rs45533131, rs62401172, ………

Stanford University
Adalimumab, Etanercept
• rs983332 at chr1:87904968
This variant is significantly associated with the efficacy of anti-TNF (Adjusted P-value: 0.000005; OR: 10.2 (2.6, 59.2)). The study is a genome-wide association study using the Illumina HapMap300
SNP chip on 89 RA patients prospectively followed after beginning of anti-TNF therapy.
• rs928655 at chr1:89622162 in GBP6
This variant is significantly associated with the efficacy of anti-TNF (Adjusted P-value: 0.00003; OR: 5.5 (1.8, 20.2)). The study is a genome-wide association study using the Illumina HapMap300 SNP
chip on 89 RA patients prospectively followed after beginning of anti-TNF therapy.
• rs13393173 at chr2:169097337 in LASS6
• rs437943 at chr4:35048493
• rs1800629 at chr6:31651010 in LTA, TNF
The TNF:(-308)G>A polymorphism is a weak marker for response to anti-TNF treatment, with A-allele carriers being significantly less likely to respond than patients with the GG genotype.
• rs10945919 at chr6:164106667
• rs854547 at chr7:94761792 in PPP1R9A
This variant is significantly associated with the efficacy of anti-TNF (Adjusted P-value: 0.000006; OR: 3.6 (1.5, 9.3)). The study is a genome-wide association study using the Illumina HapMap300 SNP
chip on 89 RA patients prospectively followed after beginning of anti-TNF therapy.
• rs854548 at chr7:94763756 in PON1, PPP1R9A
• rs854555 at chr7:94768327 in PON1
• rs868856 at chr9:27479251 in MOBKL2B
• rs3849942 at chr9:27533281
• rs774359 at chr9:27551049 in C9orf72
• rs6138150 at chr20:23795009
• rs6028945 at chr20:38254219
• rs6071980 at chr20:38301990
Stanford University
Infliximab
• rs1061622 at chr1:12175542 in TNFRSF1B
For this SNP in the TNFRSF1B gene a significant correlation was found between 196R allele carriers and low response to infliximab therapy.
• rs983332 at chr1:87904968
This variant is significantly associated with the efficacy of anti-TNF treatment (Adjusted P-value: 0.000005; OR: 10.2 (2.6, 59.2)). The study is a genome-wide association study using the Illumina HapMap300
• rs928655 at chr1:89622162 in GBP6
• rs396991 at chr1:159781166 in FCGR3A
This variant may be a useful marker to predict response to infliximab in Japanese patients with rheumatoid arthritis.
• rs13393173 at chr2:169097337 in LASS6
• rs437943 at chr4:35048493
• rs1800629 at chr6:31651010 in LTA, TNF
The TNF:(-308)G>A polymorphism is a weak marker for response to anti-TNF treatment, with A-allele carriers being significantly less likely to respond than patients with the GG genotype.
• rs10945919 at chr6:164106667
• rs854547 at chr7:94761792 in PPP1R9A
• rs854548 at chr7:94763756 in PON1, PPP1R9A
• rs854555 at chr7:94768327 in PON1
• rs3849942 at chr9:27533281
• rs774359 at chr9:27551049 in C9orf72
• rs6138150 at chr20:23795009
This variant is significantly associated with the efficacy of anti-TNF treatment (Adjusted P-value: 0.000003; OR: 11.1 (2.5, 103.3)). The study is a genome-wide association study using the Illumina
HapMap300 SNP chip on 89 RA patients prospectively followed after beginning of anti-TNF therapy.
• rs6028945 at chr20:38254219
This variant is significantly associated with the efficacy of anti-TNF treatment (Adjusted P-value: 0.0000002; OR: 11.2 (2.3, 108.1)). The study is a genome-wide association study using the Illumina
HapMap300 SNP chip on 89 RA patients prospectively followed after beginning of anti-TNF therapy.
Stanford University
Methotrexate
• rs4846051 at chr1:11777044 in MTHFR
The C allele of this variant was associated with increased risk of toxicity in African American Rheumatoid Arthritis patients receiving methotrexate.
• rs1801131 at chr1:11777063 in MTHFR
At 6 months methotrexate and folic acid therapy, of early rheumatoid arthritis patients with the MTHFR 1298AA genotype showed good improvement relative to
combined CA and AA genotypes (OR 2.3), while 1298C allele carriers developed more adverse drug events (OR 2.5) (e.g. pneumonitis, gastrointestinal ADEs, skin
and mucosal ADEs, and elevated liver
• rs1801133 at chr1:11778965 in CLCN6, MTHFR
This variant is associated with methotrexated-induced mucositis, thrombocytopenia and hepatic toxicity
In 330 patients who completed 3 months methotrexate treatment for psoriasis, no significant genotypic associations were found between clinical outcome (e.g.
efficacy, toxicity) and 50 SNPs in pathway genes for methotrexate metabolism (ATIC, FPGS, GGH, MTHFR), including 47 common ( >5% minor allele frequency)
haplotype-tagging SNPs (r(2) > 0.8) plus 3
MTHFR rs1801133, 667CT or 667TT genotypes were associated with an increased risk of methotrexate treatment discontinuation due to adverse events (relative
risk 2.01), mostly as a result of increased risk of elevated levels of liver enzyme alanine aminotransferase (relative risk 2.38) in rheumatoid arthritis patients.
In a retrospective analysis of 61 Italian patients experiencing methotrexate toxicity during treatment for acute lymphoblastic leukemia or acute promyelocytic
leukemia, carriers of the MTHFR 677TT genotype (60%) showed significantly greater drug-induced toxicity (p=0.03) compared to CC and CT genotypes.
Risk or phenotype-associated allele: CT and TT genotypes. Phenotype: The 677 CT or TT genotypes were associated with greater incidence of discontinuation of
methorexate treatment because of adverse events, mainly due to elevation of liver enzymes. Study size: 236. Study population/ethnicity: Patients who started
methorexate treatment with (n = 157) or without (n = 79) folic or folinic acid supplementation for rheumatoid arthritis. Significance metric(s): RR = 2.01 Type of
association: CO, GN.
• rs13120400 at chr4:89252551 in ABCG2
SNP is associated with clinical reponse to methotrexate in patients with psoriasis.
• rs17731538 at chr4:89274403 in ABCG2
SNP is associated with clinical reponse to methotrexate in patients with psoriasis.
• rs11545078 at chr8:64101318 in GGH
GGH 452C>T has been associated with decreased catalytic activity and higher accumulation of long-chain methotrexate-polyglutamate.
• rs1800909 at chr8:64113866 in GGH
This study found that only patients with the GGH 16C-allele and one or no copies of the GGH 452C-16T haplotype were associated with good clinical improvement
at 3 months upon treatment with methotrexate.
• rs4149081 at chr12:21269288 in SLCO1B1
Risk or phenotype-associated allele: G allele, with additive genotypic effect. Phenotype: Genome-wide analysis of 398,699 germline SNPs showed association of
the rs4149081 G allele with increased methotrexate (MTX) plasma clearance, with an additive effect per G allele (increase of 12.7 mL/min/m(2) per allele in 434
subjects), after adjusting for age, race, sex, and MTX regimen. Variants rs11045879 and rs4149081 were in linkage disequilibrium (r(2) = 1). The G allele was
associated with increased risk of gastrointestinal toxicity (mucositis) (OR = 15.3, p = 0.03). Pharmacokinetics differed by ethnicity (MTX clearance:
African>Caucasian). Study size: 434 (discovery cohort), 206 (independent validation cohort), 640 (combined cohort). Study population/ethnicity: Multiethnic children
(5.92 median age , 1.02-18.85 range) with ALL given 3,014 courses of methotrexate at 2-5 g/m(2) enrolled in Tennessee. Significance metric(s): increased MTX
clearance: p = 1.7 x 10(-9) (n = 434), p = 0.017 (n = 206), p = 6.7 x 10(-10) (n = 640); increased GI toxicity: OR = 15.3, p = 0.03. Type of association: CO; GN; PK;
ADR; TOX
Fluorescence in Situ Hybridisation (FISH)
Molecular Cytogenetic Analysis of Chromosomal Translocation
• Translocations generate novel

