Lymphomas: Molecular basics,

terms and definitions

Dr Epari Sridhar
Asst Professor
Pathology
TMC
Lymphoid neoplasms

Classification requires multiparameter

approach

Clinical features

Morphology

Immunophenotyping and

Molecular methods, in some

Both diagnostic and prognostic significance

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

vidence of remission or relapse!

Identify disease"associated findings

such as an associated virus

specific chromosomal translocation, that is useful in

su#classification!
Lymphomas Molecular testing -
Targets

Antigen receptor gene rearrangements

Non-random chromosomal abnormalities

Translocations

Numerical aberrations
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.
hromosomal !umerical "nomaly

"neuploidy: a#normal num#er of

chromosomes

Monosomy$ chromosome missing from a pair!

Denoted as %Ms&

Trisomy, tetrasomy etc$ More than t'o

chromosomes of a pair!

%Ts& for trisomy and %Tet& for tetrasomy

hromosomal structural abnormalities

Deletions: A portion of the chromosome is missing

or deleted! Denoted as sym#ol %del&

Terminal Deletion " a deletion that occurs to'ards the

end of a chromosome!

#ntercalary Deletion $ #nterstitial Deletion " a deletion

that occurs from the interior of a chromosome!

Microdeletions: An e(tremely small amount of a

chromosome is missing, possi#ly only a single gene!

Duplications %dp$dup&: Portion of the chromosome

is duplicated, resulting in e(tra genetic material!

)ene duplications or amplification

Translocations: A portion of one chromosome is

transferred to another *nonhomologous+
chromosome!
hromosomal translocations
T'o main types of translocations$

'eciprocal %non-'obertsonian& translocation:

segments from t'o different chromosomes have #een
e(changed!

'obertsonian translocation: an entire chromosome

has attached to another at the Centromere!

Balanced: even e(change of material 'ith no genetic

information e(tra or missing and ideally 'ith full
functionality

Unbalanced: Unequal e(change of material resulting in

e(tra or missing genes!
hromosomal translocations - Denotation
The International System for Human Cytogenetic Nomenclature (ISCN)

t%"()&%p*(+,&

-t. stands for translocation

%"()& denotes a translocation #et'een chromosome A

and chromosome B!

%p*(+,& denotes precise location 'ithin the chromosome

for chromosomes A and B respectively,'ith p indicating
the short arm of the chromosome, q indicating the long
arm, and the num#ers after p or q refers to regions, #ands
and su#"#ands

(amples$

Bur-itt lymphoma$ t*./01+*q21/q32+

Mantle cell lymphoma$ t*00/01+*q03/q32+

4ollicular lymphoma$ t*01/0.+*q32/q20+

hromosomal structural abnormalities

#n/ersion: A portion of the chromosome has #ro-en off,

turned upside do'n and reattached, therefore the genetic
material is inverted 'ithout loss of genetic information!
Denoted as sym#ol %in&

0aracentric: Do not include the centromere and #oth #rea-s occur

in one arm of the chromosome!

0ericentric: include the centromere and there is a #rea- point in

each arm!

'ing chromosome: A portion of a chromosome has #ro-en

off and formed a circle or ring! This can happen 'ith or
'ithout loss of genetic material!

denoted #y the sym#ol %r&

#sochromosome: 4ormed #y the mirror image copy of a

chromosome segment including the centromere!

denoted #y the sym#ol %iso&

hromosomal structural abnormalities

#nsertion:

5n a chromosomal level, refers to the insertion

of a larger sequence into a chromosome!

5n a genetic *gene+ level is the addition of one

or more nucleotide #ase pairs into a D6A
sequence!

Can #e any'here and of any si7e incorrectly

inserted into a D6A sequence of one
chromosome inserted into another!

e!g!,Is*8/0+ " insertion of part of Chr 8 into Chr 0

1ther 2uman hromosome !omenclature

9ym#ols used to designate these 'hole arm

chromosome changes are$

:;: to indicate the presence of a specific additional

autosome

:<: to indicate the a#sence of a specific autosome

:5: to indicate a missing se( chromosome

Additional =s or >s to indicate supernumerary se(

chromosomes

6um#er of chromosomes is specified, follo'ed #y a

comma and a specification of the 'hole arm
chromosome change!
'ing
chromosome
0ericentric
in/ersion
0aracentric
in/ersion
#sochromosome
#nsertion
Chromosomal structural abnormalities
Lymphomas Molecular genetic methods

3aryotyping

?imited use, especially in lymphomas

Difficult to get adequate cell gro'th esp! ?)6@?

