12th Congress of the European Hematology ... - Haematologica

0135
ROUTINE DETECTION OF CYTOGENETIC ABNORMALITIES IN B-CELL
LYMPHOPROLIFERATIVE DISORDERS USING INTERPHASE FISH TECHNIQUES
J.M. O'Connor, S.L. Barrans, K.R. Turner, B.S. Dickinson, A.S. Jack,
R.G. Owen
Leeds Teaching Hospitals NHS Trust, LEEDS, United Kingdom
Background. Specific cytogenetic abnormalities are increasingly used to
segregate patients with B-cell lymphoproliferative disorders (B-LPD) into
good and poor risk sub-groups which may influence clinical decision
making. The critical indications are in the differential diagnosis of diffuse
large B-cell lymphoma (DLBCL)/Burkitt lymphoma (BL); B-
CLL/mantle cell lymphoma (MCL); the differential diagnosis of CD5- B-
LPDs and the identification of prognostic variables such as a p53 deletion.
Conventional karyotyping significantly underestimates the incidence
of cytogenetic abnormalities in B-LPD and fails in a significant
proportion of cases. The purpose of this study was to evaluate the applicability
of interphase fluorescent in situ hybridisation (iFISH) in the routine
diagnostic laboratory. Methods. iFISH analysis was carried out on
3,126 (2,643 B-LPD and 483 myeloma) cases (excluding Myeloma IX
samples) from the Yorkshire and Humberside region presenting to
HMDS between 2003 and 2006. B-LPD samples were initially categorised
on the basis of CD5 expression, with CD5 + cases (B-CLL and
MCL) assessed for deletions of p53, ATM, 13q14, trisomy 12 and BCL-
1/IgH translocation (CD5 + CD23 – ) and CD5- cases (Burkitt/DLBCL, FL
and MZL) assessed for rearrangements of cMYC, BCL-2, IgH, MALT-
1(extranodal MZL), BCL-6 and p53 deletions. Myeloma cases were
assessed for deletion/monosomy 13 and IgH rearrangements. Cases with
an IgH rearrangement were further investigated to determine the translocation
partner. The only selection criteria used in myeloma cases was
that smears contained >10% plasma cells that had a neoplastic phenotype
by flow cytometry. iFISH was carried out on blood or marrow
smears, dab/imprint of fresh tissue, or cytospins of CSF/effusions. Probes
were used according to the manufacturer’s protocol (BCL1/IgH,
cMYC/IgH, BCL2/IgH dual colour translocation probe sets; BCL6,
cMYC, MALT-1 and IgH BreakApart's: Abbott Molecular Diagnostics
and IgH, PAX-5, BCL-3; CCND1 Split-Signal: Dako: p53/α 17, ATM/α
11, 6q21/α6, QBioGene). Results. Successful results were obtained in
2,558/2,683 (96.9%) B-LPD cases, and in 459/483 (95.0%) myeloma cases.
Abnormal results were detected in 86.1% of B-LPD cases and in
83.7% of myeloma cases. 271/1010 (26.8%) B-CLL cases were re-designated
as adverse risk because of a p53 and/or an ATM deletion; 110/391
(28.1%) DLBCL cases were re-designated as adverse risk, based on
rearrangements of either BCL-6, cMYC or BCL-2; 253/483 (52.3%)
myeloma cases have deletion/monosomy 13 and 152/483 (31.5%) an
IgH rearrangement, of these 33/483 (6.8%), had BCL-1/IgH; 22/483
(4.6%) had FGFR3/IgH; 6/483 (1.2%) had cMAF/IgH and 1/483 (0.2%)
had a cMYC/IgH translocation. The number of patients with unidentified
translocation partners (90/152, 59.2%) appears higher than that
reported in the literature and is worthy of further study. Conclusions. This
large series of cases provides evidence for the applicability and robustness
of iFISH in a routine diagnostic setting. We conclude that iFISH is
applicable to the routine assessment of patients with B-LPD or myeloma.
The majority of patients have abnormal patterns and the failure rate
is very low compared to conventional karyotyping. Interphase FISH performed
directly on smears/dabs/cytospins is rapid, reliable and relatively
inexpensive and can be integrated into diagnostic laboratories and
should ultimately allow for risk stratification in real time.
0136
COMPARISON OF PROGNOSTIC IMPACT OF CHROMOSOME 1Q21 GAIN IN PATIENTS
WITH MULTIPLE MYELOMA TREATED BY BORTEZOMIB, THALIDOMIDE AND ANY
CONVENTIONAL THERAPY
P. Nemec, 1 H. Greslikova, 1 R. Zaoralova, 1 H. Filkova, 2 V. Vranova, 2
R. Kupska, 2 J. Smejkalova, 1 A. Oltova, 2 P. Kuglik, 3 R. Hajek4 1 Myeloma Basic Research Centre, BRNO; 2 Dep. of Medical Genetics, Faculty
Hosp., BRNO; 3 Genetics & Mol. Biology, F. of Science, BRNO; 4 Dep. of Int.
Hem.-oncol., Faculty Hosp., BRNO, Czech Republic
Background. Amplification of chromosome band 1q21 as well as
increased expression of CKS1B gene in this area is a frequently mentioned
prognostic factor for patients with multiple myeloma (MM). Aims.
