Prenatal Methylmercury Exposure and Developmental Outcomes: Review of the Evidence and Discussion of Future Directions

I conducted a review of the published literature to assess the strength of the evidence for an association between prenatal exposure to methylmercury (MeHg) and subsequent child development. I identified 12 studies on this subject published since 1980. Of these, 3 were longitudinal studies—2 conducted in the Seychelle Islands, and 1 in the Faroe Islands. Nine were cross-sectional studies conducted in different countries where seafood, a source of MeHg, constituted a major part of the diet. The ages of the children studied ranged from 2 weeks to 12 years. The results of the longitudinal studies were contradictory. Researchers in the Faroe Islands identified an association between MeHg exposure and developmental effects, whereas those in the Seychelle Islands identified no such association. This inconsistency was mirrored in the results of the cross-sectional studies where there were some positive and some negative findings. It was concluded that it was not possible from currently available data to determine whether there is an association between prenatal MeHg exposure and adverse developmental effects in children. In advance of future research, consideration should be given to resolving the uncertainties surrounding exposure assessment and outcome measurement, as both elements varied between studies. It was suggested that questions of exposure assessment would benefit from the application of an expert review process. Outcome assessment would benefit from the development of theoretically based measures of specific aspects of cognitive functioning to replace the relatively crude measures of attainment and IQ currently employed in most studies. This would assist in the development of classic longitudinal studies by allowing repeated assessment over the full age range and providing data that are more readily interpretable and comparable between studies.
Organic compounds of mercury have a variety of industrial uses, and most of the data relating to the neurotoxic effects of mercury exposure have been derived from occupational populations (Chipman et al. 1995). However, in the 1950s a potential risk to the wider community was highlighted when large numbers of people living in the Minamata Bay area of Kyushu Island, Japan, developed symptoms of mercury poisoning. This well-documented incident was traced to a factory using mercuric chloride as a catalyst in the production of vinyl chloride and discharging effluent into the sea (Harada 1995). In aquatic environments, mercury is methylated by the action of common bacteria and methylmercury (MeHg), then passes up the food chain and becomes concentrated in fish and sea mammals. The heavy dependence of the Minamata inhabitants on a fish diet meant that they were subject to high levels of MeHg exposure. Both marine and freshwater fish routinely contain low levels of MeHg as a result of mercury leaching from the earth’s crust. Cases of frank poisoning have not been recorded in fish-eating populations other than those in Minamata, where special circumstances prevailed. However, the Minamata findings raised questions about whether less severe neurologic effects might occur at lower levels of exposure in populations whose diets were rich in seafood.
Given the high level of fetal abnormalities observed in Minamata (Harada 1995), particular concern focused on the potentially hazardous effects of prenatal exposure. This concern increased after an outbreak of mercury poisoning in Iraq in 1971–1972, when large numbers of people consumed bread made from grain treated with a mercury-based fungicide. Subsequent neurologic examination of 80 children born to mothers who had eaten the affected bread over a 2- to 3-month period suggested an increase in neurologic abnormalities (Amin-Zaki et al. 1974). Animal and other data have indicated that the developing fetus is more vulnerable both to exposure to neuro-toxicants such as heavy metals and to the effects of such exposure (Hanson 1997). Any adverse effects are likely to be structurally and functionally different from those seen in the exposed adult. Thus, in addition to the obvious concern for the health of those with diets high in seafood, this issue has wider public health implications in terms of the general advisability of consuming seafood during pregnancy.
The systematic investigation of potential developmental effects in children after prenatal exposure to MeHg began in the 1980s. Since that time two major longitudinal programs of work and a number of smaller cross-sectional studies have been conducted. These investigations assessed levels of prenatal exposure to MeHg in seafood-eating populations and examined subsequent developmental outcomes in children of varying ages. However, despite considerable research effort over a period of 20 years, there remains a lack of consensus on the central issue of whether exposure to MeHg derived from the routine diets of pregnant women presents a risk to their unborn children. This article contributes to the current debate on this subject with a brief description of the currently available data, some possible explanations for the inconclusive results that have so far emerged, and suggestions for potential ways forward in this contentious area of research.