chromosomes. In a translocation, a
segment from one chromosome is
transferred to a nonhomologous
chromosome or to a new site on the same
chromosome.
• The genomes of closely related species,
they can see that translocations have
occurred many times during the course of
evolution.
• Translocations that give an organism an
adaptive advantage are very rare.
• Translocations are more often associated
with negative consequences like cancer.
• In many cases, are considered to be the
primary cause of various cancers.
Nonreciprocal translocations are one-way translocations in which a

chromosomal segment is transferred to a nonhomologous chromosome. a)
An idiogram of a reciprocal translocation between chromosomes 12 and
17. b) An ideogram of a Robertsonian translocation between chromosomes
14 and 21.
Translocations Can Produce Oncogenes
• The translocation places the coding sequence of one gene (Gene B) in proximity to the regulatory
sequence for a different gene (Gene A).
• The translocation involving chromosomes 8 and 14 places the MYC proto-oncogene from
chromosome 8 under the control of the powerful immunoglobin heavy chain gene (IGH) promoter
on chromosome 14.
• The MYC protein normally signals for cell proliferation, and the translocation causes high levels
of MYC overexpression in lymphoid cells, where the IGH promoter is normally active.
• Aberrant oncogene expression from chromosomal translocation frequently leads to cellular
immortalization and clonal expansion.
Translocations Mbr (major breakpoint region, 150 bp)
Can Produce
Bcl2 Chromosome 18
Oncogenes C Chromosome 14
JH
Double strand DNA break by RAG1/2
• A rearrangement of the bcl-2 Translocation takes place in B cell precursors.
proto-oncogene on Bcl2 C t(14;18) translocation

chromosome 18 with the
immunoglobulin heavy chain
region on chromosome 14, Transformation takes place
during B cell activation in GC.
leads to deregulated BCL-2
production. bcl2 E C C 3’E
• Bcl-2 has been shown to Unregulation of Bcl2 expression by IgH enhancers
prevent programmed cell

death (apoptosis) thus Germinal center
Plasma cells
immortalizing the cell. activation

Germinal center
• The t(14;18) translocation is

characteristic of B-cell apoptosis Memory cells
lymphomas, occurring in up to
90% of follicular lymphomas.
• It is also found in 20% to 30% Germinal center Germinal center
of diffuse large B-cell IgH-Bcl2
lymphomas, where it is an activation
indicator of poor prognosis.

follicular lymphoma
Most follicular lymphoma Ig V regions contain
Apoptosis inhibited
somatic hypermutation.
• Depending on probe design (eg, the
distance between the regions
recognized) and the state of the
genomic DNA at the time of fixation,
a fused signal may appear either as
a colocalized red and green signal or
as a single yellow signal.
• When using break-apart probes,
red/green signal pairs will
occasionally appear to be slightly
separated because of the secondary
structure of the target DNA.
• (A) Interphase nuclei hybridized with the LSI IGH break apart probe (Vysis). The two
nuclei at the top display a significant dissociation of the red and green signals (arrows)
indicating the presence of a translocation affecting the IGH.
• (B) Interphase nucleus with the LSI MYC/IGH double fusion probe (Vysis). The
presence of two fused red and green signals (arrows) indicates that a translocation
t(8;14)(q24;q32) juxtaposing the MYC and IGH loci has taken place. The isolated red
and green signals point to the unrearranged MYC and IGH alleles, respectively.
• Deregulation of BCL6 either by juxtaposition next to an IG locus or by promotor substitution due
to a chromosomal translocation can be detected in about 30% of systemic diffuse-large B-cell
lymphomas (DLCBL).
• t(14;18) cytogenetically identical to that occurring in FL can lead to activation of the MALT1
oncogene. This gene is also targeted by a recurrent t(11;18)(q21;q21) present in approximately
30% of systemic marginal zone lymphomas of MALT type, which leads to fusion of the MALT1
gene with the apoptosis inhibitor-2 (API2) gene in 18q21.
Molecular Cytogenetic Analysis of Chromosomal Translocation in
Primary Cutaneous B-cell Lymphomas
• Overall, translocations affecting one IG locus are estimated to be present in at least

