Presentation on theme: "Lymphomas: Molecular basics, terms and definitions"— Presentation transcript:
1Lymphomas: Molecular basics, terms and definitions Dr Epari SridharAsst ProfessorPathologyTMC
2Lymphoid neoplasms Classification requires multiparameter approach Clinical featuresMorphologyImmunophenotyping andMolecular methods, in someBoth diagnostic and prognostic significance
3Lymphomas – molecular testing - Utility Demonstration of a clonalityreactive vs neoplastic proliferationAid in correct lymphoma diagnosisInconclusive histologic and immunophenotypic dataUseful for classification, staging, and prognosticationInformation to guide appropriate choice of therapyEvidence of remission or relapse.Identify disease-associated findingssuch as an associated virusspecific chromosomal translocation, that is useful in subclassification.
5Terms Karyotype refers to a full set of chromosomes from an individual Chromosome anomaly, abnormality or aberration reflects an atypical number or a structural abnormality in one or more chromosomes.Two basic groups: Numerical and structural anomalies.
6Chromosomal Numerical Anomaly Aneuploidy: abnormal number of chromosomesMonosomy: chromosome missing from a pair.Denoted as ‘Ms’Trisomy, tetrasomy etc: More than two chromosomes of a pair.‘Ts’ for trisomy and ‘Tet’ for tetrasomy
7Chromosomal structural abnormalities Deletions: A portion of the chromosome is missing or deleted. Denoted as symbol ‘del’Terminal Deletion - a deletion that occurs towards the end of a chromosome.Intercalary Deletion / Interstitial Deletion - a deletion that occurs from the interior of a chromosome.Microdeletions: An extremely small amount of a chromosome is missing, possibly only a single gene.Duplications (dp/dup): Portion of the chromosome is duplicated, resulting in extra genetic material.Gene duplications or amplificationTranslocations: A portion of one chromosome is transferred to another (nonhomologous) chromosome.
8Chromosomal translocations Two main types of translocations:Reciprocal (non-Robertsonian) translocation: segments from two different chromosomes have been exchanged.Robertsonian translocation: an entire chromosome has attached to another at the Centromere.Balanced: even exchange of material with no genetic information extra or missing and ideally with full functionalityUnbalanced: Unequal exchange of material resulting in extra or missing genes.
9Chromosomal translocations - Denotation The International System for Human Cytogenetic Nomenclature (ISCN)t(A;B)(p1;q2)‘t’ stands for translocation(A;B) denotes a translocation between chromosome A and chromosome B.(p1;q2) denotes precise location within the chromosome for chromosomes A and B respectively—with p indicating the short arm of the chromosome, q indicating the long arm, and the numbers after p or q refers to regions, bands and sub-bandsExamples:Burkitt lymphoma: t(8;14)(q24;q32)Mantle cell lymphoma: t(11;14)(q13;q32)Follicular lymphoma: t(14;18)(q32;q21)
10Chromosomal structural abnormalities Inversion: A portion of the chromosome has broken off, turned upside down and reattached, therefore the genetic material is inverted without loss of genetic information. Denoted as symbol ‘in’Paracentric: Do not include the centromere and both breaks occur in one arm of the chromosome.Pericentric: include the centromere and there is a break point in each arm.Ring chromosome: A portion of a chromosome has broken off and formed a circle or ring. This can happen with or without loss of genetic material.denoted by the symbol ‘r’Isochromosome: Formed by the mirror image copy of a chromosome segment including the centromere.denoted by the symbol ‘iso’
11Chromosomal structural abnormalities Insertion:On a chromosomal level, refers to the insertion of a larger sequence into a chromosome.On a genetic (gene) level is the addition of one or more nucleotide base pairs into a DNA sequence.Can be anywhere and of any size incorrectly inserted into a DNA sequence of one chromosome inserted into another.e.g.,Is(7;1) - insertion of part of Chr 7 into Chr 1
12Other Human Chromosome Nomenclature Symbols used to designate these whole arm chromosome changes are:"+" to indicate the presence of a specific additional autosome"–" to indicate the absence of a specific autosome"O" to indicate a missing sex chromosomeAdditional Xs or Ys to indicate supernumerary sex chromosomesNumber of chromosomes is specified, followed by a comma and a specification of the whole arm chromosome change.
