Sabato 27 ottobre 2012 - Pacini Editore

COmuNiCaziONi ORali
LOH analysis of WnT-activated hepatocellular
carcinomas (HCC)
E. Tamburini1 , B. Dal Bello2 , N. Campanini1 , C. Azzoni1 , L. Bottarelli1
, S. Pizzi1 , P. Soliani3 , S. Rossi4 , E.M. Silini1 1 Department of Pathology and Laboratory Medicine, Anatomic Pathology,
Azienda Ospedaliero-Universitaria di Parma, Via Gramsci, 14, Parma,
Italy; 2 Department of Anatomic Pathology and 4 VI Internal Medicine,
Fondazione IRCCS, Policlinico S. Matteo, Viale Golgi, 19, Pavia, Italy,
3 Department of General Surgery, Azienda Sanitaria locale di Ravenna,
Viale Tullo Masi, 3, Ravenna, Italy.
Background. Wnt-activated HCCs are a specific tumor entity
with distinct morphology and improved survival that are characterized
by nuclear immunoreactivity for β-catenin (CTNN1) and
overexpression of glutamine synthetase (GS) 1-3 . We evaluated
the loss of heterozygosity (LOH) profile at different chromosomal
loci in a series of HCCs according to CTNN1/GS status
to identify recurrent genetic events potentially relevant in this
specific pathways of hepatocarcinogenesis.
Materials and methods. 84 surgical-resected HCCs were considered
divided into 56 cases and 28 controls according to tissue
immunoreactivity for GS/CTNN1 expression. Analysis were performed
on DNA extracted from formalin-fixed paraffin-embedded
tissues after manual microdissection. Direct sequencing for
CTNN1 mutations and LOH analysis using a panel of 18 microsatellite
mapping at 1p, 8p, 16p, 17p and 19q, were performed 4-6 .
In tumors showing distinct areas of neoplastic progression,the
analysis was performed separately in each component.
Results. CTNN1 mutations were identified in 13/70 (19%)
HCCs: 12 in GS/CTNN1+ (24.4%), 1 in GS/CTNN1- (5%)
(p = 0.04). Identified mutations were: 1 mononucleotide deletion
and 12 missense mutations, mainly involving codons 32,
33, 37 and 45. Overall LOH frequency at main chromosomal
regions was: 8p (58%), 16p (52%), 1p (50%), 17p (48%), and
19q (32%). Losses at 8p12-p22 and 17p12-13 correlated with
InTErESSE GEnErALE
Vintage antibodies outlast the expiration date
by 10-26 years
M.C. Argentieri1 , A. Vanzati1 , C. Parravicini2 , G. Cattoretti1 1AO San Gerardo e Universitá di Milano-Bicocca, Monza (MB), 2 AO
Luigi Sacco, Milano, Italy
Primary antibodies represent an essential ancillary technique for
histopathology, both for the diagnosis and for planning the treatment
of several classes of patients.
Between 10% and 20% of the surgical specimens received by
our institutions undergo immunostaining. We currently maintain
a panel of about 150 primary antibodies, some of which are used
fairly often (anti- keratin, S-100, CD45, CD3, CD20 etc.), others
once or twice a year, although their use is not redundant. The cost
of maintaining such a large panel is aggravated by the fact that,
once they pass the expiration date written on the label, they may
not be used for diagnosis. The lab using expired reagents may be
fined for that.
The cost of the primary antibodies represents about 60% of the
budget allocated for in vitro reagents (with the exclusion of
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CTNN1 immunoreactivity (p = 0.02, p = 0.05) and mutations
(p = 0.04), conversely deletion at 1p34.2, 8p21.3-22 and
16p11.2 were associated with CTNN1 wild-type (p = 0.04,
p = 0.05, p = 0.05 respectively). Some genetic changes correlated
with clinico-pathological features of the HCCs: 8p, 17p with
age (p = 0.0006, p = 0.04); 16p, 19q with cirrhosis (p = 0.0005,
p = 0.02); 17p with vascular invasion and histologic grade
(p = 0.03, p = 0.04); 1p, 8p and 19q with nodule size > 3 cm
(p = 0.02, p = 0.04, p = 0.007 respectively). Losses at 1p21-22,
17p12-13 and 19q13.41 were associated with disease progression
(p = 0.03, p = 0.01, p = 0.03 respectively).
Conclusions. CTNN1 mutations confirm the predictive role
of immunohistochemistry for CTNN1/GS in definition Wntactivated
HCCs. Overall, chromosomal instability in the analyzed
regions is high. No specific LOH events are characteristic of the
Wnt-activated HCCs although they show a distinct genetic profile.
Indeed, LOH profiles segregate not only with different tumor
types, but also with other common clinico-pathological variables.
references
1 Dal Bello B, Rosa L, Campanini N, et al. Glutamine synthetase immunostaining
correlates with pathologic features of hepatocellular
carcinoma and better survival after radiofrequency thermal ablation.
Clin Cancer Res 2010;16:2157-66.
2 Laurent-Puig P, Legoix P, Bluteau O, et al. Genetic alterations associated
with hepatocellular carcinomas define distinct pathways of
hepatocarcinogenesis. Gastroenterology 2001;120:1763-73.
3 Audard V, Grimber G, Elie C, et al. Cholestasis is a marker for hepatocellular
carcinomas displaying beta-catenin mutations. J Pathol
2007;212:345-52.
4 Piao Z, Park C, Park JH, et al. Allelotype analysis of hepatocellular
carcinoma. Int J Cancer 1998;75:29-33.
5 Nagai H, Pineau P, Tiollais P, et al., Comprehensive allelotyping of
human hepatocellular carcinoma. Oncogene 1997;14:2927-33.
6 Austinat M, Dunsch R, Wittekind C, et al. Correlation between betacatenin
mutations and expression of Wnt-signaling target genes in
hepatocellular carcinoma. Mol Cancer 2008;7:21.
solvent, alcohol and paraffin). Replacing outdated reagents adds
up to this cost, despite a handful of well designed publications
(Tubbs, Nagle et al. 1998; Balaton, Drachenberg et al. 1999;
Vigliani and Babache 2002; Savage and DeYoung 2010) which
showed excellent performance of primary antibodies which expired
12 to 32 months before they were used.
We retrieved antibodies which were constantly kept at +4C since
having been received by two different labs, selecting the ones
dating before year 2000. Conditions for the experiment were that
they should not have been dried out, not being contaminated and
being either in the original vial or proof of the invoice could be
demonstrated.
All the antibodies shown in the table performed exceptionally
well, despite being kept for up to 26 years in the fridge.
These results were anticipated by isolated reports about antibodies
performing well 134 months after the expiration date. The
panel we chose is limited but well representative of the most used
antibodies in the daily practice. All of them are older than 134
months and all survived.
These results should prompt a revision of the recommendation
to discard primary antibodies which have passed the expiration
date. This recommendation, as we have shown, has no basis, being
much more relevant the conditions (cold ischemia, fixation,