Cannot detect Ig@ and TCA re"arrangements

Southern blot analysis

Traditional gold standard for most molecular diagnostic testing!

Aequires fresh tissue in fairly large amounts

?a#or"intensive, time"consuming method!

Aequires large percentage of a#normal cells in the sample *B<0CD+

0olymerase chain reaction %0'& methods

Direct PCA and Aeverse transcriptase *AT+ < PCA

#n-situ hybridisation %#S2&

4luorescence in situ hy#ridi7ation *4I9@+

Chromogenic in-situ hy#ridisation *CI9@+, 9ilver in-situ hy#ridisation *9I9@+

and Aapid in"situ hy#ridiation *AI9@+

#n-situ 0'

PCA in the cell on a slide, and visuali7ed in the same 'ay as in traditional I9@

Technically difficult, is often inconsistent,

6ot used in most diagnostic la#oratories!

1thers 42, Spectral 5aryotyping, Micro-array technology

Lymphomas molecular testing
targets

"ntigen receptor gene re-arrangements < Ig *Ig-,

IgE and Ig@+ F TCA *TCAG, TCAH, TCAIJK+

Southern blot analysis

4resh tissue

9lo' turn <around time

?a#our intensive

?o' analytical sensitivity

0' methods

Preferred first"line approach

Almost replaced the 9B analysis as requires less tissue and

permissi#le 'ith 44P tissues

hromosomal translocations and aneusomies$

D6A #ased and A6A transcripts *fusion genes+

Preferred methods$ 0' and 6#S2

Conventional cytogenetics
"ntigen receptor re-arrangement

Ig and TCA genes < discontinuous segments that

encode for the varia#le *L+, Moining *N+, constant *C+
and sometimes diversity *D+ regions

Diverse antigen detection capa#ility is generated #y

different synergistically acting mechanisms$

9omatic recom#ination

Complementarity"determining regions *CDAs+

In"4rame alignment of gene segments

)enetic hierarchy

Allelic e(clusion

Class s'itching
lonality assays 0' /s Southern blot
PCR Southern blot
DNA amount 1 g or less 30 g min. per probe
DNA uality!si"e #an be severely degraded$ 100%300
bp DNA
&igh uality$ &'( DNA
needed$ atleast )0 *b
DNA source Fresh or froen or P!s Fresh or froen
+estricition
en"yme digestion
Not needed +euired
,el
electrophoresis
-olyacrylamide gels$ denaturing
gradient gels . non%gel based
methods
Agarose gel reuired
Time 1 to ) days 1 to ) wee*s
Detection
methods
/luorescent dyes$ silver stain$
chemiluminescence$ radioactivity
0sually radioactivity$ less
often chemiluminescence.
Sensti"ity # cell per #$% cells #&-'& of total DNA
False negati"e Common for !-cell lymphomas(
uncommon in )-cell lymphomas
Rare
0olymerase chain reaction %0'&

In-vitro amplification of specific D6A sequences #y

primer e(tension of complementary strands of D6A

Amplifies a single or fe' copies of a piece of D6A

across several orders of magnitude, generating
thousands to millions of copies of a particular D6A
sequence

Presently, the most preferred first"line approach in

the molecular diagnostic tool
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Denaturation
OB"O.PC
Annealing
BC"QBPC

l
o
n
g
a
t
i
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n
8
B
"
.
C
P
C
PCR Cycle
PCR Cycle
!o7 of Thermal ycles opies of Target %0'
0roducts 8 "mplicons&
* ,
, 9
: ;
9 *<
= :,
< <9
,> *,>9;,=?<
:> *,>?:,?9*,;,9 %*@*>A&
E@ponential "mplification
0' Methodology

D!" based and m-'!" based

AgA rearrangements #y D6A #ased

)ene fusion < m"A6A #ased

Bualitati/e /s +uantitati/e assays

Most diagnostic assays are qualitative$ simply detect

presence or a#sence

Ruantitative < required for MAD

"ssays and detection systems

Assay design

9ingle primer set vs hemi"nested vs nested

Monople( vs multiple( reactions

Primer design

Consensus vs gene family specific

Multiple@-0'

Molecular equivalent of multitas-ing

9everal pairs of primers annealing to different target

sequences!