half of the nodal B-cell non-Hodgkin lymphomas. In contrast to systemic B-cell
lymphomas only few data exist on the presence of recurrent translocations in primary
cutaneous B-cell lymphomas (PCBCL).
• The t(14;18) translocation does not occur in PCBCL, which suggests the involvement
of different pathogenetic mechanisms compared with their nodal counterparts.
• The detection of a t(14;18) translocation in cutaneous B-cell lymphoma should

suggest the presence of systemic disease, which underlies the need for exhaustive
staging procedures.
Gene Microarray Expression Technology
• Oncogenes: BAX, BCL2L1, CASP8, CDK4, ELK1, ETS1,
HGF, JAK2, JUNB, JUND, KIT, KITLG, MCL1, MET, MOS,
MYB, NFKBIA, NRAS, PIK3CA, PML, PRKCA, RAF1,
RARA, REL, ROS1, RUNX1, SRC, STAT3, ZHX2.
• Tumor Suppressor Genes: ATM, BRCA1, BRCA2,

CDH1, CDKN2B, CDKN3, E2F1, FHIT, FOXD3, HIC1,
IGF2R, MEN1, MGMT, MLH1, NF1, NF2, RASSF1,
RUNX3, S100A4, SERPINB5, SMAD4, STK11, TP73,
TSC1, VHL, WT1, WWOX, XRCC1.
• Oncogenic & Tumor Suppressor Properties: BCR, EGF,

ERBB2, ESR1, FOS, HRAS, JUN, KRAS, MDM2, MYC,
MYCN, NFKB1, PIK3C2A, RB1, RET, SH3PXD2A,
TGFB1, TNF, TP53.
• Transcription Factors: ABL1, BRCA1, BRCA2, CDKN2A,

CTNNB1, E2F1, ELK1, ESR1, ETS1, FOS, FOXD3, HIC1,
JUN, JUNB, JUND, MDM2, MEN1, MYB, MYC, MYCN,
NF1, NFKB1, PML, RARA, RB1, REL, RUNX1, RUNX3,
SMAD4, STAT3, TGFB1, TNF, TP53, TP73, TSC1, VHL,
WT1, ZHX2.
• Epithelial-to-Mesenchymal Transition: BRCA2,

CDKN2B, CTNNB1, ERBB2, HGF, JAK2, KIT, MCL1, NF1,
RUNX3, S100A4, SMAD4, TGFB1, VHL.
• Angiogenesis: AKT1, CTNNB1, EGF, ERBB2, NF1, PML,

RUNX1, TGFB1.
• Apoptosis: BAX, BCL2, BCL2L1, BRCA1, CASP8, E2F1,

MCL1, MGMT, TNF, VHL.
• Cell Adhesion: APC, CDH1, CDKN2A, CTNNB1, KITLG,

NF1, NF2, TGFB1.
• Cell Cycle: ATM, BRCA1, BRCA2, CCND1, CDK4,

CDKN1A, CDKN2A, CDKN2B, CDKN3, E2F1, HGF,
MEN1, STK11, TP53.
• Chemotaxis, Cell Migration & Motility: HRAS, JAK2,

MET, NF1, NF2, PRKCA, SERPINB5, STAT3.
• DNA Damage & Repair: ABL1, APC, ATM, BRCA1,

BRCA2, CDKN1A, MEN1, MGMT, MLH1, PML, TP53,
TP73, XRCC1.
Non-Hodgkin lymphoma (NHL)
• Non-Hodgkin lymphoma (NHL) is a heterogeneous, complex, and progressive clonal expansion of B-, T-lymphocytes and rarely
NK-cells or their precursors.
• Our taxonomy of lymphomas, which is based mostly on histopathology and immunophenotyping, includes about 30 distinct
entities arising from diverse cells types.
• The genetic complexity of lymphomas probably explains the clinical diversity with traditional methods and genomic expression
analysis.
• Microarrays technique is effective in deciphering this clinical diversity.
• A number of published studies identify gene expression signatures for major non-Hodgkin lymphoma types and subtypes, and
uncover gene expression patterns that correlate with various characteristics of non-Hodgkin lymphoma.
• Mature T-cell and NK-cell neoplasms • Mature B-cell neoplasms

– Mycosis fungoides (MF) – Cutaneous marginal zone B-cell lymphoma (MALT-type)
– Variants of MF – Primary cutaneous follicle center lymphoma
• Pagetoid reticulosis (localized disease) – Growth patterns
• Folliculotropic, syringotropic, granulomatous variants
• Follicular
– Subtype of MF
• Granulomatous slack skin • Follicular and diffuse
– Sezary syndrome • Diffuse
– CD30+ T-cell lymphoproliferative disorders of the skin – Cutaneous diffuse large B-cell lymphoma, leg type
• Lymphomatoid papulosis – Cutaneous diffuse large B-cell lymphoma, others
• Primary cutaneous anaplastic large cell lymphoma – Intravascular large B-cell lymphoma
– Subcutaneous panniculitis-like T-cell lymphoma – Lymphomatoid granulomatosis
– Primary cutaneous peripheral T-Cell lymphoma (PTL), – Chronic lymphocytic leukemia
unspecified
– Subtypes of PTL – Mantle cell lymphoma
• Primary cutaneous aggressive epidermotropic CD8+ – Burkitt lymphoma
T-cell lymphoma (provisional) • Immature hematopoietic malignancies
• Cutaneous gamma/delta-positive T-cell lymphoma – Blastic NK-cell lymphoma CD4+/CD56+ hematodermic
(provisional) neoplasm
• Primary cutaneous CD4+ small/medium-sized –
pleomorphic T-cell lymphoma (provisional) Precursor lymphoblastic leukemia/lymphoma
– Extranodal NK/T-cell lymphoma, nasal type • T-lymphoblastic lymphoma
• Hydroa vacciniforme-like lymphoma (variant) • B-lymphoblastic lymphoma
– Adult T-cell leukemia/lymphoma – Myeloid and monocytic leukemias
– Angioimmunoblastic T-cell lymphoma • Hodgkin lymphoma
Current Microarray Technology
Tissu Lysis mRN cDNA

