Disciplina de Dermatologie, Universitatea de Medicin Victor Babe Timioara

METODE DE DIAGNOSTIC MOLECULAR SI GENETIC IN DERMATOLOGIE

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.

Genome Evolution

DNA
Transcription

RNA

Protein
Translation

Selection

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.

Flow cytometric immunophenotyping

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.

Flow cytometric immunophenotyping

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 tumorspecific 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 Bcells. 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 Progenitor (or pre-pro) B cells Early Pro (or pre-pre)-B cells Late Pro (or pre-pre)-B cells Large Pre-B cells Small Pre-B cells Immature B cells Mature B cells

Heavy chain germline undergoes D-J rearrangement undergoes V-DJ rearrangement is VDJ rearranged is VDJ rearranged is VDJ rearranged is VDJ rearranged

Light chain germline germline germline germline undergoes V-J rearrangement VJ 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 () and beta () fragments (in / T cells, the halves are gamma () and delta () 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 chain, the variable region is called V and the constant region is called C; on the chain they are called V and C 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

DNA molecule (chromosome)

Chemical bases A T 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

Most of the population At least 1 percent of the population

G to C

Common sequence

Variant 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 U G C G U U A U A C G U A A

T A C G C A A T A T G C A T T

DNA

mRNA

Genes to Proteins II

Genes to Proteins III

Methionine Arginine Ribosome tRNA Threonine Tyrosine

mRNA A U G C G U U A U A C G Methionine Tyrosine Threonine U A A Stop

Codons: AUG=Methionine=Start CGU=Arginine UAU=Tyrosine ACG=Threonine UAA=Stop

Arginine

Protein Folding and Function

Amino acid chain grows

and folds

into a 3-D structure.

SNPs in Coding Regions

No Changes in Protein
GAC

mRNA

DNA SNP C to G

GAG

CUG

CUC

RNA Codon CUG to CUC

CUG CUC

Leucine

Protein Leucine to Leucine

Leucine

No change in shape

SNPs in Coding Regions

Subtle, Harmless Changes in Protein
CTA

DNA SNP A to C

CTC

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

SNPs in Coding Regions

DNA SNP T to A
C A A

mRNA
GAU GUU

RNA Codon GAU to GUU

GAU GUU

Aspartic acid

Protein Aspartic acid to Valine

Valine

Change in shape

PCR Requirements

Magnesium chloride: .52.5mM Buffer: pH 8.3-8.8 dNTPs: 20-200M Primers: 0.1-0.5M DNA Polymerase: 1-2.5 units Target DNA: 1 g

Gel electrophoresis

Heterozygous = having two different alleles for a single trait.

Wild type Mutant

Homozygous = having identical alleles for a single trait.

SNPs in Coding Regions

Subtle Changes in Proteins That Only Switch on Under Certain Conditions
Smoking Switched-on genes

Pattern A Many years later

Pattern B 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 SNP profile A Does Not Respond to Standard Drug Treatment SNP profile B

SNP profile E

SNP profile C

SNP profile D

Gene TNFa IL-1a IL-2 IL-4 IL-6 Chromosome: 6; Location: 6p21.3 Chromosome: 2; Location: 2q14 Chromosome: 4; Location: 4q26-q27 Chromosome: 5; Location: 5q31.1 Chromosome: 7; Location: 7p21

SNP rs2228088, rs3179060, rs35131721, rs4645843, rs1800620, rs1800618, rs11574936 , rs3783588, rs55910084, rs1801715, rs3783581, rs17562, rs17561, rs61538608, rs20540, rs3783531, rs1051753, rs2069763, rs3087209, rs4986964, rs56279116, rs55743996, rs35648164, rs71645915, rs34280821, rs2069830, rs11544633, rs56383910, rs34012176, rs71708959, rs2069860, rs13306435, rs34709428, rs2069849, rs1803205, rs71745371, rs34012639, rs55780930, rs2230052, rs56272177, rs35990253, rs55691228, rs56043315, rs1042154, rs1042155,

