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Prepublished online as a Blood First Edition Paper on June 5, 2003; DOI 10.1182/blood-2002-12-3817.
Blood, 1 October 2003, Vol. 102, No. 7, pp. 2321-2333 Leukemia in twins: lessons in natural historyFrom the Leukaemia Research Fund Centre, Institute of Cancer Research, London, United Kingdom.
Identical infant twins with concordant leukemia were first described in 1882, and since that time many such pairs of infants and older children have been described. It has long been recognized that this situation offers a unique opportunity to identify aspects of the developmental timing, natural history, and molecular genetics of pediatric leukemia in general. We reviewed both the older literature and more recent molecular biologic studies that have uncovered the basis of concordance of leukemia. Molecular markers of clonality, including unique, genomic fusion gene sequences, have provided unequivocal evidence that twin pairs of leukemia have a common clonal origin. The only plausible basis for this, first suggested more than 40 years ago, is that following initiation of leukemia in one twin fetus, clonal progeny spread to the co-twin via vascular anastomoses within a single, monochorionic placenta. This explanation has been endorsed by the identification of clonotypic gene fusion sequences in archived neonatal blood spots of individuals who subsequently developed leukemia. These analyses of twin leukemias have thrown considerable light on the natural history of disease. They reveal a frequent prenatal origin and an early or initiating role for chromosome translocations. Further, they provide evidence for a variable and often protracted latency and the need, in childhood acute lymphoblastic leukemia (ALL)/acute myeloblastic leukemia (AML), for further postnatal exposures and/or genetic events to produce clinical disease. We argue that these insights provide a very useful framework for attempts to understand etiologic mechanisms. (Blood. 2003;102:2321-2333)
Around 3 to 4 per 1000 live births produce a clone of 2 genetically identical twins. These twins are monozygotic, being derived from a single fertilized ovum followed by splitting of the early embryo. In contrast, fraternal or nonidentical twins are simultaneously derived from independent, fertilized ova and have the same genetic diversity as non-twinned siblings. The common genetic heritage of monozygotic twins is instantly recognizable in their striking similarity of appearance, and, principally for this reason, they have been singled out in almost all human cultures, historical and contemporary, as deserving of special attention. This unique status is reflected in a rich mythology, literature, and art.1,2 In some respects, this interest has been relatively trivial, focusing, as did Shakespeare, on the comic potential of mistaken identity. But twins also raise more fundamental philosophical and ethical issues. They challenge our precepts of identity, individuality, and fate. Recently, their "natural" origins have been used, in our view erroneously, to legitimize the prospect of human reproductive cloning. Their common inheritance also raises questions of importance to medicine and, in particular, provides an opportunity to explore vulnerability to disease. Not surprisingly, therefore, twins have been much used, and occasionally abused, in biomedical research. In 1875, Francis Galton, cousin of Charles Darwin, introduced the idea of exploiting the shared genetic identity of monozygotic twins to assess the relative effect of heredity and environment—or nature versus nurture—in the development of unique characteristics, both normal and pathologic.3 Galton's evidence was anecdotal, but it persuaded him of the omnipotence of genetic predetermination. The effect of Galton is difficult to exaggerate. Not only did he initiate more than 100 years of productive medical research on twins, but he also spawned and advocated the eugenics movement that led inexorably to the pseudo-scientific justification for Nazi fascism and Mengele's sadistic experimentation on twins in Auschwitz.1 Contrary to what is often quoted, Galton did not introduce the "classical" twin research method of comparing concordance rates of disease or phenotype in identical versus nonidentical twins. This method came about 50 years later when the zygosity and genetic status of these 2 different twin types was recognized.4 In the modern era of human genetics, very many human traits and medical conditions have been subject to twin pair analysis.5,6 Some human disorders have close to 100% concordance in identical twins; this concordance applies to co-inheritance of mutant genes that are dominant and highly penetrant, for example, in Huntington chorea.7 Most diseases or traits (but not all) show a concordance rate in identical twins in the broad range of 5% to 75% and some 2 to 5 times higher than in fraternal twins. Examples include the following: tuberculosis,8 other infectious diseases,9 autoimmune diseases,10 autism,11 obesity,12 some adult cancers,13 cardiovascular diseases,5 cognitive abilities,14 self-esteem,15 and appreciation of musical pitch.16 In the cancers, concordance rates for monozygotic (versus dizygotic) twins vary markedly for subtypes, being high for prostate and breast cancers, low for lung cancer.13 These data have been generally taken to indicate that most of our ailments (as well as many of our talents) are a consequence of complex genotype-environment interactions. In some cases, such as type 1 diabetes,17 alleles involved in inherited susceptibility have been uncovered, and many more relevant gene polymorphisms can be anticipated with the advent of the human genome completion and new high-throughput single nucleotide polymorphism (SNP) assays. Although all of these studies enforce the notion that inherited genetics contributes to disease or trait susceptibility, interpretation is not without difficulty.5,18 Aside from ascertainment bias and the possibility that monozygotic twins may be more likely to share the same postnatal environmental exposures than fraternal twins, the in utero placental environment is usually different in the 2 twin types, and this difference could influence subsequent risk of disease. This situation turns out to be of some considerable relevance to leukemia in twins.