Current Evidence
A search of relevant databases [Medline (http://omni.ac.uk/medline), Toxfile (http://www.dialog.com), Embase (http://www.embase.com), Cancerlit (http://cancerweb.ncl.ac.uk/cancernet/cancerlit/), Biosis Previews (http://www.biosis.org.uk), SciSearch (http://www.bids.ac.uk), and the Web of Science (http://wok.mimas.ac.uk)], using keywords such as “methylmercury,” “prenatal exposure,” “child development,” and “neurobehavior,” revealed that 12 investigations have been published thus far on this subject, all of which have been conducted since 1980.These investigations have used either neurologic examinations, developmental rating scales, or psychological tests to evaluate postnatal neurologic effects in children prenatally exposed to MeHg. Nine of the studies were cross-sectional (Table 1), in the sense that a sample of children was tested on one occasion and associations between test results and a measure of prenatal exposure to MeHg were examined. (Cordier et al. 2002; Grandjean et al. 1999b; Kjellstrom 1991; Marsh et al. 1995b; McKeown-Eyssen et al. 1983; Murata et al. 1999b; Steuerwald et al. 2000; Stewert et al. 2003; Weihe et al. 2002). Of these cross-sectional studies, however, one consisted of a form of follow-up, in that children were tested at 6–7 years of age and divided into comparison groups on the basis of exposure data collected from their mothers soon after birth (Crump et al. 1998; Kjellstrom 1991). In addition the study of Stewert et al. (2003) comprised a cross-sectional element of a larger longitudinal investigation concerned primarily with the adverse effects of prenatal exposure to polychlorinated biphenyls (PCBs). During the course of this investigation, possible interactions between PCB and MeHg exposure as determinants of adverse health effects were also investigated. Sample sizes in the cross-sectional studies ranged from 43 to 351 and response rates from 64 to 99% (Table 1).
Three studies were longitudinal in design (Table 2) and followed children from birth, at intervals, for several years. Of these, the Faroe Islands study (Budtz-Jorgensen et al. 1999, 2000, 2002; Grandjean et al. 1992, 1995, 1997, 1998, 1999a, 2001a, 2001b, 2002a, 2002b, 2003; Murata et al. 1999a, 2002a, 2002b; Weihe et al. 1996) commenced in 1986 with a cohort of newborn infants who were subsequently tested at ages 12 months, 7 years, and 14 years. Two further longitudinal studies were carried out in the Seychelle Islands. The first commenced in 1987 with a sample of newborn infants who were followed up at age 5 weeks, 66 months, and 108 months (Cernichiari et al. 1995a; Davidson et al. 2000; Marsh et al. 1995a; Myers et al. 1995a, 1995b; Shamlaye et al. 1995). This was considered a pilot study by the authors, although a large amount of data was collected of a good standard and was therefore reported. A subsequent study commenced in the Seychelle Islands in 1989 with a sample of newborns who were followed up at ages 6.5 months, 19 months, 29 months, 66 months, and 108 months (Axtell et al. 1998, 2000; Cernichiari et al. 1995b; Cox et al. 1999; Crump et al. 2000; Davidson et al. 1995a, 1995b, 1998, 1999b, 2001; Myers et al. 1995c, 1995d, 1997, 2000, 2003; Palumbo et al. 2000). Sample sizes in the longitudinal studies were larger than those in the cross-sectional studies (Tables 1 and 2), although there was inevitable attrition over the periods of longitudinal investigation (Table 2).
Exposure assessment.