e A
Amplificati
on
Optical
Fluorescent
Detection
Labeling
Scanning
Image Analysis
Data Analysis
• In standard microarrays, the probes are attached via surface engineering to a solid surface by a
covalent bond to a chemical matrix (via epoxy-silane, amino-silane, lysine, polyacrylamide or others).
• The solid surface can be glass or a silicon chip, in which case they are colloquially known as an Affy
chip when an Affymetrix chip is used.
• The core principle behind microarrays is hybridization between two DNA strands, the property of
complementary nucleic acid sequences to specifically pair with each other by forming hydrogen bonds
between complementary nucleotide base pairs.
• A high number of complementary base pairs in a nucleotide sequence means tighter non-covalent
bonding between the two strands.
• After washing off of non-specific bonding sequences, only strongly paired strands will remain hybridized.
• So fluorescently labelled target sequences that bind to a probe sequence generate a signal that
depends on the strength of the hybridization determined by the number of paired bases, the
hybridization conditions (such as temperature), and washing after hybridization.
• Two-color microarrays or two-channel
microarrays are typically hybridized
with cDNA prepared from two samples
to be compared (e.g. diseased tissue
versus healthy tissue) and that are
labeled with two different fluorophores.
• Fluorescent dyes
commonly used for cDNA
labeling include Cy3, which
has a fluorescence
emission wavelength of 570
nm (corresponding to the
green part of the light
spectrum), and Cy5 with a
fluorescence emission
wavelength of 670 nm
(corresponding to the red
part of the light spectrum).
• The two Cy-labeled cDNA
samples are mixed and
Microarray analysis
hybridized to a single
microarray that is then
scanned in a microarray
scanner to visualize
fluorescence of the two
fluorophores after excitation
with a laser beam of a
defined wavelength.
• Relative intensities of each
fluorophore may then be
used in ratio-based analysis
to identify up-regulated and
down-regulated genes.
Repression Induction
• Components of DNA Microarray image analysis are
– (1) Grid Alignment Problem,
– (2) Foreground Separation,
– (3) Quality Assurance,
– (4) Quantification and
– (5) Normalization.
• Variations in microarray image Data processing:
– grid geometry,
– foreground and background intensity,
– spot morphology
Examples of accurate (top) and inaccurate

(bottom) foreground separation
Right image shows variations of spots; a regular

spot, an inverse spot or a ghost shape, a
spatially deviating spot inside of a grid cell, a
spot radius deviation, a tapering spot or a comet
shape, spot with a hole or a doughnut shape, a
partially missing spot and a scratched spot.
Hierarchical clustering
• Microarray data sets are commonly very large, and analytical precision is influenced
by a number of variables. Statistical challenges include taking into account effects of
background noise and appropriate normalization of the data.
• For statistical analysis and visualization of gene expression data a large number of
commercial and non-commercial software tools have been developed (e.g., Gene
Spring, Gene Cluster, Cluster, and Treevoew, SAM and dCHIP).
• Hierarchical clustering output as dendogram or tree attached to a heatmap
representation of the clustered matrix
• Clustering aims at grouping objects, such as genes, together, according to some
measure of similarity, so that objects within one group or cluster are more similar to
each other than to objects in other groups. It is a mean to visualize patterns of gene
expression in the data.
Hierarchical clustering
• The final, computational form, of the
Pearson correlation coefficien:
• To return to the context of hierarchical

clustering, a Pearson correlation
coefficient must be computed for
every possible gene comparison.
• When clustering an entire genome of
6,000 or more genes this can mean a
considerable number of comparisons
must be performed, yet the results
can provide valuable generalizations
about the genes' relationships.
Data base
• DNA arrays enable the simultaneous analyses of expression of

thousands of genes, making it possible to evaluate expression
profiles in a temporal fashion within a single sample, or to
compare profiles among samples.
• The power of this technology comes from the enormous
amount of data it generates, although thus far it has been
difficult to utilize the technology to its full potential because
data-mining technology has not reached the same level of
sophistication.
• To fully exploit this technology in the future, it will be crucial to
construct standardized public databases that can sort and
store the enormous quantity of data for reference and
comparison for scientists worldwide.
Data base
• Standardization
• Microarray data is difficult to exchange due to the lack of standardization in

platform fabrication, assay protocols, and analysis methods. This presents an
interoperability problem in bioinformatics. Various grass-roots open-source
projects are trying to ease the exchange and analysis of data produced with
non-proprietary chips:
– For example, the "Minimum Information About a Microarray Experiment" (MIAME)

checklist helps define the level of detail that should exist and is being adopted by
many journals as a requirement for the submission of papers incorporating
microarray results. But MIAME does not describe the format for the information, so
while many formats can support the MIAME requirements, as of 2007 no format
permits verification of complete semantic compliance.
– The "MicroArray Quality Control (MAQC) Project" is being conducted by the US
Food and Drug Administration (FDA) to develop standards and quality control
metrics which will eventually allow the use of MicroArray data in drug discovery,
clinical practice and regulatory decision-making.[15]
– The MGED Society has developed standards for the representation of gene
expression experiment results and relevant annotations.
Data base
• There are major data bases of publicly available

information:
– Gene Bank,
– EMBL,
– GEO,
– NCBI at NIH,
– DNA Data Bank of Japan,
– HuGENet,
– Stanford Microarray Database
MICROARRAY EXPRESSION PROFILING
• Diffuse large B-cell lymphoma (DLBCL) is one disease in which

attempts to define subgroups on the basis of morphology have
largely failed owing to diagnostic discrepancies arising from inter
and intra-observer irreproducibility.
• Diffuse large B-cell lymphoma is an aggressive malignancy of
mature B lymphocytes, with an annual incidence of over 25,000
cases, accounting for roughly 40% of cases of non-Hodgkin's
lymphoma.
• Patients with DLBCL have highly variable clinical courses: although
most patients respond initially to chemotherapy, fewer than half of
the patients achieve a durable remission.
• Although a combination of clinical parameters is currently used to
assess a patient's risk profle, these prognostic variables are
considered to be proxies for the underlying cellular and molecular
variation within DLBCL.
MICROARRAY EXPRESSION PROFILING
IDENTIFICATION OF:
 genes involved in pathogenesis / progression, localization / spread, etc
 novel genes / disease categories (“class discovery”)
 known cell lineage, differentiation stage, etc (“class prediction”)
 genes involved in sensitivity / resistance
 risk factors / prognostic groups
 novel molecular targets for therapy

