DNA from Potentally Oncogenic Viruses Is Not Detected in Guthrie Cards from Chlidren Who Later Developed Acute Lymphoblastic Leukaemia.
Authors: Gustafsson Britt , Bogdanovic Gordana , Jernberg Asa Gustafsson , Broliden Kristina , Priftakis Peter 
Published: October 2019

Abstract
INTRODUCTION
Acute lymphoblastic leukemia (ALL) is the most frequent childhood malignancy in the western countries. Epidemiological reports and animal observations suggest a possible association between maternal viral infections during pregnancy and subsequent development of childhood ALL. A maternal viral infection during pregnancy, which is transferred to the foetus in a critical time during foetal hematopoesis, could initiate a preleukemic clone. A postnatal molecular event, during maximum stress on lymfocyte precursor proliferation, could then expand the preleukemic clone. A possible infectious agent should be able to cross the placenta and induce genomic instability in the foetal lymphocytes, without causing severe foetal abnormalities. Candidate viruses could be the Human Polyoma viruses (JC Virus and BK virus), Human Parvovirus B 19, Human Herpes virus 6 (HHV 6), Epstein-Barr virus (EBV) or Cytomegalo virus (CMV). In order to investigate if children who later develop ALL were prenatally infected with these viruses, Guthrie cards taken at birth were analysed by PCR.
PATIENTS, MATERIAL AND METHODS
Capillary blood routinely collected at 3–5 days of age and stored on Guthrie cards, were retrieved from 54 patients who later developed ALL. A total of 50 of these children had been diagnosed as pre-B ALL (either CD10+, CD20+, FAB LI or L2) and four were diagnosed as T-ALL (CD3+ or CD8+). The median age of diagnosis was 5 years (range from 9 months to 17 years, mean 5,9 years). These children had been admitted for treatment at four different Paediatric Oncology Swedish Centres from 1980–2001. The control group were 47 healthy controls matched for age and birth place.
DNA was extracted from the original Guthrie cards, using Minimal Essential Medium (MEM) to eluate the blood. Presence of BKV and JCV DNA were analysed by a nested PCR, amplifying a 176 and 173 bp segment respectively. Presence of Parvovirus B19 DNA was analysed by the NS1-PCR, amplifying a 284 bp segment. Presence of HHV 6 DNA was analysed by a nested PCR, amplifying a 173 bp segment. Presence of EBV DNA was analysed by a nested PCR, amplifying a 208 bp segment. Presence of CMV DNA was analysed by a Real Time PCR. To exclude the possibility of negative results due to failure to extract DNA, all samples were tested by a HLA-DQ PCR.
RESULTS AND DISCUSSION
In order to detect if a viral in utero infection could initiate the development of ALL we analysed presence of BKV, JCV, HHV 6, Parvorirus B19 and CMV by PCR in from Guthrie cards at birth, in 54 patents that later developed ALL and 47 matched healthy control patients. No viral DNA was detected in the samples from ALL patients or in the healthy controls. All samples contained amplifiable DNA when tested by HLA-DQ PCR. Thus, it is less likely that childhood ALL is associated to an in utero infection of BKV, JCV, HHV 6, Parvorirus B19, EBVor CMV. However, it is not possible to exclude an association with a latent utero infection with very low levels of circulating virus at birth. In view of the epidemiological evidence between childhood ALL and infection, the search for a viral etiology must continue.