Permits the simultaneous analysis of multiple

targets in a single sample!

Multiplex Ligation-epenent !ro"e

#mplification %or ML0"&: multiple targets to #e
amplified using only a single pair of primers!
!ested 0'

Increases the specificity of D6A amplification and

more successful in specifically amplifying long D6A
products!

T'o sets of primers are used in t'o successive

reactions!

In the first PCA, one pair of primers is used to generate

D6A products, 'hich may contain products amplified from
non"target areas!

The products from the first PCA are then used as template
in a second PCA, using one *Shemi"nestingS+ or t'o
different primers 'hose #inding sites are located *nested+
'ithin the first set, thus increasing specificity!
Buantitati/e 0' %B-0'&

Measures the specific amount of target D6A *or

A6A+!

9pecial thermal cyclers are used that monitor the

amount of product during the amplification!

Quantitative Real-Time PCR *RAT"PCA+$

measures the amount of amplified product #y
using fluorescence dye tagged primers!
'e/erse Transcription 0' % 'T-0'&

Aeverse transcri#e and amplify A6A to

D6A!

Before the PCA reaction, conversion of A6A

to cD6A is done #y a reverse transcriptase
en7yme!
Methylation-specific 0' %MS0&

Identifies patterns of D6A methylation at Cp)

*cytosine"guanine+ islands!

)isulphite con/ersion " converts unmethylated

cytosine #ases to uracil, 'hich is complementary to
adenosine in PCA primers!

T'o amplifications are then carried out$

5ne primer set anneals to D6A 'ith cytosines

*corresponding to methylated cytosine+, and

5ther set anneals to D6A 'ith uracil *corresponding to

unmethylated cytosine+!
0' Methodology - 0roduct
detection system
Simple gel electrophoresis

Most fre+uently employed

Based on si7e " agarose and polyacralymide

Aequires ethidium #romide staining and UL illumination

@y#ridisation 'ith la#elled pro#es

PA) allo's superior resolution and preferred for small

PCA products

Cannot achieve single #ase resolution

6ot quantitative

Insensitive in detecting small monoclonal popln esp! in the

#ac-ground of polyclonal population
0' Methodology 1ther detection
systems

Comple( gel electrophoresis

Denaturing gradient gel electrophoresis *D))+

Temperature gradient gel electrophoresis *T))+

@eteroduple( analysis in mutation detection enhancing gels

9ingle strand conformational polymorphism analysis *99CP+

9olution #ased methods

Colorimetric, fluorescent and chemiluminscent

?ess commonly used

apillary electrophoresis Cith automated fluorescent D!"

fragment analysis %E4S( 4eneScan&

Considered to superior of all #ecause of high sensitivity and

high throughput

Method of choice
2eteroduple@ analysis

9equence variations in dsD6A can cause #ends in

the dou#le heli(, or even alter the #asic structure of
the heli( and thus restricts the mo#ility of the same
in the media!

A mismatch #et'een the t'o strands of D6A in a

duple( can produce a more radical -in- in the
structure, producing a heteroduple( species 'hich
can easily #e resolved from the homoduple( #y
electrophoresis!

heteroduple( products 'ill result in a smear of slo'