Construction of a specialized DNA microarray
• Recent technical and analytical advances make it practical to quantitate the
expression of thousands of genes in parallel using complementary DNA microarrays.
• To apply this method to questions in normal and malignant lymphocyte biology,
Alizadeh et al designed a specialized microarray, the `Lymphochip‘, by selecting
genes that are preferentially expressed in lymphoid cells and genes with known or
suspected roles in processes important in immunology or cancer.
• Because of the suspected importance of the germinal centre B cell to the genesis of
non-Hodgkin's lymphomas, 12,069 out of the 17,856 cDNA clones on this microarray
were chosen from a germinal centre B-cell library.
• Alizadeh et al included an additional 2,338 cDNA clones from libraries derived from
DLBCL, follicular lymphoma (FL), mantle cell lymphoma and chronic lymphocytic
leukaemia (CLL).
• Finally, Alizadeh et al added clones representing a variety of genes that are induced
or repressed during B- and T- lymphocyte activation by mitogens or cytokines and a
curated set of 3,186 genes of importance to lymphocyte and/or cancer biology.
• About a quarter of the genes included in this microarray were represented by two or
more different cDNA clones, providing internal controls for the reproducibility of gene
expression quantitation.
• 1.8-million measurements of gene expression were made in 96 normal and malignant
lymphocyte samples using 128 Lymphochip microarrays.
Ash A. Alizadeh, Distinct types of diffuse large B-cell lymphoma identifed by gene expression profling, NATURE, VOL 403, 3 FEBRUARY 2000
• A hierarchical clustering
algorithm was used to
group genes on the basis
of similarity in the pattern
with which their expression
varied over all samples.
• The data are shown in a
matrix format, with each
row representing all the
hybridization results for a
single cDNA element of the
array, and each column
representing the measured
expression levels for all
genes in a single sample.
• To visualize the results, the
expression level of each
gene (relative to its median
expression level across all
samples) was represented
by a colour, with red
representing expression
greater than the mean,
green representing
expression less than the
mean, and the colour
intensity representing the
magnitude of the deviation
from the mean.
Ash A. Alizadeh, Distinct types of diffuse large B-cell lymphoma identifed by gene expression profling, NATURE, VOL 403, 3 FEBRUARY 2000
• The results paints a
complex, but remarkably
ordered, picture of the
variation in gene
expression patterns in
lymphoid malignancies,
with large sets of genes
displaying coordinate
expression in related
biological samples.
• The coloured bars on the
right indicate clusters of
coordinately expressed
genes that we
operationally defend as
gene expression
`signatures'.
• A gene expression
signature was named by
either the cell type in which
its component genes were
expressed (for example,
the `T-cell' signature) or
the biological process in
which its component genes
are known to function (for
example, the `proliferation'
signature).
Gene expression profiling in diffuse large B-cell
lymphoma
• Subclassification of DLB-CL was done by tissue
specific microarrays and selected into prognostic
subgroups based on cellular origin:
– germinal center B-cell –like (GCB-like)
– activated B-cell like (ABC-like)
– type 3 or primary mediastinal B-cell
lymphoma (PMBL)
• The DLB-CL subgroups are distinguished from
each other by the differential expression of
hundreds of different genes, and these genes
relate to each subgroup to separate stage of B cell
differentiation and activation. These molecular
differences suggest that DLB-CL subgroups (GBC,
ABC, and PMBL) should be considered as
separate diseases
• Patients with gene expression profiling of GCB
have a significantly better survival than the
patients with gene expression profiling of ABC. The
type 3 has a poor clinical outcome which is similar
to the ABC subgroup.
• Alizadeh et al., created a “fuzzy neural network” for
the precise prediction of survival of patients with
DLB-CL. In this model four genes are identified
(CDIU, AA800/551, AA805661, and IRF4) that
could be used to predict prognosis with 93%
accuracy.
Gene expression profiling in diffuse large B-cell
lymphoma
• Patients with low expression CD10
have a poor prognosis.
• Patients with high CD10 and
AA807551 high expression and low
expression of AA805611 genes have
poor prognosis.
• The gene expression profiling (GEP)
of GCB includes many markers of
germinal center differentiation
(e.g.CD10, CD38, A-myb, OGG1,
HGAL, Bcl-6,Bcl-7A, and LMO2).
• The ABC GEP includes genes: IREL
(MUM1/LSIRF), CCND2, SYCA3,
FLIP, Bcl-Xl, Bcl-2, BLIMP1, and
XBP1, and absent expression of Bcl-
6 gene.
• The expression of Bcl-2, CCND2 and
SYCA3 correlated with short survival.
• The expression of LMO2, Bcl-6 and
lymph node signature correlated with
prolonged survival
Gene expression profiling of follicular
lymphomas
• About 70% FLs have indolent clinical course, 10% of FLs may transform into DLB-CL with
more aggressive clinical course.
• A number of gene signatures have
been identified: “81 gene predictor
signature”, “37 genes signature”, and
“indicator genes” signature.
• Investigators from NCI have
discovered two subsets of genes
“survival-associated signatures named
IR-l, and IR-2 , whose expression is
linked to survival advantage in
patients with follicular lymphomas.
• The overexpression of the immune
response-1(IR-1) signature correlated
with good prognosis, while immune
response-2(IR-2) signature correlated
with poor prognosis.
Vladimir Baltić, Milan Baltić, Arch Oncol 2007;15(1-2):28-35.

