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

12 th Congress of the European Hematology Association
combined with dexamethasone compared to 2-DG alone (Figure 1d).
Conclusions. Since glycoltic alterations are observed under sole 2-DG
treatment, but had no effect on cell viability, these changes are not sufficient
to explain the strong effect of the co-administration. Although the
mode of action of the combined effects of GC and 2-DG remains to be
delineated, the strong effect on the kinetics of apoptosis induction in two
independent childhood ALL models makes this combination a promising
candidate for an improved treatment option for childhood acute
lymphoblastic leukemia and perhaps other lymphoid malignancies.
1129
A MODIFIED DHPLC ASSAY FOR A RAPID SCREENING OF JAK2 EXON 14 MUTATION
E. Albiero, M. Bernardi, D. Madeo, M. Ruggeri, F. Rodeghiero
San Bortolo Hospital, VICENZA, Italy
Background. V617F mutation in exon 14, within JH2 domain of the
JAK2 gene, occurs in 90% of patients with polycythemia vera (PV) and
50% essential thrombocythemia (ET). The mutated clone contributes to
the myeloid lineage but sometimes it is found in a very low proportion
of circulating leukocytes, requiring sensitive assays for its detection. The
molecular basis of V617F negative myeloproliferative diseases is
unknown, but two novel nucleotide changes in the JH2 domain of the
JAK2 gene have been recently described. The allele-specific (AS)-PCR is
still the most sensitive assay for the detection of V617F; nevertheless,
this conventional method is unable to identify other mutations. DHPLC
could be an attractive alternative for the detection of new mutations, but
a previous study reported a sensitivity of 50% in identifying V617F positive
cases. Aim. Development of a novel, fast and high-throughput Temperature
Modulated Heteroduplex Analysis (TMHA) DHPLC assay for
the detection of mutations in the JAK2 exon 14. Methods. 160 PV and 174
ET patients, diagnosed according to WHO criteria, were tested by AS-
PCR for the V617F mutation. 141 (88%) PV and 94 (54%) ET patients
were positive. V617F negative cases (19 PV, 80 ET) and 10 randomly
selected heterozygous V617F positive patients were studied with
TMHA DHPLC searching for mutations in JAK2 exon 14. For the detection
of the heteroduplex by DHPLC, partial denaturation of the amplicon
was obtained by temperature modulation. In the case of JAK2
V617F, the melting profile of the region of interest was very unstable (Figure
1, A), hence a short stretch of nucleotides (GC-clamp) was added to
the forward primer, creating an additional stable domain at the 5’ end
of the fragment, resulting in amplicon stabilization. All amplifications
were performed under standard conditions using the Jak2F clamp (5’-cgcccgccgccgccctgcatctttattatggcagagaga-3’)
and Jak2R (5’-cactgacacctagctgtgatcc-3’)
primers. For the heteroduplex creation sample:wild-type
JAK2 ratio was of 3:1. 7 µL of mixed products were loaded on a WAVE
fragment analysis system. Results. The new predicted melting curve with
GC clamp was uniform along the target sequence (Figure 1 B). The presence
of the V617F mutation in positive PV samples was confirmed by
an abnormal profile at 60°C (Figure 1 C).
Figure 1.
Serial dilutions of 100% homozygous sample allowed a 7-10% detection
rate of purified granulocytes. V617F positive controls were missed
by DHPLC in the absence of the clamp. No additional allelic variants
were found, confirming the very low frequency of mutations other than
416 | haematologica/the hematology journal | 2007; 92(s1)
V617F. Conclusions. A significative proportion of myeloproliferative disorder
patients remains without an identified genetic defect. At variance
with conventional methods, like AS-PCR or restriction enzyme digestion,
DHPLC is suitable in identifying genetic abnormalities outside the
specific mutational hot-spot. The addition of a GC-clamp to the amplicon
allowed us to employ this technology performing a rapid, cheap,
reliable and sensitive assay for the detection of allelic variant involving
JAK2 exon 14. These preliminary results need to be confirmed in a larger
patients population.
