Lymphomas: Molecular basics, terms and definitions

1. Lymphomas: Molecular basics, terms and definitions Dr Epari Sridhar Asst Professor Pathology TMC

2. Lymphoid neoplasms • Classification requires multiparameter approach • Clinical features • Morphology • Immunophenotyping and • Molecular methods, in some • Both diagnostic and prognostic significance

3. Lymphomas – molecular testing - Utility • Demonstration of a clonality • reactive vs neoplastic proliferation • Aid in correct lymphoma diagnosis • Inconclusive histologic and immunophenotypic data • Useful for classification, staging, and prognostication • Information to guide appropriate choice of therapy • Evidence of remission or relapse. • Identify disease-associated findings • such as an associated virus • specific chromosomal translocation, that is useful in subclassification.

4. Lymphomas – Molecular testing - Targets • Antigen receptor gene rearrangements • Non-random chromosomal abnormalities • Translocations • Numerical aberrations

5. Terms • Karyotyperefers to a full set of chromosomes from an individual • Chromosome anomaly, abnormality or aberrationreflects an atypical number or a structural abnormality in one or more chromosomes. • Two basic groups: Numerical and structural anomalies.

6. Chromosomal Numerical Anomaly • Aneuploidy: abnormal number of chromosomes • Monosomy: 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

7. Chromosomal 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 amplification • Translocations: A portion of one chromosome is transferred to another (nonhomologous) chromosome.

8. Chromosomal 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 functionality • Unbalanced:Unequal exchange of material resulting in extra or missing genes.

9. Chromosomal 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-bands • Examples: • Burkitt lymphoma: t(8;14)(q24;q32) • Mantle cell lymphoma: t(11;14)(q13;q32) • Follicular lymphoma: t(14;18)(q32;q21)

10. Chromosomal 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’

11. Chromosomal 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

12. Other 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 chromosome • Additional Xs or Ys to indicate supernumerary sex chromosomes • Number of chromosomes is specified, followed by a comma and a specification of the whole arm chromosome change.

13. Chromosomal structural abnormalities Isochromosome Paracentric inversion Ring chromosome Pericentric inversion Insertion

14. Lymphomas – Molecular genetic methods • Karyotyping • Limited use, especially in lymphomas • Difficult to get adequate cell growth esp. LGNHL • Cannot detect IgH and TCR re-arrangements • Southern blot analysis • Traditional gold standard for most molecular diagnostic testing. • Requires fresh tissue in fairly large amounts • Labor-intensive, time-consuming method. • Requires large percentage of abnormal cells in the sample (5–10%) • Polymerase chain reaction (PCR) methods • Direct PCR and Reverse transcriptase (RT) – PCR • In-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 PCR • PCR in the cell on a slide, and visualized in the same way as in traditional ISH • Technically difficult, is often inconsistent, • Not used in most diagnostic laboratories. • Others – CGH, Spectral karyotyping, Micro-array technology

15. Lymphomas – molecular testing –targets • Antigen receptor gene re-arrangements – Ig (Igk, Igλ and IgH) & TCR (TCRγ, TCRβ, TCRα/δ) • Southern blot analysis • Fresh tissue • Slow turn –around time • Labour intensive • Low analytical sensitivity • PCR methods • Preferred first-line approach • Almost replaced the SB analysis as requires less tissue and permissible with FFPE tissues • Chromosomal translocations and aneusomies: DNA based and RNA transcripts (fusion genes) • Preferred methods: PCR and FISH • Conventional cytogenetics

16. Antigen receptor re-arrangement • Ig and TCR genes – discontinuous segments that encode for the variable (V), joining (J), constant (C) and sometimes diversity (D) regions • Diverse antigen detection capability is generated by different synergistically acting mechanisms: • Somatic recombination • Complementarity-determining regions (CDRs) • In-Frame alignment of gene segments • Genetic hierarchy • Allelic exclusion • Class switching

18. Clonality assays – PCR vs Southern blot

19. Polymerase chain reaction (PCR) • In-vitro amplification of specific DNA sequences by primer extension of complementary strands of DNA • Amplifies 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 sequence • Presently, the most preferred first-line approach in the molecular diagnostic tool

20. 3’ 3’ 5’ Denaturation 95-98ºC 5’ 5’ 3’ 3’ 5’ Annealing 50-65ºC 5’ 3’ 5’ 3’ 3’ 5’ 3’ 5’ 3’ Elongation 75-80ºC 5’ 5’ 3’ 3’ 5’ 5’ 3’ 3’ 5’ 3’ 5’ 5’ 3’ 5’ 3’ PCR Cycle

21. PCR Cycle

22. Exponential Amplification

23. PCR Methodology • DNA –based and m-RNA based • AgR rearrangements by DNA based • Gene fusion – m-RNA based • Qualitative vs quantitative assays • Most diagnostic assays are qualitative: simply detect presence or absence • Quantitative – required for MRD • Assays and detection systems • Assay design • Single primer set vs hemi-nested vs nested • Monoplex vs multiplex reactions • Primer design • Consensus vs gene family specific

24. Multiplex-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.

25. Nested PCR • Increases 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.

26. Quantitative 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.

27. Reverse 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.

28. Methylation-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), and • Other set anneals to DNA with uracil (corresponding to unmethylated cytosine).