Gene expression profiling of follicular
lymphomas
• The immune response-1 signature included genes encoding T-cells markers (CD7, CD8B1, ITK,
LEF, and STAT4, osteopontin, MRCOX2, turgen, GRO1, GRO2, NKCT4, LEU13, IFN2, NCF4,
CUB C1s, C1qr, TCRbeta, TCRteta, TNF1beta, TNFalfa1, JUNB, FOSGA-beta, p75NTR) and
genes that are highly expressed in macrophages (ACTN1, and TNFRF13B).
• The immune response-2 signature included genes known to be preferentially expressed in
macrophages, dendritic cells or both (TLR5, FCCR1A, SEP1o, LOM, and CAR1).
The FLs signature contains genes

upregulated in aggressive phase disease
that are involved:
•in cell cycle (CCNE2, CCNA2, CDK2,

CHEK1, MCM7)
•DNA synthesis (TOP2A, POLO3A, HMGA1,
POLE2, GMPS, CTPS);
•increased metabolism (FRSB, RARS, HK2,
LDH2);
•signal transduction (FR2B, HCFCR1,
PIK4CA, MAPK1)
•and genes derived from the reactive infiltrate of
T cells and macrophages (CD3D, CXCLI2,
TM4SF2).
Vladimir Baltić, Milan Baltić, Arch Oncol 2007;15(1-2):28-35.

Comparative Genomic Hybridization
• 1q32.1:
– 1-chromosome 1
– q- long arm Morphology
– 3- 3rd region
– 2- 2nd band
– 1- 1st subband
• Prior to entering mitosis and undergoing division, a cell must
exactly replicate its genome by synthesizing a new copy of each
chromosome, using the existing DNA as a template.
• During this process several types of large-scale chromosomal
alterations can occur.
• Cancer is fundamentally a disease of genomic alteration.
– Cancer cells typically carry many genomic alterations that confer on tumors
their distinctive abilities (such as the capacity to proliferate and
metastasize, ignoring the normal signals that block cellular growth and
migration) and liabilities (such as unique dependence on certain cellular
pathways, which potentially render them sensitive to certain treatments that
spare normal cells).
• The development of solid tumors is associated with the acquisition of complex genetic alterations
that modify normal cell growth and survival.
• Cancer is a stepwise process, typically requiring accumulation of mutations in a number of genes.
• Many of these changes involve gains and/or losses of parts of the genome: Amplification of an
oncogene or deletion of a tumor suppressor gene are considered as important mechanisms for
tumorigenesis.
• Regions of DNA can be deleted or fail to be replicated, resulting in a loss at that chromosomal
locus.
• Conversely, regions can be duplicated or multiplied, resulting in a gain of copy number or
amplification.
• Furthermore, entire segments of chromosomes can be inappropriately fused with other
chromosomes in a process called translocation.
Comparative Genomic Hybridization
• Powerful experimental tool
• Screens the entire genome for gains and losses in DNA

material in one experiment
• Variants:
– Conventional CGH
– Array based CGH
Comparative Genomic Hybridization (CGH)
The basic procedure of CGH begins with purifying genomic DNA from samples of cancerous and normal
control tissue (e.g., lymphocytes from a healthy individual).
These two DNA preparations are labeled with different fluorochromes which will emit light at easily
distinguishable wavelengths–generally red and green. The samples are then mixed and allowed to 94
competitively hybridize to immobilized normal DNA.
Equal amounts of biotin-labeled tumor DNA and digoxigenin-labeled normal reference DNA are hybridized to normal
metaphase chromosomes
The tumor DNA is visualized with fluorescein and the normal DNA with rhodamine
The signal intensities of the different fluorochromes are quantitated along the single chromosomes
The over-and underrepresented DNA segments are quantified by computation of tumor/normal ratio images and
average ratio profile
In regions where there are no amplifications or deletions in the cancer genome, binding of both samples will be equal,
and the equal emission of light from both fluorochromes will result in a perceived yellow fluorescence.
However, where there are losses in copy number in the cancer DNA, the color with which the normal DNA was labeled
(e.g., red) will predominate. Similarly, in regions of DNA copy number gain, the color with which the tumor DNA was
labeled (e.g., green) will be apparent.
The UCSF-CCC primarily uses bacterial
artificial chromosomes (BACs). While the
human haploid genome has 3 billion pairs
of bases, each BAC consists of a smaller
100-200 kilobase (kb) region of the DNA.
These BAC clones are grown in bacteria,
purified, and spotted onto slides by a
robot. In carcinogenesis, researchers
compare tumor samples to normal
samples in order to examine
hypothesized cancer related aberrations
of the genome.
1.2Array CGH
Technical consideration in array CGH
Specimen preparation
• Differences in quality of cell lines, frozen/fresh/fixed tissue
• Heterogeneous specimens
• Extraction of DNA
Data analysis
• Significance of signal ratios
Factors influencing the success of array CGH
(2005) Nature Genetics, 37:s11-17

Daniel Pinkel & Donna G Albertson
Conventional CGH Array CGH
• Advantages
• Advantages
– Archived material
– Does not require culture of
cells for karyotypic – Improved resolution 1MB
analysis – Technical ease
– Archived material • Disadvantages
• Disadvantages – >30-50% of cells need to
– Limited resolution -10-20 M have an abnormality
– >30-50% of cells need to – Requires microdissection
have an abnormality
– Requires microdissection
Melanocytic Neoplasia
• Benign lesions -Nevi

– Junctional, intradermal, and compound nevi
– Blue nevi (including cellular and epithelioid)
– Spitz nevi
– Congenital nevi
– Other
• Malignant -Melanomas
Intradermal Melanocytic Nevus
Blue
Nevus
Spitz Nevus
Melanoma
2 y/o girl with lesion on the rt knee
Expert Dermatopathology diagnosis
• Spitz nevus
• Spitz tumor of uncertain malignant potential
• Spitzoid melanoma (Breslow depth 4.5 mm)
• 2.3%-25% of cases with diagnostic discrepancies
• Ambiguous lesions with overlapping criteria
– Atypical Spitz nevi
– Atypical blue nevi
– Recurrent melanocytic nevi
– Nevi in acral, genital or mammary line regions
– Mechanically irritated nevi
– Nevoid melanoma
• Interobserver variability
Benign or malignant?
• Under/overtreatment of patients
• Most common reason for medical malpractice in dermatopathology
• A test that helps dx accuracy would have a significant impact on patient

care
Kornstein, et al. Arch Pathol and Lab Med, 2007

Gerami, et al, Am J Surg Pathol, 2009
CGH in Melanocytic lesions
• 54 benign nevi
– 27 Spitz nevi
– 19 Blue nevi
– 7 Congenital nevi
• 132 MM
– 22 Acral location
– 108 non-Acral
Bastian B et al, Am J Pathol 2003