migrating products
0' - techni+ues

#ndications

AgA gene rearrangements

9pecific translocations

MAD detection and monitoring

Suitable specimens$

9mall tissue #iopsies including BMB( F BMA,

cells scrapedJmicrodissected from slides and
specimens

Both fresh and 44P tissue samples

0' techni+ues laboratory
factors

9ignificant interla#oratory variation in the

senstivity of clonal detection 'ith the paraffin
fi(ed tissues

4actors affecting

4i(atives" formalin is the #est/ mercuric fi(atives and

Bouin fluid are least suita#le

Decalcification < DTA is #etter than formic acid

Methods of e(traction of D6A

E4S( 4enescan

Advantages

5#viates la#our intensive gel preparation and

e(posure to ha7ardous UL light and ethidium
#romide

9peed $ read out requires hardly 3C minutes

Automatic data processing and electronic

storage

Achieves single #ase pair resolution

Compara#le sensitivity

?imitations

@ighly e(pensive

6ot resistant for false positives

0' Sensiti/ity D Specificity

Sensiti/ity

Case selection

6ature of sample *nature of #ac-ground cellularity+

Type of detection methods used

Specificity

?ineage infidelity < 'ell recogni7ed in

lympho#lastic lymphomas

Clonality does not al'ays equate 'ith malignancy

nor vice versa
0' T ell clonality testing -
#ndications

T"cell lymphomas are often difficult to diagnose

Difficult to distinguish from the #enign reactive T

cell proliferations #y immunophenotyping

Molecular assay for clonality $ targeting TC$%

an TC$& receptor genes

TC$% is preferre ue to the simplicity of the

structure
0' T ell clonality testing
TC$% rearrangements

Primers com#ination of LG and NG < detects all possi#le

rearrangements " 4our L regions and five N regions

PA) is good enough for separation of products and

detection #ut 2eteroduple@ analysis and E4S best

Rualitative sensitivity$ Tide reported range *QC"0CCD+

Tith multiple primers PA) achieves .C"OCD and high resolution li-e
heteroduple( analysis or C)9 can reach upto 0CCD

Analytical sensitivity

Aoutine PA) < 0"BD/ Much higher 'ith C)9

Test specificity and positive predictive value

Aange from 8C"0CCD

LoC in lymphobastic lymphomas,

@igh false positivity in inflammatory dermatoses, sometimes in

plasma cell dyscrasias and @odg-in lymphomas
CD*+*'#, #-.F, c.o /0( C1#2#'-,*+.F, c.o /0
PCR for )CR3 gene rearrangement
0' T ell clonality testing
TC$& gene rearrangements

?ess often used due to comple(ity of the gene

structure

Difficult for consensus primers

Rualitative sensitivity $ range BC".CD

Ruantative sensitivity$ 2"BD

Clonal detection range may #e increased #y

as much as 2CD if used along 'ith TCAG
0' ) ell clonality testing

Ig@ gene testing is the principal approach

Rualitative sensitivity$ UBCD to virtually 0CCD "

depends upon case mi(, primer details and
detection methods

4alse negatives are -no'n to occur in 4?, MV? and

D?BC?s

4alse positives reported in AIT?s and PTC?

Clinical utility for tests is e(tremely rare

specially in diagnosing composite B cell lymphomas

for determining t'o different clones of B"cells
0' assays - pitfalls

Com#ination of technical and #iological factors and

interpretation errors

4alse positive rates

ontaminations

(cessive amplification cycles

Inorganic D6A e(traction methods

Pseuodoclonality$ selective oligoclone amplification due to

insufficient sample

Inappropriate Ag) rearrangements

4alse negative rates

9ampling errors

D6A and A6A degradation

PCA design

Biological factors

By using comple( multiple( assays 'ith advanced

methods of detection increases the chance of
detection of clonality in #enignJreactive conditions
Molecular ata shoul ne'er "e reporte in
isolation from all other clinicopathological
factors in each case
StandardiEation of 0' assays

Multicentre uropean colla#orative studies have

#een instituted to optimise and standardi7e the
PCA assays for purpose of clonality studies in
lymphoma clonality testing < (iome ) concerte
action

Involves the use of *>? standardised primers in a

series of 0. multiple( reations

Product detection either #y heteroduple@ or

automated 4enescan analysis7
6luorescence in situ hybridiEation
%6#S2&

Allo's detection of #oth structural and numerical

chromosomal a#normalities

Considered superior to PCA methods for detection

of translocations and aneusomys

6ot 'idely used in the routine diagnostic

evaluation of paraffin"em#edded #iopsies,

Technically more demanding *perception+

Uncertainties regarding diagnostic thresholds and result

interpretation!
6#S2

Types

Metaphase 4I9@

Interphase 4I9@ < for solid T(s and 44PT can #e used

!rinciple

Lisuali7ation of #ound of flourochlorome tagged D6A

fragments to complementary target genomic region

!ro"es$ t'o types for translocation

Dual fusion pro#es

9uperior due to lac- of false positivity

Brea-"apart

)ives a#normal results for variant translocations also

Do not detect the other gene involved

!ro"es$ t'o types for detection of copy num#er changes

?ocus specific

Chromosome enumeration *centromeric or pericentromeric

satellites+
Courtesy: JMD November 2000, Vol. 2, No. 4
4etaphase F5S/ 5nterphase F5S/
Normal cells
Abnormal cells
3
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e
m
a
t
i
c

f
i
g
s
Dual fusion probes
4rea* apart probes
A 4
A$4 5 Two different cases of 4ur*itt lymphoma showing fusion
signals for 6g&!#'7# 8as shown by arrows9
6#S2 #nterpretation

