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 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.

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
Paracentric
inversion
Pericentric inversion
Isochromosome
Insertion
Ring chromosome

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
DNA amount
1 µg or less
30 µg min. per probe
DNA quality/size
Can be severely degraded, bp DNA
High quality, HMW DNA needed, atleast 20 kb
DNA source
Fresh or frozen or PBs
Fresh or frozen
Restricition enzyme digestion
Not needed
Required
Gel electrophoresis
Polyacrylamide gels, denaturing gradient gels & non-gel based methods
Agarose gel required
Time
1 to 2 days
1 to 2 weeks
Detection methods
Fluorescent dyes, silver stain, chemiluminescence, radioactivity
Usually radioactivity, less often chemiluminescence.
Senstivity
1 cell per 103 cells
1%-5% of total DNA
False negative
Common for B-cell lymphomas; uncommon in T-cell lymphomas
Rare

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

21. PCR Cycle

22. Exponential Amplification
No. of Thermal Cycles
Copies of Target (PCR Products = Amplicons) 1 2 4 3 8 16 5 32 6 64 20
1,048,576 30
1,073,741,824 (1x109)

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 Suitable specimens:
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 Limitations
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
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 %
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 Principle Probes: two types for translocation
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. Normal cells
Abnormal cells
Schematic figs
Dual fusion probes
Break apart probes

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.

51. FISH -Interpretation Choosing proper area for Evaluation
Preferably areas
richest in abnormal cells
bright, distinct signals and
low background in which individual nuclei are clearly distinguishable
But screening of entire area is essential
For 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.

52. Area to be avoided for interpretation

53. FISH -Interpretation
Awareness of nuclear truncation artefacts induced by sectioning
Should distinguish from loss of chromosome
Establishment of cut off values for different probes and all signal patterns
Complex chromsomal abnormalities

54. FISH - Applications
Detection of numerical and structural chromosomal abnormalities
Identification of marker chromosomes (rearranged chromosomes of uncertain origin)
Detection of gene deletions and gene amplifications
Detection of early relapse or minimal residual disease
Identification of the origin of bone marrow cells following stem cell transplantation

55. FISH - Advantages
Rapid technique, and large numbers of cells can be scored in a short period
Efficiency of hybridisation and detection for is high for structural and numerical abnormalities
Can be applicable in scant cellular specimens (post Tx samples and hypocellular samples
Permits direct correlation of cytogenetic and morphologic features, enabling pathologists to differentiate malignant from benign conditions in equivocal cases

56. FISH - Limitations
Restricted to those abnormalities that can be detected with currently available probes
Only one or few abnormalities can be assessed simultaneously
Cytogenetic data can be obtained only for the target chromosomes;
Not a good screening tool for heterogenous diseases
Requires fluorescence microscopy

57. Lymphomas - Gene expression profiling
Offers the prospects of future refining the lymphoma sub-classification at molecular level
May provide prognostic data and potential for novel targeted therapies
Presently a research tool and requires fresh tissue
Technique:
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 genes
Confocal microscopy along with computerised image analysis system measure the emission spectra
Signal intensity at each spot is proportional to the level of gene expression
Large data generated can be investigated by using mathematical algorithims

58. Gene expression profiling - Utility
DLBCL
3 distinct subgroups based on differential expression of 1000 genes
Germinal centre-like, activated B-cell like and 3rd distinct group, represents heterogenous group
Germinal centre signature was shown to have better survival rates
Further supervised analysis – five differential gene expression profiles
Differential gene expression – early and late or advanced stages
CLL
Overexpression of ZAP 70 – aggressive course
Mantle cell lymphoma
Enables prediction of poor prognosis group
Follicular lymphoma
Tranformation to DCBCL characterized by altered gene expression profile
Reports of prediction for response to rituximab therapy

59. Thank You