IL-8 IL-12

Chromosome: 4; Location: 4q13-q21 Chromosome: 5; Location: 5q31.1-q33.1

IL-13
IL-17 IL-22 IL-23 VEGF

Chromosome: 5; Location: 5q31

Chromosome: 6; Location: 6p12 Chromosome: 12; Location: 12q15 Chromosome: 12; Location: 12q13.3 Chromosome: 6; Location: 6p12

rs55733734, rs56035208, rs34255686, rs34654684, rs20541, rs56258826,

rs17880588, rs17878530, rs2227507, rs61937689, rs11465746, rs11171806, rs71772333, 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 This variant is significantly associated with the efficacy of anti-TNF (Adjusted P-value: 0.000004; OR: 6.8 (1.7, 40.3)). 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. rs437943 at chr4:35048493 This variant is significantly associated with the efficacy of anti-TNF (Adjusted P-value: 0.000004; OR: 4.6 (1.8, 12.3)). 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. 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 This variant is significantly associated with the efficacy of anti-TNF (Adjusted P-value: 0.0000003; OR: 4.6 (1.8, 12.3)). 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. 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 This variant is significantly associated with the efficacy of anti-TNF (Adjusted P-value: 0.000003; OR: 8.5 (2.6, 36.5)). 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. rs854555 at chr7:94768327 in PON1 This variant is significantly associated with the efficacy of anti-TNF (Adjusted P-value: 0.000002; OR: 4.6 (1.8, 12.3)). 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. rs868856 at chr9:27479251 in MOBKL2B This variant is significantly associated with the efficacy of anti-TNF (Adjusted P-value: 0.0000005; OR: 4.9 (1.8, 14.0)). 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. rs7046653 at chr9:27480967 in MOBKL2B This variant is significantly associated with the efficacy of anti-TNF (Adjusted P-value: 0.0000005; OR: 4.9 (1.8, 14.0)). 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. rs2814707 at chr9:27526397 in MOBKL2B This variant is significantly associated with the efficacy of anti-TNF (Adjusted P-value: 0.000002; OR: 5.2 (1.8, 16.7)). 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. rs3849942 at chr9:27533281 This variant is significantly associated with the efficacy of anti-TNF (Adjusted P-value: 0.000005; OR: 5.0 (1.7, 15.8)). 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. rs774359 at chr9:27551049 in C9orf72 This variant is significantly associated with the efficacy of anti-TNF (Adjusted P-value: 0.0000006; OR: 5.4 (1.9, 17.3)). 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. rs6138150 at chr20:23795009 This variant is significantly associated with the efficacy of anti-TNF (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 (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. rs6071980 at chr20:38301990 This variant is significantly associated with the efficacy of anti-TNF (Adjusted P-value: 0.000003; OR: 7.6 (1.9, 44.6)). 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

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 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 treatment (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. 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 This variant is significantly associated with the efficacy of anti-TNF treatment (Adjusted P-value: 0.000004; OR: 6.8 (1.7, 40.3)). 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. rs437943 at chr4:35048493 This variant is significantly associated with the efficacy of anti-TNF treatment (Adjusted P-value: 0.000004; OR: 4.6 (1.8, 12.3)). 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. 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 This variant is significantly associated with the efficacy of anti-TNF treatment (Adjusted P-value: 0.0000003; OR: 4.6 (1.8, 12.3)). 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. rs854547 at chr7:94761792 in PPP1R9A This variant is significantly associated with the efficacy of anti-TNF treatment (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 This variant is significantly associated with the efficacy of anti-TNF treatment (Adjusted P-value: 0.000003; OR: 8.5 (2.6, 36.5)). 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. rs854555 at chr7:94768327 in PON1 This variant is significantly associated with the efficacy of anti-TNF treatment (Adjusted P-value: 0.000002; OR: 4.6 (1.8, 12.3)). 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. rs868856 at chr9:27479251 in MOBKL2B This variant is significantly associated with the efficacy of anti-TNF treatment (Adjusted P-value: 0.0000005; OR: 4.9 (1.8, 14.0)). 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. rs7046653 at chr9:27480967 in MOBKL2B This variant is significantly associated with the efficacy of anti-TNF treatment (Adjusted P-value: 0.0000005; OR: 4.9 (1.8, 14.0)). 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. rs2814707 at chr9:27526397 in MOBKL2B This variant is significantly associated with the efficacy of anti-TNF treatment (Adjusted P-value: 0.000002; OR: 5.2 (1.8, 16.7)). 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. rs3849942 at chr9:27533281 This variant is significantly associated with the efficacy of anti-TNF treatment (Adjusted P-value: 0.000005; OR: 5.0 (1.7, 15.8)). 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. rs774359 at chr9:27551049 in C9orf72 This variant is significantly associated with the efficacy of anti-TNF treatment (Adjusted P-value: 0.0000006; OR: 5.4 (1.9, 17.3)). 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. 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 rs1801133 at chr1:11778965 in CLCN6, MTHFR 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 rs1801133 at chr1:11778965 in CLCN6, MTHFR 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. rs1801133 at chr1:11778965 in CLCN6, MTHFR 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. rs1801133 at chr1:11778965 in CLCN6, MTHFR 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 Can Produce Oncogenes