29. PCR Methodology - Product detection system Simple gel electrophoresis • Most frequently employed • Based on size - agarose and polyacralymide • Requires ethidium bromide staining and UV illumination • Hybridisation with labelled probes • PAGE allows superior resolution and preferred for small PCR products • Cannot achieve single base resolution • Not quantitative • Insensitive in detecting small monoclonal popln esp. in the background of polyclonal population

30. PCR Methodology – Other detection systems • Complex gel electrophoresis • Denaturing gradient gel electrophoresis (DGGE) • Temperature gradient gel electrophoresis (TGGE) • Heteroduplex analysis in mutation detection enhancing gels • Single strand conformational polymorphism analysis (SSCP) • Solution based methods • Colorimetric, fluorescent and chemiluminscent • Less commonly used • Capillary electrophoresis with automated fluorescent DNA fragment analysis (CEGS; GeneScan) • Considered to superior of all because of high sensitivity and high throughput • Method of choice

31. Heteroduplex 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

32. PCR - techniques • Indications • AgR gene rearrangements • Specific translocations • MRD detection and monitoring • Suitable specimens: • Small tissue biopsies including BMBx & BMA, cells scraped/microdissected from slides and specimens • Both fresh and FFPE tissue samples

33. PCR – techniques – laboratory factors • Significant interlaboratory variation in the senstivity of clonal detection with the paraffin fixed tissues • Factors affecting • Fixatives- formalin is the best; mercuric fixatives and Bouin fluid are least suitable • Decalcification – EDTA is better than formic acid • Methods of extraction of DNA

34. CEGS; Genescan • Advantages • Obviates labour intensive gel preparation and exposure to hazardous UV light and ethidium bromide • Speed : read out requires hardly 30 minutes • Automatic data processing and electronic storage • Achieves single base pair resolution • Comparable sensitivity • Limitations • Highly expensive • Not resistant for false positives

35. PCR – Sensitivity & Specificity • Sensitivity • Case selection • Nature of sample (nature of background cellularity) • Type of detection methods used • Specificity • Lineage infidelity – well recognized in lymphoblastic lymphomas • Clonality does not always equate with malignancy nor vice versa

36. PCR – T Cell clonality testing - Indications • T-cell lymphomas are often difficult to diagnose • Difficult to distinguish from the benign reactive T cell proliferations by immunophenotyping • Molecular assay for clonality : targeting TCRγ and TCRβ receptor genes • TCRγ is preferred due to the simplicity of the structure

37. PCR – T Cell clonality testing TCRγ rearrangements • Primers combination of Vγ and Jγ – detects all possible rearrangements - Four V regions and five J regions • PAGE is good enough for separation of products and detection but Heteroduplex analysis and CEGS – best • Qualitative 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 sensitivity • Routine PAGE – 1-5%; Much higher with CEGS • Test specificity and positive predictive value • Range from 70-100% • Low in lymphobastic lymphomas, • High false positivity in inflammatory dermatoses, sometimes in plasma cell dyscrasias and Hodgkin lymphomas

38. CD24251: 19/F, c/o HL; CE17159:24/F, c/o HL PCR for TCRγ gene rearrangement

40. PCR – T Cell clonality testing TCRβ gene rearrangements • Less often used due to complexity of the gene structure • Difficult for consensus primers • Qualitative sensitivity : range 50-80% • Quantative sensitivity: 2-5% • Clonal detection range may be increased by as much as 20% if used along with TCRγ

41. PCR – B Cell clonality testing • IgH gene testing is the principal approach • Qualitative sensitivity: >50% to virtually 100% - depends upon case mix, primer details and detection methods • False negatives are known to occur in FL, MZL and DLBCLs • False positives reported in AITLs and PTCL • Clinical utility for tests is extremely rare • Especially in diagnosing composite B cell lymphomas for determining two different clones of B-cells

42. PCR assays - pitfalls • Combination of technical and biological factors and interpretation errors • False positive rates • Contaminations • Excessive amplification cycles • Inorganic DNA extraction methods • Pseuodoclonality: selective oligoclone amplification due to insufficient sample • Inappropriate AgG rearrangements • False negative rates • Sampling errors • DNA and RNA degradation • PCR design • Biological factors

43. By using complex multiplex assays with advanced methods of detection increases the chance of detection of clonality in benign/reactive conditions Molecular data should never be reported in isolation from all other clinicopathological factors in each case

44. Standardization 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 action • Involves the use of 107 standardised primers in a series of 18 multiplex reations • Product detection either by heteroduplex or automated Genescan analysis.

45. Fluorescence in situ hybridization (FISH) • Allows detection of both structural and numerical chromosomal abnormalities • Considered superior to PCR methods for detection of translocations and aneusomys • Not widely used in the routine diagnostic evaluation of paraffin-embedded biopsies, • Technically more demanding (perception) • Uncertainties regarding diagnostic thresholds and result interpretation.

46. FISH • Types • Metaphase FISH • Interphase FISH – for solid Txs and FFPET can be used • Principle • Visualization of bound of flourochlorome tagged DNA fragments to complementary target genomic region • Probes: two types for translocation • Dual fusion probes • Superior due to lack of false positivity • Break-apart • Gives abnormal results for variant translocations also • Do not detect the other gene involved • Probes: two types for detection of copy number changes • Locus specific • Chromosome enumeration (centromeric or pericentromeric satellites)

47. Metaphase FISH Interphase FISH Courtesy: JMD November 2000, Vol. 2, No. 4

48. Abnormal cells Schematic figs Dual fusion probes Break apart probes Normal cells

49. A,B – Two different cases of Burkitt lymphoma showing fusion signals for IgH/CMYC (as shown by arrows) A B

50. FISH – Interpretation • Acquire 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 and • the complex nature of genetic arrangements seen in some lymphoid neoplasms. • Should have a HE stained slide at your hand.