The first case report of concordant leukemia in twin children appeared in the German literature in 1882.19 This anecdote was followed considerably later by a few case reports beginning in the 1930s and subsequent reviews on the topic in the 1960s and 1970s.20-25 All told, more than 70 same-sex or known monozygotic twin pairs with concordant disease have been recorded in variable detail. These cases are listed in their historical sequence in Figures 1 and 2, which illustrates the ages at diagnosis in each twin pair and the leukemia subtypes. Concordant leukemia in unlike sex or known dizygotic twin pairs is exceedingly rare.25 Because of the rarity of the condition, the limited availability of suitable databases, possible ascertainment biases, and uncertain zygosity status in the early case pairs, there is no very accurate calculation of the concordance rates in monozygotic twins. In an article published in 1964, MacMahon and Levy22 provided the first estimates of concordance rates for childhood leukemia and also drew the important inference that their data strongly suggested a prenatal origin of the disease (or, as they conceded, a prezygotic, genetic influence). All told, a number of surveys (Table 1), albeit involving only small numbers of twin pairs with leukemia, suggest that the concordance rate for acute leukemia (including both acute lymphoblastic leukemia [ALL] and acute myeloblastic leukemia [AML]) in children aged birth to 15 years is between 5% and 25%. Infant ALL is a biologically and clinically distinct disease from childhood ALL.26 It would be more appropriate, therefore, to consider concordance rates separately for infant versus childhood ALL. If infant pairs are removed from the series reported (Table 1), the concordance rate for ALL remains at around 10%. Note, however, that in very few pairs of twins concordant for ALL does presentation occur after the age of 6 years (Figure 2; see also Figure 7). This information suggests that the concordance rate for the common variant of B-cell precursor ALL in the 2- to 5-year age incidence peak is higher than 10%, perhaps around 15%. For infants, there is no firm estimate of concordance, but it is clearly considerably higher than that in older children. Note that infant pairs (< 1 year at diagnosis) constitute around half of the reported concordant pairs (Figure 2); this finding contrasts with their proportional representation among singleton cases diagnosed with leukemia of approximately 5%. In reviewing the published data prior to 1970, both Zuelzer and Cox25 and Keith et al27 concluded that concordance rate for infant leukemia (< 1 year) was close to 100%. We discuss this issue further in "Concordance rates in twins, latency, and the need for a second, postnatal `hit'."