A rearrangement of the bcl-2 proto-oncogene on chromosome 18 with the immunoglobulin heavy chain region on chromosome 14, leads to deregulated BCL-2 production. Bcl-2 has been shown to prevent programmed cell death (apoptosis) thus immortalizing the cell. The t(14;18) translocation is characteristic of B-cell lymphomas, occurring in up to 90% of follicular lymphomas. It is also found in 20% to 30% of diffuse large B-cell lymphomas, where it is an indicator of poor prognosis.

Mbr (major breakpoint region, 150 bp) Bcl2 Chromosome 18

C JH Double strand DNA break by RAG1/2

Chromosome 14

Translocation takes place in B cell precursors. C

Bcl2

t(14;18) translocation

Transformation takes place during B cell activation in GC. E C C 3E

bcl2

Unregulation of Bcl2 expression by IgH enhancers

Plasma cells Germinal center activation Germinal center

apoptosis

Memory cells

Germinal center IgH-Bcl2 activation

Germinal center

follicular lymphoma Most follicular lymphoma Ig V regions contain somatic hypermutation. Apoptosis inhibited

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 Mycosis fungoides (MF) Variants of MF Pagetoid reticulosis (localized disease) Folliculotropic, syringotropic, granulomatous variants Subtype of MF Granulomatous slack skin Sezary syndrome CD30+ T-cell lymphoproliferative disorders of the skin Lymphomatoid papulosis Primary cutaneous anaplastic large cell lymphoma Subcutaneous panniculitis-like T-cell lymphoma Primary cutaneous peripheral T-Cell lymphoma (PTL), unspecified Subtypes of PTL Primary cutaneous aggressive epidermotropic CD8+ T-cell lymphoma (provisional) Cutaneous gamma/delta-positive T-cell lymphoma (provisional) Primary cutaneous CD4+ small/medium-sized pleomorphic T-cell lymphoma (provisional) Extranodal NK/T-cell lymphoma, nasal type Hydroa vacciniforme-like lymphoma (variant) Adult T-cell leukemia/lymphoma Angioimmunoblastic T-cell lymphoma

Mature B-cell neoplasms Cutaneous marginal zone B-cell lymphoma (MALT-type) Primary cutaneous follicle center lymphoma Growth patterns Follicular Follicular and diffuse Diffuse Cutaneous diffuse large B-cell lymphoma, leg type Cutaneous diffuse large B-cell lymphoma, others Intravascular large B-cell lymphoma Lymphomatoid granulomatosis Chronic lymphocytic leukemia Mantle cell lymphoma Burkitt lymphoma Immature hematopoietic malignancies Blastic NK-cell lymphoma CD4+/CD56+ hematodermic neoplasm Precursor lymphoblastic leukemia/lymphoma T-lymphoblastic lymphoma B-lymphoblastic lymphoma Myeloid and monocytic leukemias Hodgkin lymphoma

Current Microarray Technology

Tissue Lysis mRNA

cDNA
Amplification

Optical Detection
Scanning

Fluorescent Labeling

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 twochannel 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 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 upregulated and downregulated genes.

Microarray analysis

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.