Acquire e(perience of normal and a#normal signal patterns

for each pro#e applied,

using negative tissues *eg! reactive lymph nodes+ and relevant

positive samples *eg! lymphomas -no'n to contain the a#normality
under investigation+!

5ther factors to #e a'are of$

the architecture of the tissue, including local variations in neoplastic

cell content, fi(ation, and cellularity 'ithin the section/

6uclear truncation and

the comple( nature of genetic arrangements seen in some

lymphoid neoplasms!

9hould have a @ stained slide at your hand!

6#S2 -#nterpretation
hoosing proper area for E/aluation

Prefera#ly areas

richest in a#normal cells

#right, distinct signals and

lo' #ac-ground in 'hich individual nuclei are clearly distinguisha#le

But screening of entire area is essential

4or the presence of su#clonal changes that might #e of diagnostic

and prognostic importance, e!g!, the presence of t*./01+ only in a
su#population might indicate transformation into a more aggressive
lymphoma!

#reas of nuclear o'erlapping *ith inistinct nuclear

outlines an high cell ensity - shoul "e a'oie+
Area to be avoided for interpretation
6#S2 -#nterpretation

A'areness of nuclear truncation

artefacts induced #y sectioning

9hould distinguish from loss of chromosome

sta#lishment of cut off values for

different pro#es and all signal patterns

Comple( chromsomal a#normalities

6#S2 - "pplications

Detection of numerical and structural chromosomal

a#normalities

Identification of mar-er 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 #one marro' cells

follo'ing stem cell transplantation
6#S2 - "d/antages

Aapid technique, and large num#ers of cells can

#e scored in a short period

fficiency of hy#ridisation and detection for is high

for structural and numerical a#normalities

Can #e applica#le in scant cellular specimens

*post T( samples and hypocellular samples

Permits direct correlation of cytogenetic and

morphologic features, ena#ling pathologists to
differentiate malignant from #enign conditions in
equivocal cases
6#S2 - Limitations

Aestricted to those a#normalities that can #e

detected 'ith currently availa#le pro#es

5nly one or fe' a#normalities can #e assessed

simultaneously

Cytogenetic data can #e o#tained only for the

target chromosomes/

6ot a good screening tool for heterogenous diseases

Aequires fluorescence microscopy

Lymphomas - 4ene e@pression
profiling

5ffers the prospects of future refining the lymphoma su#"

classification at molecular level

May provide prognostic data and potential for novel targeted

therapies

Presently a research tool and requires fresh tissue

Technique$

Co"hy#ridisation of differentially flourochrome la#elled A6A or cD6A

of tumour and normal tissue 'ith a cD6A chip *lymphochip+

The chips contain ro#otically arranged -no'n cD6As from hundreds

to thousands of genes

Confocal microscopy along 'ith computerised image analysis

system measure the emission spectra

9ignal intensity at each spot is proportional to the level of gene

e(pression

?arge data generated can #e investigated #y using mathematical

algorithims
4ene e@pression profiling - Utility

D?BC?

3 distinct su#groups #ased on differential e(pression of 0CCC genes

)erminal centre"li-e, activated B"cell li-e and 3rd distinct group,

represents heterogenous group

)erminal centre signature 'as sho'n to have #etter survival rates

4urther supervised analysis < five differential gene e(pression profiles

Differential gene e(pression < early and late or advanced stages

C??

5vere(pression of VAP 8C < aggressive course

Mantle cell lymphoma

na#les prediction of poor prognosis group

4ollicular lymphoma

Tranformation to DCBC? characteri7ed #y altered gene e(pression

profile

Aeports of prediction for response to ritu(ima# therapy

)han6 7ou