1130
ANTITUMOR EFFECTS OF SOME ANIMAL AND PLANT NUCLEASES AND THEIR
POLYETHYLENE GLYCOL (PEG)-CONJUGATES
J. Soucek, 1 J. Matousek, 2 M. Zadinova, 3 D. Hlouskova, 3 P. Pouckova3 1 Institute of Hematology and Blood Transf, PRAGUE 2, Czech Republic; 2 2Institute
of Animal Physiology and Gene, LIBECHOV, Czech Republic; 3 Institute of
Biophysics and Informatics, PRAGUE, Czech Republic
Aims. The antitumor and immunosuppressive effects of Bovine pancreatic
ribonuclease (RNase A), Bovine seminal ribonuclease (BS-RNase),
wheat leaf neutral ribonuclease (WLN-RNase) and Mung been nuclease
(PhA) both in vitro and in vivo were studied. Method. RNase A and PhA
enzymes were obtained from comercial sources whereas BS RNase and
WLN-RNase were isolated in our laboratories from bull seminal vesicles
and weat leaves. Antiproliferative activity was tested on ML-2 cell line
and immunosuppressive effect on human lymphocytes in mixed lymphocyte
culture. The antitumor activity in vivo was tested in athymic
nude mice bearing human melanoma tumor. Results. The free RNase A
and PhA-nuclease exerted very low antiproliferative and immunosuppressive
activity in comparison to the effect of BS-RNase. Immunosuppressive
effect of WLN-nuclease reached the same level as that of BS-
RNase. However, in the experiments in vivo a significant decrease of the
tumor size was observed in the mice treated with WLN- and PhA-nucleases.
Furthermore conjugate of PhA with PEG injected seven times at the
dose of 10 µg i.p. showed identical antitumor activity as that of BS-
RNase. The tumor size after treatment with PEG-RNase A decreased by
70%. The both free or PEG-conjugated BS-RNases injected into the
human melanoma tumors intraperitoneally decreased the tumor size
by the same way. The strongest aspermatogenic, embryotoxic and
immunosuppressive effects, and antigenicity were proved by BS-RNase
enzyme. The smallest side effects were demonstrated by PhA nuclease.
Conclusion. This paper shows for the first time antitumor activity of
some polymer conjugated plant RNases and their potential use as agents
in treatmant of human malignancies.
Work supported by the Grant Agency of the Czech Republic no. 523/04/0755
and 521/06/1149. The autors are also grateful for the supporting to I RP I APG
no. AVOZ 50450515 and partly by the Grant no. RA 8033-3 (Ministry of
Health, Czech Republic) and by the Grant League against Cancer Prague.
1131
FARNESYLTRANSFERASE INHIBITORS AS A NEW THERAPEUTIC APPROACH IN ACUTE
LYMPHOBLASTIC LEUKEMIA
A.B. Sarmento-Ribeiro, 1 C. Costa, 2 J.E. Casalta, 2 C. Andrade, 2
A. Oliveira, 3 D. Moreira, 3 A.C. Gonçalves, 1 V. Alves, 2 T. Silva, 2
M. Dourado1 1 Faculty of Medicine and CIMAGO, COIMBRA, Portugal; 2 Faculty of Medicine,
COIMBRA, Portugal; 3 University of Aveiro, AVEIRO, Portugal
Adult acute leukemias remain formidable therapeutic challenge. Only
70% of adults with newly diagnosed adult acute lymphoblastic
leukemias (ALLs) achieve complete remission (CR) after cytotoxic induction
chemotherapy. Although these CRs may be prolonged in 35% to
40% of younger adults, the remainder have a relapse and die. The prognosis
is particularly poor in Philadelphia chromosome (Ph1) disease.
Thus, new approaches are needed to improve the outcome for adults
with refractory leukemias. Improved understanding of signal transduction
pathways has resulted in identification of a panoply of potential
therapeutic targets. Novel agents in the early phase of clinical testing
include farnesyltransferase inhibitors (FTIs), are also being investigated
as potential therapies. FTIs are drugs that target the farnesyltransferase
enzyme. This enzyme initially developed to inhibit the prenylation necessary
for Ras activation, adds the farnesyl moiety to Ras protein, among
other targets, enabling Ras to attach to the cell membrane. It is believed
that inhibition of this enzymatic activity would interfere with Ras function,
altering cell signaling in tumor cells. FTIs have been developed and