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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.
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 / FISH target(s)/ follicular L
if neg. : / t(14;18)(q32;q21) and variants
t(3;14)(q27;q32) and variants / IgH/BCL2 or BCL2
BCL6/3q27
mantle cell L
if neg. : / t(11;14)(q13;q32)
t(12;14)(p13;q32) and t(6;14)(p21;q32) / IgH/CCND1
CCND2 and CCND3
Burkitt L / t(8;14)(q24;q32) and variants / MYC/8q24
diffuse large cell L
aggressive or child
? follicular L transf. / t(3;14)(q27;q32) and variants
t(8;14)
t(14;18) / BCL6/3q27 (or IgH ?)
MYC/8q24
IgH/BCL2
/ primitive mediastinal B cell L / (+9p) / (JAK2)
/ informative / no / marginal zone L :
MALT L / t(11;18)(q21;q21) or t(14;18)(q32;q21)
or t(3;14)(p13;q32) / MALT1/18q21
FOXP1/3p13
nodal/leukemic MZL / +3,+18
/ splenic MZL / del(7q) +3,+18
/ B / lymphoplasmocytic L / t(9;14)(p13;q32) / PAX5/9p13
/ Waldenström / del(6q), (+4 and del(13q))
adult
child (<18years) / other / other Ig rearrangement
+12,i(9p) / IgH/14q32
not informative / yes / according to clinical and morphological features
/ / anaplastic large cell L / t(2;5)(p23;q35) or t(2;other) / ALK/2p23
/ informative / no / γδ T L / i(7)(q10) / i(7)(q10)
T / other / TCR genes rearrangement / TRα/TRδ/14q11, TRβ/7q34, TRγ/7p14
not informative / yes / according to clinical and morphological features