Only rare cases of concordant leukemia have been recorded in adults,28 and the rate is probably less than 1%. An exception to this may be in chronic lymphocytic leukemia (CLL) for which several twin pairs are recorded.29,30 If the concordance rate is indeed higher than is expected by chance, then this rate is likely to be linked to the recognized familial or inherited genetic factors in CLL.31 The adult leukemia data are, however, entirely anecdotal, and it would be worthwhile to analyze concordance more systematically as has been done for childhood acute leukemia. The concordance rate for other hematologic malignancies is also unknown, except for Hodgkin disease in young adults in which it is approximately 5%.32 Four pairs of identical twins concordant for Langerhans cell histiocytosis (or histiocytosis X), a very rare clonal neoplastic disorder, have been reported.33
Historically, most researchers favored the conventional view that concordance in twins reflected a shared, inherited, or genetic susceptibility. There were, however, several facts that sat uneasily with this interpretation. First, the concordance rate, at least for infants, appeared to be extraordinarily high. Nothing like this rate was seen with other pediatric cancers in which, aside from the well-recognized familial, inherited component in retinoblastoma, the concordance rate is only around 2%.34 Diagnosis of leukemia in infant twin pairs was also remarkably synchronous in most cases (Figure 1). Additionally, although there were no reliable data on the frequency of concordant leukemia in dizygotic infant twins or non-twinned siblings, it appeared to be very low; not what one would expect if familial and highly penetrant leukemia-susceptible genes were involved. Several researchers speculated that a common placental environment of monozygotic twins might contribute in some way toward concordance of leukemia. In 1962, Wolman35 suggested that "the disease may have originated in one twin within the uterus and have been transmitted to the other through the conjoined circulation?" This suggestion, as it turns out, was correct, but the idea was largely ignored until it was resurrected and developed more fully in a letter to the Lancet in 1971 from Bayard Clarkson and Ed Boyse36 at the Memorial Sloan-Kettering Institute. Clarkson, a leukemia physician, was aware of the rare but striking instances of leukemia in twins. Boyse, an immunologist, was familiar with the observations of Ray Owen37 on blood chimerism in twin cattle in which dizygotic twins share a single placenta. This situation had been exploited by Peter Medawar (Billingham et al38) to discover neonatal immunologic tolerance and subsequent allograft acceptance, a finding that led to Medawar, with MacFarlane Burnet, being awarded the Nobel Prize for Medicine in 1960. Clarkson and Boyse pointed out that monozygotic human twins were likely in most cases to be blood chimeras also. They, literally, have shared blood. This reasoning, in turn, suggested a very plausible, nongenetic (inheritance-based) explanation for concordance of leukemia—that it arises as a single leukemia in one twin in utero and then spreads, via intraplacental anastomoses, to the other twin.
Placental status and blood chimerism in monozygotic twins depends on the timing of embryo splitting, and not all monozygotic twins share a single or monochorionic placenta39 (Figures 3-4). Embryos that split within the first 3 days after ovum fertilization develop separate, dichorionic placentas (Figure 3). Embryo splitting after day 3 but before day 7 results in separate diamniotic embryos, developing in a single monochorionic placenta (Figure 4A), and 60% of monozygotic twins are in this category. The occasional very late (14-day plus) splitting of an embryo results in conjoined (or "Siamese") twins. Dizygotic twins, in contrast, are always dichorionic. Occasionally, however, the 2 placentae can fuse with some exchange of cells, and around 8% of dichorionic twins have blood group chimerism.40
German pathologists in the 19th century discovered that a variety of vascular anastomoses exist within monochorionic placentas39 (Figure 4B). These anastomoses provide the anatomic traffic route for blood cell passage. They are also the cause of pathology as recognized in the twin-twin transfusion syndrome.41 This problem arises in some 20% of monozygotic, monochorionic twin pairs and accounts for around 15% to 20% of perinatal mortality in twins. Unequal blood distribution resulting from asymmetry of vascular anastomoses produces reciprocal polycythemia and anemia and, in some cases, cardiac, neurologic, and renal complications. The syndrome was first recorded by Herlitz42 in 1941 but is presumably inherent to twinning, a possible case being recorded in much earlier artistic representation (Figure 5).43
The intraplacental transfer hypothesis, therefore, had a sound anatomical basis, but it was nevertheless radical, implicating as it did a prenatal clonal origin in one twin followed by what was in effect a metastasis to another individual. Clarkson and Boyse36 suggested that this prenatal, monoclonal explanation might be proven by a demonstration of shared, nonconstitutive, cytogenetic abnormalities in leukemic cells from pairs of twins.