14Lymphomas – Molecular genetic methods KaryotypingLimited use, especially in lymphomasDifficult to get adequate cell growth esp. LGNHLCannot detect IgH and TCR re-arrangementsSouthern blot analysisTraditional gold standard for most molecular diagnostic testing.Requires fresh tissue in fairly large amountsLabor-intensive, time-consuming method.Requires large percentage of abnormal cells in the sample (5–10%)Polymerase chain reaction (PCR) methodsDirect PCR and Reverse transcriptase (RT) – PCRIn-situ hybridisation (ISH)Fluorescence in situ hybridization (FISH)Chromogenic in-situ hybridisation (CISH), Silver in-situ hybridisation (SISH) and Rapid in-situ hybridiation (RISH)In-situ PCRPCR in the cell on a slide, and visualized in the same way as in traditional ISHTechnically difficult, is often inconsistent,Not used in most diagnostic laboratories.Others – CGH, Spectral karyotyping, Micro-array technology
15Lymphomas – molecular testing –targets Antigen receptor gene re-arrangements – Ig (Igk, Igλ and IgH) & TCR (TCRγ, TCRβ, TCRα/δ)Southern blot analysisFresh tissueSlow turn –around timeLabour intensiveLow analytical sensitivityPCR methodsPreferred first-line approachAlmost replaced the SB analysis as requires less tissue and permissible with FFPE tissuesChromosomal translocations and aneusomies: DNA based and RNA transcripts (fusion genes)Preferred methods: PCR and FISHConventional cytogenetics
16Antigen receptor re-arrangement Ig and TCR genes – discontinuous segments that encode for the variable (V), joining (J), constant (C) and sometimes diversity (D) regionsDiverse antigen detection capability is generated by different synergistically acting mechanisms:Somatic recombinationComplementarity-determining regions (CDRs)In-Frame alignment of gene segmentsGenetic hierarchyAllelic exclusionClass switching
18Clonality assays – PCR vs Southern blot DNA amount1 µg or less30 µg min. per probeDNA quality/sizeCan be severely degraded, bp DNAHigh quality, HMW DNA needed, atleast 20 kbDNA sourceFresh or frozen or PBsFresh or frozenRestricition enzyme digestionNot neededRequiredGel electrophoresisPolyacrylamide gels, denaturing gradient gels & non-gel based methodsAgarose gel requiredTime1 to 2 days1 to 2 weeksDetection methodsFluorescent dyes, silver stain, chemiluminescence, radioactivityUsually radioactivity, less often chemiluminescence.Senstivity1 cell per 103 cells1%-5% of total DNAFalse negativeCommon for B-cell lymphomas; uncommon in T-cell lymphomasRare
19Polymerase chain reaction (PCR) In-vitro amplification of specific DNA sequences by primer extension of complementary strands of DNAAmplifies a single or few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequencePresently, the most preferred first-line approach in the molecular diagnostic tool
22Exponential Amplification No. of Thermal CyclesCopies of Target (PCR Products = Amplicons)1243816532664201,048,576301,073,741,824 (1x109)
23PCR Methodology DNA –based and m-RNA based AgR rearrangements by DNA basedGene fusion – m-RNA basedQualitative vs quantitative assaysMost diagnostic assays are qualitative: simply detect presence or absenceQuantitative – required for MRDAssays and detection systemsAssay designSingle primer set vs hemi-nested vs nestedMonoplex vs multiplex reactionsPrimer designConsensus vs gene family specific
24Multiplex-PCR Molecular equivalent of multitasking Several pairs of primers annealing to different target sequences.Permits the simultaneous analysis of multiple targets in a single sample.Multiplex Ligation-dependent Probe Amplification (or MLPA): multiple targets to be amplified using only a single pair of primers.
25Nested PCRIncreases the specificity of DNA amplification and more successful in specifically amplifying long DNA products.Two sets of primers are used in two successive reactions.In the first PCR, one pair of primers is used to generate DNA products, which may contain products amplified from non-target areas.The products from the first PCR are then used as template in a second PCR, using one ('hemi-nesting') or two different primers whose binding sites are located (nested) within the first set, thus increasing specificity.
26Quantitative PCR (Q-PCR) Measures the specific amount of target DNA (or RNA).Special thermal cyclers are used that monitor the amount of product during the amplification.Quantitative Real-Time PCR (QRT-PCR): measures the amount of amplified product by using fluorescence dye tagged primers.
27Reverse Transcription PCR ( RT-PCR) Reverse transcribe and amplify RNA to DNA.Before the PCR reaction, conversion of RNA to cDNA is done by a reverse transcriptase enzyme.
28Methylation-specific PCR (MSP) Identifies patterns of DNA methylation at CpG (cytosine-guanine) islands.Bisulphite conversion - converts unmethylated cytosine bases to uracil, which is complementary to adenosine in PCR primers.Two amplifications are then carried out:One primer set anneals to DNA with cytosines (corresponding to methylated cytosine), andOther set anneals to DNA with uracil (corresponding to unmethylated cytosine).