This study was aimed at comparison of prognostic impact of 1q21 gain
in three selected groups of patients with diagnosed MM based on treatment
regiment. Methods. Plasma cells were identified by cytoplasmic
light-chain fluorescence in situ hybridisation (cIg-FISH), 1q21 amplifica-
12 th Congress of the European Hematology Association
tion (Amp1q21) utilizing the 1q21/1p36 DNA probe. Amp1q21 was taken
such as detection of one or more additional signals of 1q21 DNA
probe. Cut-off level for presence of Amp1q21 was established to 20%.
Patients with Amp1q21 and patients lacking Amp1q21 of each group
were statistically correlated with clinical parameters. Up to date we have
carried out analysis of 66 (n=66) patients. This group of patients with
median of follow'up 8,6 months (range: 0,3-30,4) was divided according
to the undergone therapy into 3 groups: C-group comprises 17 patients
treated by any conventional therapy; T-group comprises 27 patients treated
by thalidomide; V-group comprises 22 patients treated by Bortezomib.
The response and other parameters such as time to progression (TTP) and
overall survival (OS) were assigned by IMWG criteria. Results. Amp1q21
was found in 62.1% of all 66 patients. Percentage of patients with
Amp1q21 in C/T/V-groups were as follows: 64.7%/40.7%/86.4%,
respectively. Clinical parameters valid for patients with Amp1q21 (listed
in C/T/V order) were as follows: overall response rate (ORR) 42.8%
/83.3%/50%; TTP 8.8/12.1/8.0 months; OS 16.1/6.6 / not yet reached for
V-group. The same parameters valid for patients lacking Amp1q21: ORR
33.3%/80%/66.7%; TTP not yet reached for C-group / 8.2/not yet
reached for V-group; OS not yet reached for all groups. TTP median of
patients with Amp1q21 vs. patients lacking 1q21 was: 8.2 vs. 12.1 months
(p=0.269), OS 6.6 vs. not yet reached (p=0,072) in thalidomide group. We
didn't find any other significant differences between patients with/without
Amp1q21 and their parameters in V- and C-group. Summary and conclusions.
Our results suggest that patients with Amp1q21 treated by
thalidomide show a trend towards the worst prognosis based on overall
survival. We are currently investigating whether or not our findings will
be confirmed on a larger cohort of patients with longer follow-up.
Supported by Monoclonal Gammopathy and Multiple Myeloma Basic
Research Centre (LC 06027), Masaryk University, Czech republic.
0137
ROUTINE DETECTION OF CYTOGENETIC ABNORMALITIES IN B-CELL LYMPHOPROLIFERA-
TIVE DISORDERS USING INTERPHASE FISH IN PARAFFIN EMBEDDED TISSUE
S. Barrans, K. Turner, S. O'Connor, B. Dickinson, R. Owen, A. Jack
Leeds Teaching Hospitals NHS Trust, LEEDS, United Kingdom
Specific cytogenetic abnormalities are increasingly being used in the
differential diagnosis of B-cell lymphoproliferative disorders (B-LPD) and
to segregate patients into risk sub-groups which may influence clinical
decision making. Paraffin embedded tissue (PET) is often the only diagnostic
material available for analysis and it is therefore essential that
molecular techniques are applicable in this setting. The critical indications
for performing FISH on PET are in the differential diagnosis of diffuse
large B-cell lymphoma (DLBCL)/Burkitt lymphoma (BL); B-
CLL/mantle cell lymphoma (MCL); confirming the diagnosis of follicular
lymphoma (FL), especially where BCL2 is negative; and in extranodal
marginal zone lymphoma (ExMZL). The purpose of this study was to
evaluate the applicability of interphase fluorescent in situ hybridization
(iFISH) on PET in the routine diagnostic laboratory. Paraffin iFISH analysis
was carried out on 329 B-LPD cases diagnosed in HMDS between
2003 and 2006, where only a paraffin block was available for analysis.
Cases were investigated based on the provisional diagnosis following
histology and immunohistochemistry. 221 cases with a differential diagnosis
of BL/DLBCL were investigated for rearrangements of cMYC,
BCL2, and BCL6; 22 FL and 81 ExMZL were assessed for t(14;18) and
BCL6 rearrangements and IgH and MALT1 rearrangements respectively.
6 cases with a differential diagnosis of B-CLL/MCL were investigated
for BCL1 rearrangements. Cases with an IgH rearrangement were
further investigated to determine the translocation partner. An adaptation
of our ‘fresh’ iFISH protocol was used, with an additional enzymatic
digestion with Sigma Protease XXIV and a 90C pre-denaturation step.
We have validated this method by comparison with iFISH on fresh tissue
and PCR on the same sample. Probes were used according to the
manufacturer's protocol (BCL1/IgH, cMYC/IgH, BCL2/IgH dual colour
translocation probe sets; BCL6, cMYC, MALT-1 and IgH BreakApart's:
Abbott Molecular Diagnostics and IgH, PAX-5, BCL-3; CCND1 Split-Signal:
Dako). Successful results were obtained in 318/329 (96.6%) of cases,
which is comparable to fresh material. 89/221 BL/DLBCL cases had
a cMYC rearrangement as the sole abnormality and a final diagnosis of
BL was made based on this basis. The remaining 132 cases were classed
as DLBCL and had either a t(14;18), a BCL6 rearrangement, additional
cMYC rearrangements, or extra copies of any combination of the probes
assessed. The t(14;18) was seen in 8/12 FL-common type and 1/8 FL-
BCL2neg cases had an unbalanced BCL2 rearrangement. 4/6 B-CLL/MCL
cases had rearrangement of BCL1 and were therefore re-classified as
MCL. 10 cases of ExMZL had rearrangement of MALT1, and 10 had IgH
haematologica/the hematology journal | 2007; 92(s1) | 49