Belgian society of human genetics permanent working group :
Belgian cytogenetic group
in hematology and oncology (BCG-HO)
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RECOMMENDATIONS FOR THE CYTOGENETIC ANALYSIS
OF NON HODGKIN LYMPHOMA SPECIMENS
TYPE OF SAMPLE
Lymph node, tumoral biopsy, bone marrow or peripheral blood if invaded.
Most invaded sample PREFERRED.
CULTURE TIMES
Solid samples (lymph node, biopsy): short term (overnight with increased exposure to colcemid and/or
24h) cultures are recommended using a cell concentration of at least (3 to) 6 x106 cells/ml media. If
there is enough material, other cultures such as stimulated (see below) long term (72h) can also be
beneficial.
Bone marrow and blood samples: usually stimulated (TPA if B cell, PHA if T cell lymphoma, both if
unknown) long term (72h) culture.
CHROMOSOME ANALYSIS
In the section below the general rules are outlined concerning the number of mitoses analysed.
Important is to look at metaphases with poor chromosome morphology although normal cells with
better chromosome morphology might be present in excess.
The laboratory procedures may vary according to whether the sample is diagnostic or follow up, on
the initial cytogenetic findings and the WHO classification (if known).
DIAGNOSTIC SPECIMENS
If a normal karyotype is obtained at least 20 metaphases should be analysed in order to exclude a
clonal abnormality. FISH analysis may be required depending on the clinical criteria and results of
molecular analysis, especially to detect diagnostic and/or prognostic chromosomal alterations.
If an abnormal karyotype is obtained a sufficient number of cells should be analysed to establish
clonality and identify clonal evolution. If a specific chromosomal alteration is detected, at least 2
metaphases are necessary to ascertain the clonality for structural aberrations or trisomies, and 3 in
case of monosomies. It may not be appropriate to characterise all abnormalities if the karyotype is
very complex.
2/3
FOLLOW UP SPECIMENS
If the karyotype was normal at diagnosis chromosome analysis is not deemed contributive for
remission specimens. At relapse analysis should follow the recommendations for diagnostic samples.
If an abnormal karyotype was detected at diagnosis it is preferable to screen at least 30 cells for the
pertinent abnormalities rather than do a full analysis of 10-20 cells. Interphase FISH could also be
performed if an informative probe is available.
FISH ANALYSIS
The general guidelines outlining the circumstances where FISH analysis is appropriate should be
followed.
In addition to these general recommendations an NHL FISH strategy orientated by cytogenetic
findings and morphological subgroups is recommended (Table here below).
FISH in Non Hodgkin Lymphoma
3/3
(excluding chronic lymphoproliferative disorders, and lymphoblastic lymphoma which has to be considered as acute lymphoblastic leukaemia : see specific
recommendations)
patient
lineage
karyotype
FISH ?
lymphoma subtype
recurrent prognostic rearrangement
follicular L
informative
no
B
adult
child (<18years)
not informative
yes
informative
no
not informative
yes
T
t(14;18)(q32;q21) and variants
if neg. : t(3;14)(q27;q32) and variants
mantle cell L
t(11;14)(q13;q32)
if neg. : t(12;14)(p13;q32) and
t(6;14)(p21;q32)
Burkitt L
t(8;14)(q24;q32) and variants
diffuse large cell L
t(3;14)(q27;q32) and variants
aggressive or child
t(8;14)
? follicular L transf.
t(14;18)
primitive mediastinal B cell L
(+9p)
marginal zone L :
MALT L
t(11;18)(q21;q21) or
t(14;18)(q32;q21)
or t(3;14)(p13;q32)
nodal/leukemic MZL
+3,+18
splenic MZL
del(7q) +3,+18
lymphoplasmocytic L
t(9;14)(p13;q32)
Waldenström
del(6q), (+4 and del(13q))
other
other Ig rearrangement
+12,i(9p)
FISH target(s)
IgH/BCL2 or BCL2
BCL6/3q27
IgH/CCND1
CCND2 and CCND3
MYC/8q24
BCL6/3q27 (or IgH ?)
MYC/8q24
IgH/BCL2
(JAK2)
MALT1/18q21
FOXP1/3p13
PAX5/9p13
IgH/14q32
according to clinical and morphological features
anaplastic large cell L
γδ T L
other
t(2;5)(p23;q35) or t(2;other)
i(7)(q10)
TCR genes rearrangement
according to clinical and morphological features
ALK/2p23
i(7)(q10)
TRα/TRδ/14q11,
TRβ/7q34, TRγ/7p14