29PCR Methodology - Product detection system Simple gel electrophoresisMost frequently employedBased on size - agarose and polyacralymideRequires ethidium bromide staining and UV illuminationHybridisation with labelled probesPAGE allows superior resolution and preferred for small PCR productsCannot achieve single base resolutionNot quantitativeInsensitive in detecting small monoclonal popln esp. in the background of polyclonal population
30PCR Methodology – Other detection systems Complex gel electrophoresisDenaturing gradient gel electrophoresis (DGGE)Temperature gradient gel electrophoresis (TGGE)Heteroduplex analysis in mutation detection enhancing gelsSingle strand conformational polymorphism analysis (SSCP)Solution based methodsColorimetric, fluorescent and chemiluminscentLess commonly usedCapillary electrophoresis with automated fluorescent DNA fragment analysis (CEGS; GeneScan)Considered to superior of all because of high sensitivity and high throughputMethod of choice
31Heteroduplex analysis Sequence variations in dsDNA can cause bends in the double helix, or even alter the basic structure of the helix and thus restricts the mobility of the same in the media.A mismatch between the two strands of DNA in a duplex can produce a more radical kink in the structure, producing a heteroduplex species which can easily be resolved from the homoduplex by electrophoresis.heteroduplex products will result in a smear of slow migrating products
32PCR - techniques Indications Suitable specimens: AgR gene rearrangementsSpecific translocationsMRD detection and monitoringSuitable specimens:Small tissue biopsies including BMBx & BMA, cells scraped/microdissected from slides and specimensBoth fresh and FFPE tissue samples
33PCR – techniques – laboratory factors Significant interlaboratory variation in the senstivity of clonal detection with the paraffin fixed tissuesFactors affectingFixatives- formalin is the best; mercuric fixatives and Bouin fluid are least suitableDecalcification – EDTA is better than formic acidMethods of extraction of DNA
34CEGS; Genescan Advantages Limitations Obviates labour intensive gel preparation and exposure to hazardous UV light and ethidium bromideSpeed : read out requires hardly 30 minutesAutomatic data processing and electronic storageAchieves single base pair resolutionComparable sensitivityLimitationsHighly expensiveNot resistant for false positives
35PCR – Sensitivity & Specificity Case selectionNature of sample (nature of background cellularity)Type of detection methods usedSpecificityLineage infidelity – well recognized in lymphoblastic lymphomasClonality does not always equate with malignancy nor vice versa
36PCR – T Cell clonality testing - Indications T-cell lymphomas are often difficult to diagnoseDifficult to distinguish from the benign reactive T cell proliferations by immunophenotypingMolecular assay for clonality : targeting TCRγ and TCRβ receptor genesTCRγ is preferred due to the simplicity of the structure
37PCR – T Cell clonality testing TCRγ rearrangementsPrimers combination of Vγ and Jγ – detects all possible rearrangements - Four V regions and five J regionsPAGE is good enough for separation of products and detection but Heteroduplex analysis and CEGS – bestQualitative sensitivity: Wide reported range (60-100%)With multiple primers PAGE achieves 80-90% and high resolution like heteroduplex analysis or CEGS can reach upto 100%Analytical sensitivityRoutine PAGE – 1-5%; Much higher with CEGSTest specificity and positive predictive valueRange from %Low in lymphobastic lymphomas,High false positivity in inflammatory dermatoses, sometimes in plasma cell dyscrasias and Hodgkin lymphomas
40PCR – T Cell clonality testing TCRβ gene rearrangementsLess often used due to complexity of the gene structureDifficult for consensus primersQualitative sensitivity : range 50-80%Quantative sensitivity: 2-5%Clonal detection range may be increased by as much as 20% if used along with TCRγ
41PCR – B Cell clonality testing IgH gene testing is the principal approachQualitative sensitivity: >50% to virtually 100% - depends upon case mix, primer details and detection methodsFalse negatives are known to occur in FL, MZL and DLBCLsFalse positives reported in AITLs and PTCLClinical utility for tests is extremely rareEspecially in diagnosing composite B cell lymphomas for determining two different clones of B-cells
42PCR assays - pitfallsCombination of technical and biological factors and interpretation errorsFalse positive ratesContaminationsExcessive amplification cyclesInorganic DNA extraction methodsPseuodoclonality: selective oligoclone amplification due to insufficient sampleInappropriate AgG rearrangementsFalse negative ratesSampling errorsDNA and RNA degradationPCR designBiological factors
43By using complex multiplex assays with advanced methods of detection increases the chance of detection of clonality in benign/reactive conditionsMolecular data should never be reported in isolation from all other clinicopathological factors in each case
44Standardization of PCR assays Multicentre European collaborative studies have been instituted to optimise and standardize the PCR assays for purpose of clonality studies in lymphoma clonality testing – Biomed 2 concerted actionInvolves the use of 107 standardised primers in a series of 18 multiplex reationsProduct detection either by heteroduplex or automated Genescan analysis.