Both before and following the Clarkson-Boyse letter, several papers appeared reporting karyotyping investigations on single pairs of identical twins with acute leukemia.44-49 Some of these investigations recorded a sharing of a chromosome marker, compatible with a single cell or clonal origin. These data were, however, equivocal; the quality of cytogenetic studies at the time was less than ideal, and it was not generally appreciated that consistent chromosome markers are shared by entirely independent leukemias of the same subtype. Clearly, cytogenetic evidence for the single-clone idea would only be strongly supportive if the markers were very uncommon and/or if they were complex. One more recent cytogenetic report on concordant AML in monozygotic twins (diagnosed at age 3 and 4 years) found that the leukemic cells did share the karyotype +8, inv16, +21.50 In this case, it could well be that this pattern of chromosome abnormalities developed rapidly in one twin, in utero. Further in this section, we describe a recent twin pair in whom the leukemic cells shared a remarkable pattern of chromosomal instability indicative of a prenatal clonal origin. Unambiguous evidence for a common clonality can be derived from molecular markers of clonal uniqueness. There are several candidate markers in this respect, although each has limitations (Table 2). A significant finding in this direction was provided by a short report that a pair of infant twin's leukemic cells appeared to share the same or similar IGH gene rearrangement, that is, same-sized restriction fragment in a Southern blot.51 This evidence was limited, however, by the fact that only one restriction enzyme was used, and, furthermore, the twins were conjoined with shared vascular connections after birth as well as before. IGH and TCR can provide supportive evidence for a common clonal origin of twin leukemias (below), but unequivocal evidence comes from the use, as clonal markers, of leukemia fusion genes generated by chromosome translocation.
Chimeric fusion genes are formed by normal, error-prone repair of DNA double-strand breaks.52,53 The critical breaks occur in clustered intronic regions. The boundaries of the breakpoint regions are circumscribed by functional requirements of the resultant hybrid protein, and the breakpoint cluster region itself can vary from a few base pairs to 100 kilobase (kb) or more in length, depending on the gene. Within these breakpoint regions of the paired genes, the breaks can be widely scattered, although not entirely randomly and with significant microclustering.54 Critically, each patient's leukemic cells have, in each partner gene and in the resultant fusion gene junction, a unique or clonotypic genomic breakpoint and fusion sequence (Figure 6).52-54 This sequence then provides a highly specific and stable marker of the leukemic clone and a stringent test for the clonal relationship of leukemic populations in a pair of twins. If the leukemic cells of twins were found to share the same acquired clonotypic breakpoint, then this would indicate a monoclonal origin of their leukemia. An important caveat is, however, that clonotypic fusion genes are only ideal markers in this context if they are very early or initiating events in leukemogenesis. If they were postnatal, secondary events, then they would have different breakpoints regardless of the clonal origins of the leukemias.