Gains: 6p Losses: Gains: 11p: 11% Losses:
1q 9p 7q: 2% 0%
7p 9q
7q 10q
8q 10p
17q 6q •The 7 cases were all Spitz
20q 11q nevi (no progression to MM
at 7 yrs FU)

• Spitz nevi generally show no genetic changes by conventional CGH, but a
subset can show an 11p gain. In contrast, melanomas show a number of
cytogenetic abnormalities, including deletions of chromosomes 9, 10, 6q and
8p, and amplifications of chromosomes 7, 8q, 6p, 1q, 20, 17, and 2
GTPase HRas also known as transforming protein p21 is an enzyme that in

humans is encoded by the HRAS gene. The HRAS gene is located on the
short (p) arm of chromosome 11 at position 15.5
GTPase HRas is involved in regulating cell division in response to growth
factor stimulation.
HRAS is in the Ras family, which also includes three other proto-
oncogenes: KRAS, RRAS and NRAS.
Chromosomal Aberrations and Spitz Nevi
• Chromosomal analyses may be of

assistance in classifying histologically
ambiguous lesions
• 3/17 (18%) Spitz nevi with gain on 11p by
CGH
• Melanomas do not show 11p gain
Wynnis L. Tom et al,

Bastian B et al, Am J Pathol 2000 Journal of the American Academy of Dermatology
Bastian B et al, J Invest Pathol 1999 March 2011(Vol. 64, Issue 3,Pages 559-572)
Neoplasms arising in congenital nevi
Several types of melanocytic tumors can develop in

congenital nevi:
– Simulants of superficial spreading melanoma
– Simulants of nodular melanoma (proliferative nodules)
– True melanomas
• It may be difficult to separate them

CGH in congenital nevi
CMN CMN simulating CMN simulating True MM arising

SSM NM in CMN
No changes No changes Multiple DNA Multiple DNA

gains and gains and
losses losses
Whole chromosome Partial
abnormalities = chromosome
defects in abnormalities =
chromosomal DNA breaks
segregation
Group I Group II Group III
Benign cellular blue Histopathologically Histopathologically

nevi ambiguous lesions malignant lesions
11 cases (Atypical blue nevi) 7 cases
11 cases
Group I:
•Small lesions
•Uniform, oval melanocytes
•Small nucleoli
•No atypia
•No mitoses

11 cases
Group II (ambiguous
because of the presence
of one or more of the
following):
•Multinodular
growth pattern
•Deep penetration
•Focus of increase
cellularity
•Focal necrosis
•Mild to moderate
nuclear atypia
•>1 mitoses / HPF

11 cases
Group III (clearly identifiable

features of malignancy):
•Multiple foci of necrosis
•Ulceration
•Irregular dermal growth
pattern
•Marked nuclear atypia and
pleomorphism
3 mitoses / HPF
•Atypical mitoses

(Atypical blue nevi)
CGH
No abnormalities 3/11 (27%) 7/7 (100%)

1 to 3 Average of 8
abnormalities/case abnormalities/case
Unusual pattern: Pattern similar to
regular primary
-Gain of chr 9q, loss of
melanomas:
chr 3q
None of the 11 cases
behaved aggressively
Presence of more than 3 chromosomal aberrations is suggestive of melanoma
Summary of chromosomal abnormalities
Benign Atypical Malignant

lesions lesions lesions
1.Common nevi 1.Atypical Spitz 1.Malignant

•No abnormalities •No abnormalities 85% Melanoma
•Gain of 11p ~15% •Multiple abnormalities
•Rare: multiple
2.Spitz nevi (ave 6) in 96%
abnormalities
•No abnormalities 85%
•Gain of 11p ~15% 2.Atypical Blue
nevus
•No abnormalities 63%
3.Cellular blue •1-3 abnormalities 27% 2.Acral
nevi Melanoma
•No abnormalities 3.Proliferative •Multiple abnormalities
nodules in CN (ave 10) in 100%
4.Congenital •Whole chromosome •Focused amplifications
nevi abnormalities 66%
•No abnormalities
• 1q31 (COX2)
CGH date from • 4q12 (KIT)
melanomas • 7q34 (BRAF)
• 6p35 (RREB1)
• 6q23 (MYB1)
• 6 cen
• 7 cen
• 9p31 (p16)
• 10 cen
• 11q13 (CCND1)
• 17q25 (TK1)
• 17q21 (RARA)
• 17 cen
• 20q13 (ZNF217)
Gerami et al. Am J Surg Pathol 2009

When to use molecular testing
• Not in all cases!
• Ambiguous melanocytic tumors
– Atypical Spitz tumors, etc
• Other unusual melanomas (eg. lentiginous Newman et al. Mod
Pathol 2009)
• For staging in melanomas with associated nevi (Newman et al.
Mod Pathol 2009)
Disadvantages of CGH
• Requires a significant amount of tumor tissue