45Fluorescence in situ hybridization (FISH) Allows detection of both structural and numerical chromosomal abnormalitiesConsidered superior to PCR methods for detection of translocations and aneusomysNot widely used in the routine diagnostic evaluation of paraffin-embedded biopsies,Technically more demanding (perception)Uncertainties regarding diagnostic thresholds and result interpretation.
46FISH Types Principle Probes: two types for translocation Metaphase FISHInterphase FISH – for solid Txs and FFPET can be usedPrincipleVisualization of bound of flourochlorome tagged DNA fragments to complementary target genomic regionProbes: two types for translocationDual fusion probesSuperior due to lack of false positivityBreak-apartGives abnormal results for variant translocations alsoDo not detect the other gene involvedProbes: two types for detection of copy number changesLocus specificChromosome enumeration (centromeric or pericentromeric satellites)
47Metaphase FISHInterphase FISHCourtesy: JMD November 2000, Vol. 2, No. 4
49A,B – Two different cases of Burkitt lymphoma showing fusion signals for IgH/CMYC (as shown by arrows)AB
50FISH – InterpretationAcquire experience of normal and abnormal signal patterns for each probe applied,using negative tissues (eg. reactive lymph nodes) and relevant positive samples (eg. lymphomas known to contain the abnormality under investigation).Other factors to be aware of:the architecture of the tissue, including local variations in neoplastic cell content, fixation, and cellularity within the section;Nuclear truncation andthe complex nature of genetic arrangements seen in some lymphoid neoplasms.Should have a HE stained slide at your hand.
51FISH -Interpretation Choosing proper area for Evaluation Preferably areasrichest in abnormal cellsbright, distinct signals andlow background in which individual nuclei are clearly distinguishableBut screening of entire area is essentialFor the presence of subclonal changes that might be of diagnostic and prognostic importance, e.g., the presence of t(8;14) only in a subpopulation might indicate transformation into a more aggressive lymphoma.Areas of nuclear overlapping with indistinct nuclear outlines and high cell density - should be avoided.
53FISH -InterpretationAwareness of nuclear truncation artefacts induced by sectioningShould distinguish from loss of chromosomeEstablishment of cut off values for different probes and all signal patternsComplex chromsomal abnormalities
54FISH - ApplicationsDetection of numerical and structural chromosomal abnormalitiesIdentification of marker chromosomes (rearranged chromosomes of uncertain origin)Detection of gene deletions and gene amplificationsDetection of early relapse or minimal residual diseaseIdentification of the origin of bone marrow cells following stem cell transplantation
55FISH - AdvantagesRapid technique, and large numbers of cells can be scored in a short periodEfficiency of hybridisation and detection for is high for structural and numerical abnormalitiesCan be applicable in scant cellular specimens (post Tx samples and hypocellular samplesPermits direct correlation of cytogenetic and morphologic features, enabling pathologists to differentiate malignant from benign conditions in equivocal cases
56FISH - LimitationsRestricted to those abnormalities that can be detected with currently available probesOnly one or few abnormalities can be assessed simultaneouslyCytogenetic data can be obtained only for the target chromosomes;Not a good screening tool for heterogenous diseasesRequires fluorescence microscopy
57Lymphomas - Gene expression profiling Offers the prospects of future refining the lymphoma sub-classification at molecular levelMay provide prognostic data and potential for novel targeted therapiesPresently a research tool and requires fresh tissueTechnique:Co-hybridisation of differentially flourochrome labelled RNA or cDNA of tumour and normal tissue with a cDNA chip (lymphochip)The chips contain robotically arranged known cDNAs from hundreds to thousands of genesConfocal microscopy along with computerised image analysis system measure the emission spectraSignal intensity at each spot is proportional to the level of gene expressionLarge data generated can be investigated by using mathematical algorithims
58Gene expression profiling - Utility DLBCL3 distinct subgroups based on differential expression of 1000 genesGerminal centre-like, activated B-cell like and 3rd distinct group, represents heterogenous groupGerminal centre signature was shown to have better survival ratesFurther supervised analysis – five differential gene expression profilesDifferential gene expression – early and late or advanced stagesCLLOverexpression of ZAP 70 – aggressive courseMantle cell lymphomaEnables prediction of poor prognosis groupFollicular lymphomaTranformation to DCBCL characterized by altered gene expression profileReports of prediction for response to rituximab therapy