The most common chromosome translocation in infant leukemia results in the fusion of the MLL gene at 11q23 with a variety of partner genes, but principally AF4 in ALL.55 Following the cloning of the MLL gene, it was shown that MLL gene breakpoints, as indicated by restriction site mapping, were scattered throughout an 8.3-kb breakpoint region (introns between exons 5 to 11) and individualistic.56,57 Taking advantage of these data, Ford et al58 showed that, in 3 pairs of identical twin infants with concordant ALL, each pair shared the same MLL gene rearrangements as indicated by identical-sized restriction fragments (in Southern blots) of MLL with multiple enzyme digests. The rearrangements were, as anticipated, absent from nonblood cells and were also absent from remission samples and were, therefore, acquired and not constitutive. Subsequently, 2 additional infant twin pairs with shared, identical MLL fusions were recorded.59,60 Clarkson and Boyse36 regarded the very short latency and near synchronous diagnosis of leukemia in identical twin infants as the plausible outcome of in utero leukemogenesis. However, they speculated that leukemia in noninfant children would most likely be postnatal in origin. This idea appears to have embraced the (false) premise that leukemogenesis is a single hit phenomenon with short latency but also begs the question of what might underlie cases of concordant ALL in older children. The studies of Ford et al58 of infant twins were part of a concerted attempt to identify twin pairs via a worldwide collaborative survey to resolve this issue of clonal origins. The prior hypothesis here was that the common (c) form of childhood ALL might involve a minimum of 2 independent genetic hits, one before and one after birth.61 Initiated in the 1980s, the Leukaemia Research Fund (LRF) twin survey ("Appendix") has, to date, collected 19 pairs of concordant ALL (18 pairs) or AML (1 pair), and we have informative molecular data for clonality status on 11 of these. For all these pairs for which we have molecular data on clonality, monozygosity was confirmed using microsatellite markers.62 The subtypes and ages of these cases are represented in Figure 7. Of these 19 pairs, 3 had MLL gene fusions (the pro-B ALL infant cases nos. 1-3, in red in Figure 7). Fourteen pairs had a common B-cell precursor (CD10+) ALL, and, of these, 5 (nos. 11, 12, 14, 15, and 16; Figure 7) had TEL-AML1 fusion. Only one had a hyperdiploid karyotype (no. 17; Figure 7). In the remaining 8 cALL pairs, the chromosome karyotype was nonascertained or "normal" except for pair no. 5. In this pair, there was a discordant karyotype: 46XY, 2p- in one twin; 46XY, t(3;16)(p13;p13), 12p+, ?del (14q) in the other. The most common chromosome translocation in ALL is the t(12;21), resulting in TEL(ETV6)-AML1(RUNX1) fusion.63,64 Some 25% of ALL have this marker,65 and the age distribution of positive cases at diagnosis mirrors that of the major 2- to 5-year incidence peak of ALL.66 Of those twin pairs in the LRF series, 5 had a TEL-AML1 fusion (marked * in Figure 7). Four of these have been molecularly characterized by cloning67 or long-distance polymerase chain reaction (PCR) methods and sequencing.68-70 The key observation was that within each of the 4 pairs of twins the same, identical, or clonotypic TEL and AML1 breakpoints were present (Figure 8). Pair no. 11 had a very asynchronous diagnosis, age 5 and 14 years. In the latter twin, it was possible to demonstrate the presence of the clonotypic TEL-AML1 fusion sequence in an archived bone marrow slide taken 9 years before diagnosis (and considered at the time to be hematologically normal) when her twin sister was diagnosed.69 This observation was important as it suggested that during a protracted period of postnatal latency, the leukemia or preleukemia was widely disseminated. The first of these TEL-AML1-positive twin pairs to be reported (no. 12; Figure 7) also shared a clonotypic IGH DN-J sequence.67
A common clonal origin was also documented for a pair of twins with T-cell malignancy at age 9 and 11 years (pair no. 13 in Figure 7). In this case, the malignant blood cells shared a common, clonal T-cell receptor-
Somewhat surprisingly, only one pair of twins in the LRF series (pair no. 17 in Figure 7) was known to have the most common genetic abnormality in childhood ALL—hyperdiploidy. Triploidy of the same chromosomes, as in this pair, could not be taken, by itself, as firm evidence for a common clonality, but this pair shared unique TCR In 7 of the "early" pairs of cALL (Figure 7), we had no informative molecular data on clonality and limited availability of DNA. The IGH rearrangements all appeared to be different within pairs in Southern blot and PCR analysis, but, in the absence of sequencing, this cannot be considered as evidence against a common clonal origin. The children in pair no. 19 were infants with AML. Their leukemic cells had an extraordinarily diverse clonal and intraclonal karyotype.73 Detailed analysis by fluorescence in situ hybridization (FISH) revealed that a stem line with alterations in 6 chromosomes (with deletions, duplications, and an insertion) was common to the twins' leukemic blasts, but the further and extensive subclonal chromosomal abnormalities were distinct. In this case, chromosome instability generated a rapid evolution of intraclonal diversity leading to leukemogenesis, and the twin comparison provided a distinction between the initiating, prenatal events and subsequent postnatal genetic divergence of the clones.
