Title: Validation of a DNA FISH biomarker set in a random Barrett’s esophagus surveillance
population.
Pacha A.F1,2 , Rygiel A.M1,2, Rosmolen W.M1, Elzer B1, Verhulst H1, Visser M1, ten Kate
F.J.W.3 Hutten B.A4, Mallant-Hent R.C5,11 , Naber A.H.J6,11, van Oijen A.H.A.M7,11, Baak
L.C8,11, Scholten P9,11, C.J.M. Böhmer C.J.M10,11, Bergman J.J1,11 Krishnadath K.K1,2,11.
1)
Department of Gastroenterology and Hepatology, 2) Center for Experimental Molecular Medicine ,
Department of Pathology, 4)Clinical Epidemiology, Biostatistics and Bioinformatics, Academic
Medical Center, Amsterdam, The Netherlands, 5)Department of Gastroenterology, Flevoziekenhuis,
Almere, 6)Department of Gastroenterology Tergooiziekenhuizen te Hilversum, 7)Department of
Gastroenterology, Medisch Centrum Alkmaar, 8)Department of Gastroenterology, Onze Lieve Vrouwe
Gasthuis, Amsterdam, 9)Department of Gastroenterology, Sint Lucas Andreas Ziekenhuis,
Amsterdam,10)Department of Gastroenterology, Spaarneziekenhuis, Hoofddorp. 11)The Amsterdam
Gastroenterological Association.
3)
The current surveillance programmes of Barrett’s esophagus (BE) patients have proven to be
inadequate. Several of the previously identified biomarkers in BE can be efficiently assessed
using DNA-fluorescent in situ hybridization (DNA FISH) on brush cytology specimens of BE
patients. Previously, we showed in an academic setting that a DNA FISH marker set
consisting of centromeric DNA probes specific for chromosome 7, 17, and the locus specific
probes for Her-2(17q11.2-12), 20q13.2, C-myc (8q24.12-13) have high sensitivity and
specificity for diagnosing HGD and EAC. Additionally, the combination of CEP 7, 17, and
p53 (17p13.1), p16 (9p21) proved to be potentially prognostic. To validate a diagnostic and
“potentially” prognostic DNA FISH marker set in an unselected community based BE
surveillance population. Between January 2007 until November 2010, prospectively collected
brush cytology specimens of 200 unselected BE patients from six regional hospitals in the
Amsterdam/North Holland region were evaluated for the seven FISH markers. All patients
underwent upper GI endoscopy for routine surveillance of BE following the 4-quadrant
standard biopsy protocol. Biopsies were processed routinely for histopathological staging of
dysplasia. Brush specimens were taken from the normal squamous epithelium and the Barrett
segment. FISH for the seven biomarker set was carried out on the cytology specimens.
Results were evaluated manually and by an automated FISH microscope using spot scoring
software. Cut off values were determined in the cytology samples of the normal squamous
mucosa. Finally, FISH results were correlated with histological stage. In the cohort of 200
patients, 173 patients were diagnosed with IM, 24 patients with LGD and 3 patients with
HGD. The most frequent cytogenetic abnormality through the sequence of dysplasia was loss
of p16: IM (53/173) 30%, LGD (17/24) 71%, HGD (3/3) 100%, (Figure1). Loss of P53 was
observed in two cases of IM (1%), in 2 LGD (25%) and in one HGD (33%). Trisomy and/or
tetrasomy of chromosome 17 and 7 were present in 8 (5%) and 6 (3%) of IM cases, in 5
(20%) and 2 (8%) of LGD cases respectively. Two out of three cases of HGD had trisomy of
chromosome 17. There was no abnormality seen with 20q13.2, and Her-2 in the IM and LGD
cases. All HGD cases showed gain of C-myc and Her-2, while in only two out of three cases
gain of 20q was observed. For the prognostic markers p16 and p53 and CEP 7 and 17 there is
a significant trend (P<0.05, Trend test) with an increasing frequency of genetic abnormalities
through the sequences of dysplasia. The true prognostic potential of these markers will be
determined after prospective follow up of the cohort. C-myc, 20q and Her-2 are confirmed as
highly sensitive and specific diagnostic markers as they were seen only in HGD.
Frequency distributions of genetic abnormalities as found for the seven FISH marker set in 200 BE
patients; IM (n=173), LGD (n=24), HGD (n=3)
100
IM
LGD
HGD
90
80
% of BE patients
70
60
50
40
30
20
10
0
Loss of p16
loss of p53
gain of cep17
gain of cep7
gain of 20q
gain of Cym
Gain of Her2