• Requires 30-50% pure tumor cells
• Does not allow histologic correlation
• Cannot detect tumor subpopulations
• These disadvantages can be overcome by FISH analysis
• The diagnostic accuracy employing clinical and histological features alone ranges
from 50% to 75%, but reaches 80% when morphologic features are combined with
the immunophenotypic or genotypic characterization of tumor cells.
• Immunohistochemical (IHC) identification of the tumor cell phenotype plays such a
crucial and invaluable role in the diagnostic work-up of cutaneous lymphoma (CL)
that it can be regarded as a mandatory step in establishing the correct diagnosis.
• The final diagnosis has always to be based on integrative synopsis of all clinical,
histopathological,IHC, and molecular biological findings.
TEST
1. Imunohistochimic pot fi evidentiat
urmatorii markeri?
• A. Nucleari
• B. Intracitoplasmatici
• C. Membranari
• D. Genetici
• Raspuns: A, B, C
2. Diagnosticul clonalitatii este utila in?
• A. Sifilisul tertiar
• B. Melanom
• C. Depistarea unei populatii de limfocite T clonale in
cazul micozisului fungoid
• D. Depistarea unei populatii de keratinocite clonale in
cazul epiteliomului bazocelular
• Raspuns: C
3. PCR (polymerase chain reation)
reprezinta?
• A. marcarea genelor imunohistochimic
• B. amplificarea exponentiala a unei sectiunii de pe gena
• C. kariotipare
• D. reactie imuno-enzimatica
• Raspuns: B
4. Produsii de reactie PCR sunt evaluati
prin?
• A. electroforeza
• B. westernblott
• C. la microscopul cu fluorescenta
• D. vitropresiune
• Raspuns: A
5. Depistarea SNP (single nucleotide
polymorphism) sunt utile pentru?
• A. raspunsul terapuetic in cazul unor medicamente
• B. incadrarea intr-o grupa de risc
• C. depistarea unor afectiuni latente
• D. evolutie si prognostic
• Raspuns: A, B, C, D
6. Tehnica FISH (Fluorescence in Situ
Hybridization)?
• A. depisteaza SNP
• B. identifica translocatiile
• C. foloseste doua tipuri de probe: dual-fusion si break-
apart
• D. evalueaza genomul in totalitatea lui
• Raspuns: B, C
7. Care sunt avantajele tehnicii
microarray?
• A. depsiteaza gene implicate in patogeneza, progresie,
localizare si raspandirea tumorilor
• B. descopera posibile noi tinte terapeutice
• C. se poate efectua si pe markeri extracelulari
• D. procesarea resultatelor obtinute este foarte simpla
• Raspuns: A, B
8. Tehnica microarray poate depista
semnaturi specifice pentru?
• A. sifilis si alte BTS
• B. metabolism
• C. ciclu celular si sinteza ADN
• D. diferite subtipuri de limfoame si tumori
• Raspuns: C, B, D
9. Tehnica hibridizarii genomice
comparative depisteaza?
• A. Deletii
• B. Insertii (duplicatii)
• C. SNP-uri
• D. Translocatii
• Raspuns: A, B, D
10. Tehnica hibridizarii genomice comparative
este utila in diagnosticul diferential al
urmatoarelor afectiuni?
• A. Melanom si nevul Spitz
• B. Zona Zoster si Dermatita atopica
• C. Melanom si nevi benigni
• D. Melanom si angiosarcoame
• Raspuns: A, C

LP 6 TesteMoleculareGeneticeDermatologie

Încărcat de

Informații document

Titlu original

Drepturi de autor

Formate disponibile

Partajați acest document

Partajați sau inserați document

Opțiuni de partajare

Vi se pare util acest document?

Este necorespunzător acest conținut?

Drepturi de autor:

Formate disponibile

LP 6 TesteMoleculareGeneticeDermatologie

Încărcat de

Drepturi de autor:

Formate disponibile

Disciplina de Dermatologie, Universitatea de Medicină Victor Babeş Timişoara

METODE DE DIAGNOSTIC MOLECULAR SI

• Tumor Suppressor Genes: ATM, BRCA1, BRCA2,

• Oncogenic & Tumor Suppressor Properties: BCR, EGF,

• Transcription Factors: ABL1, BRCA1, BRCA2, CDKN2A,

• Epithelial-to-Mesenchymal Transition: BRCA2,

• Angiogenesis: AKT1, CTNNB1, EGF, ERBB2, NF1, PML,

• Apoptosis: BAX, BCL2, BCL2L1, BRCA1, CASP8, E2F1,

• Cell Adhesion: APC, CDH1, CDKN2A, CTNNB1, KITLG,

• Cell Cycle: ATM, BRCA1, BRCA2, CCND1, CDK4,

• Chemotaxis, Cell Migration & Motility: HRAS, JAK2,

• DNA Damage & Repair: ABL1, APC, ATM, BRCA1,

• Some antibodies do not work with sections

Stage Heavy chain Light chain

• Processes for TCR formation are similar to those

• The TCR alpha chain is generated by VJ recombination,

Gene 1 Coding region Protein 1

Gene 2 Coding region Protein 2

= Variations in DNA that cause no changes

= Variations in DNA that cause harmless changes

= Variations in DNA that cause latent effects

SNP marks Gene A SNP may cause Gene B

Lysine side chain Lysine

Methionine Tyrosine Stop

Amino acid chain grows

into a 3-D structure.

Leucine Protein Leucine

Aspartic acid Protein Glutamic acid

Slight change in shape

Aspartic acid Protein Valine

Heterozygous = having two

Homozygous = having identical

= SNPs causing latent effects

Individual SNP Profiles Are Sorted

SNP profile A SNP profile B

SNP profile E SNP profile C

IL-2 Chromosome: 4; Location: 4q26-q27 rs1051753, rs2069763, rs3087209, ………

IL-8 Chromosome: 4; Location: 4q13-q21 rs1803205, rs71745371, ………

IL-17 Chromosome: 6; Location: 6p12 rs17880588, rs17878530, ………

IL-22 Chromosome: 12; Location: 12q15 rs2227507, ………

VEGF Chromosome: 6; Location: 6p12 rs25648, rs45533131, rs62401172, ………

• Translocations generate novel

Nonreciprocal translocations are one-way translocations in which a

Double strand DNA break by RAG1/2

• A rearrangement of the bcl-2 Translocation takes place in B cell precursors.

proto-oncogene on Bcl2 C t(14;18) translocation

• Bcl-2 has been shown to Unregulation of Bcl2 expression by IgH enhancers

prevent programmed cell

immortalizing the cell. activation

• The t(14;18) translocation is

of diffuse large B-cell IgH-Bcl2

lymphomas, where it is an activation

indicator of poor prognosis.

• Overall, translocations affecting one IG locus are estimated to be present in at least

• The detection of a t(14;18) translocation in cutaneous B-cell lymphoma should

• Tumor Suppressor Genes: ATM, BRCA1, BRCA2,

• Oncogenic & Tumor Suppressor Properties: BCR, EGF,

• Transcription Factors: ABL1, BRCA1, BRCA2, CDKN2A,

• Epithelial-to-Mesenchymal Transition: BRCA2,

• Angiogenesis: AKT1, CTNNB1, EGF, ERBB2, NF1, PML,

• Apoptosis: BAX, BCL2, BCL2L1, BRCA1, CASP8, E2F1,

• Cell Adhesion: APC, CDH1, CDKN2A, CTNNB1, KITLG,

• Cell Cycle: ATM, BRCA1, BRCA2, CCND1, CDK4,