In this LRF twin series, there were 2 sets of triplets with ALL (pairs no. 16 and no. 18 in Figure 7), and these triplets were particularly informative. In pair no. 16, 2 monozygotic twins that had shared a single placenta developed ALL, with a shared TEL-AML1 fusion but distinctive secondary genetic changes (see "Concordance rates in twins, latency, and the need for a second postnatal `hit'").68 The third co-twin was dizygotic, developed in a separate placenta, and remains leukemia free by molecular screening. In set no. 18, all 3 twins were monozygotic (we assume a fourth twin was lost in utero), and all 3 developed ALL. No TEL-AML gene was present in these patients, but their leukemic cells shared clonotypic IGH sequences.74 These data suggest that both monozygosity and placental status may be critical for risk of concordant leukemia. All the patients in the LRF series (Figure 7) with known placental status (17 of 19) had a single placenta. One concordant pair of monozygotic infant twins with ALL was reported to have had independent (dichorionic) placentae in utero.59 As around 8% of dizygotic, dichorionic twins have blood cell chimerism,40 possibly based on occasional placental fusion, and something similar may have occurred in this pair. Overall, these data have provided compelling evidence that concordant leukemia at any age in identical twin children is due to a clonal origin in utero. An important corollary is that postnatal latency following prenatal initiation can be very variable and occasionally protracted (ie, up to 14 years).
Concordant infant ALL or concordant childhood common, B-cell precursor ALL in identical twins is no different biologically, clinically, or in its age incidence (Figure 2) from ALL in singletons, and there is no basis for considering that leukemia might be uniquely initiated in utero only in the context of twinning. Therefore, the conclusion from the twin studies that leukemia can originate prenatally must apply more generally to pediatric patients. The crucial question, however, is how often is this likely to be the case? A prenatal origin of infant ALL is not surprising, given the very young age of patients (average = about 6 months); indeed some cases of 11q23/MLL fusion gene-positive cases are diagnosed neonatally75,76 or, in one case, in a stillborn fetus.77 Moreover, because monozygotic infant twins discordant for leukemia appear to be extremely rare, this finding suggests that, when an infant who happens to be a twin has ALL, it will inevitably have been of prenatal origin. This suggestion should then apply also to leukemia in singleton infants. The same logic does not apply to leukemia in older children. If we accept that the concordance rate for common B-cell precursor ALL in noninfant children in the 2- to 5-year age incidence range is, say, 10%, then it follows that there is 90% discordance. This discordance could arise via 2 different mechanisms. One possibility, favored by Clarkson and Boyse,36 is that most of leukemias in twin children (> 1 year), as well as in singleton children, are initiated postnatally, setting a lower limit of approximately 10% on the cases in twins that can be concordant. Alternatively, and as some earlier commentators on twin leukemia suggested,22,78 leukemia in non-twin children might be initiated in utero but requires an essential postnatal exposure and/or genetic (or stochastic) event for which discordance was the norm in twins. The latter would imply that childhood cALL has a somewhat different biology and natural history than infant ALL, but this is already evident from molecular genetics and from the twin data with the highly variable postnatal latency and asynchronous diagnostic dates. One test that would distinguish these alternatives would be to demonstrate that, in cases of discordant identical twin children with ALL, the healthy co-twin usually does have the clonotypic TEL-AML1 fusion gene in his or her blood, despite the absence of overt disease. This idea, to our knowledge, has not been examined; our attempts to do so, to date, have been thwarted by logistical and ethical difficulties. The significance of the modest concordance rate of ALL in twin children has, however, been resolved by a different tactic.
For the twin leukemia interpretation to be correct, then the clonal progeny of the transformed fetal "preleukemic" cells, have to be present in circulating blood before birth. If this is the case, then it should be possible to identify them directly. This identification has now been achieved by retrospective scrutiny of archived neonatal blood spots or Guthrie cards. These cards have been used for many years for genetic screening for inborn errors of metabolism such as phenylketonuria (PKU).79 They are also a source of reasonably intact constitutive DNA that can be amplified by PCR to reveal inherited mutations80 or exogenous viral sequences.81 Neonatal blood is usually taken during the first week of life by heel prick, and each spot is approximately 30 µL in volume with approximately 30 000 mononuclear cells. Therefore, if leukemic or preleukemic cells with a unique fusion gene sequence were to be present in at least 1 cell per 30 000 in blood, then they might be detectable by sensitive PCR methods with clonotypic primers specifically designed for each patient's leukemic cells. We first showed that this was possible using MLL-AF4 genomic sequences in 3 cases of non-twinned ALL in patients aged 2 months, 6 months, and 24 months.82 Encouraged by this result, we next examined the neonatal blood spots of a pair of twins diagnosed at age 3 years with ALL who shared an identical TEL-AML1 sequence (no. 14; Figure 7). Four segments of one blood spot for each twin was examined,70 and 2 of 4 blood spots for each patient registered a TEL-AML1 signal verified by sequencing (Figure 9).
Analysis of Guthrie cards of non-twinned children (aged 2-6 years) with ALL showed that most did have detectable, clonotypic TEL-AML1 sequences at birth.70 Some blood spots were negative, but this result is uninterpretable: It could mean that these cases were postnatal in origin, but, equally, it could indicate that in some prenatal cases, there are less than 1 in 30 000 leukemia cells in the blood. We regard the latter as a likely explanation in at least some "negative" cases, particularly, as in positive cases, the number of leukemic cells per spot is clearly low with some negative segments of spots. These data have now been backed up with the detection of clonotypic IGH sequences in blood spots of children with ALL, including cases of ALL that have the common hyperdiploid karyotype.83-85 In one case, there was evidence that triploidy of chromosome 14 (with 3 unique rearranged IGH alleles) was present in the blood spot.86 In a recent study, 50% of AML cases investigated had clonotypic AML1-ETO fusion sequences in their neonatal blood spots, including 2 children who were older than 10 years at diagnosis.87 It might have been anticipated that not all subtypes of pediatric leukemias would involve prenatal initiation, and this appears to be the case. Few if any cases of pre-B ALL with t(1;19) E2A-PBX1 have positive neonatal blood spots,88 and no pairs of identical twins concordant for that subtype of ALL have been described. These data have 3 important implications: first, they provide direct confirmation of the prenatal interpretation for the concordant leukemias in twins. Second, they verify that this early developmental origin in fetal hemopoiesis applies to most, although probably not all, pediatric leukemias. And third, they indicate that discordance of ALL in older children can be ascribed in large measure, if not entirely, to the requirement for one or more additional postnatal events following in utero initiation. Guthrie cards have proven to be an extraordinarily rich resource for leukemia research. It is, therefore, somewhat ironic that when Bob Guthrie was developing his blood spot technique (for PKU screening) in the 1950s, he was asked to stop working on it because of its lack of any relevance to leukemia (D. Pinkel, personal communication to M.F.G., October 2002).
The concordance rates of leukemia in twins have important implications for our understanding of leukemogenesis in general. A very high concordance rate for infants implies that, by the time that the twins are born, the process of leukemogenesis is sufficiently complete that a clinical diagnosis is inevitable. This might signify that an MLL fusion gene is, in itself (in the appropriate stem cell type), sufficient for overt leukemia development. The corollary would be that MLL fusion genes have a powerful deregulatory effect on gene expression.89 This effect might be achieved by MLL fusion proteins disrupting gene transcription in a broad fashion, say by simultaneously corrupting 2 or more signal pathways in cells. An alternative and perhaps more plausible possibility is that, once formed, an MLL fusion protein somehow promotes the inevitable accumulation of additional genetic changes via, for example, very rapid clonal expansion, genetic instability, or inhibition of DNA damage repair. Either way, initiation of leukemogenesis via MLL gene fusion appears to be tightly coupled | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Abstract
Introduction
(TCR
DN-J and IGH DN-J sequences, indicating that, in this case also, the leukemia was probably initiated prenatally.