With the exception of the longitudinal Faroe Islands study and the study of Steuerwald et al. (2000), all studies used maternal hair as the vehicle for measuring prenatal exposure. In the case of the two longitudinal Seychelle Islands studies and six of the cross-sectional studies (Kjellstrom 1991; Marsh et al. 1995b; McKeown-Eyssen et al. 1983; Steuerwald et al. 2000; Stewert et al. 2003; Weihe et al. 2002), hair samples were taken from the mothers at parturition. However, three of the cross-sectional studies used hair samples taken from the mother and/or the child at the time the child was tested (Cordier et al. 2002; Grandjean et al. 1999b; Murata et al. 1999b). In the study by Grandjean et al. (1999b), for example, exposure was assessed by reference to MeHg concentrations in maternal hair samples and, where this was not available, by reference to concentrations in the child’s hair at the time of testing. Dietary habits and social circumstances had changed little in the community during the previous years. For this reason maternal hair samples were judged likely to be representative of parturition samples. In addition child and maternal hair concentrations were highly correlated. Thus, where it was necessary to use child hair samples, these were also regarded as representing an adequate proxy measure for maternal exposure at parturition The longitudinal Faroe Islands study and the cross-sectional study of Steuerwald et al. (2000) used samples of cord blood as well as maternal hair to assess levels of prenatal exposure. All studies reported details of quality control measures for the analysis of samples. However, the specific form of mercury included in the measurement was not always clear, and a number of different exposure metrics were employed (Tables 1 and 2).
Outcome measurements.
A variety of outcome measures were used that included neurologic examination, developmental rating scales, neuropsychological tests, and attainment tests. Although this variety was accounted for partly by the differing ages of the children, necessitating different forms of assessment, variation also occurred between studies in terms of the tests used for children of the same age group. All studies used tests or rating scales that were established, published assessment tools. With the exception of the Seychelle Islands studies, information on testing procedures provided in the published reports was rather limited and therefore difficult to evaluate. All studies reported control of some potential effect modifiers such as socioeconomic status, ethnicity, and parental IQ, but the particular factors selected for inclusion varied between studies. All studies used multivariate analysis techniques.
Three of the 12 studies, namely, the longitudinal Faroe Islands study and the studies of Kjellstrom (1991) and Grandjean et al. (1999b), reported a statistically significant relationship between prenatal exposure to MeHg and at least one developmental outcome. In the case of the Faroe Islands study, significant associations were observed for the group as a whole, at 7 years of age, between MeHg in cord blood and some, but not all, of the outcome measures on six neurobehavioral tests. Many of these associations were of borderline statistical significance. However, a dose–response relationship between cord blood MeHg and some test outcomes was demonstrated in a subgroup with the highest MeHg levels. Initial analysis in the Kjellstrom study (1991) did not indicate any significant associations between test outcomes and MeHg. However, exclusion of one outlier whose mother had particularly high MeHg levels resulted in the emergence of associations between MeHg and six test outcomes (Crump et al. 2000; Kjellstrom 1991). In the study by Grandjean et al. (1999b), an association between MeHg exposure was observed for three test outcomes. No discernible pattern was evident in these results in terms of effects on particular tests. Three further studies reported some positive associations between MeHg exposure and developmental outcomes, although all qualify their conclusions and express some reservations about the strength of their findings. In two of these studies, children were under 3 years of age, and assessments were in the form of neurologic examination. Steuerwald et al. (2000) reported that examination of children at 2 weeks of age showed that those with higher prenatal exposures had slightly lower overall neurologic scores, although there was no discernible pattern of suboptimal findings. Similarly, McKeown-Eyssen et al. (1983) carried out neurologic examinations on Cree Indian children between 12 and 30 months of age and found an association between MeHg exposure and the prevalence of abnormal muscle tone reflexes in males only. The authors note the mildness of the abnormality, which they consider to be of doubtful clinical significance. Cordier et al. (2002), in a study of children 9 to 12 years of age, found an association between scores on one test and MeHg exposure, but this did not appear in separate analysis of the highest exposure subgroup. Moreover, results from one test showed a positive association with MeHg exposure. This type of counterintuitive result also occurred in a study of children 7–12 years of age (Weihe et al. 2002) and in the main Seychelle Islands study when children were assessed at 66 months (Davidson et al. 1998). Neither of these studies or the first Seychelle Islands study report associations between other test results and MeHg exposure. Similarly, the results of Marsh et al. (1995b) and Murata et al. (1999b) with children between 6 and 7 years of age indicated no associations between psychological test scores and MeHg exposure. Murata et al. (1999b), however, demonstrated an effect of MeHg exposure on two neurophysiologic measures. Finally, the study of Stewert et al. (2003) of younger children 38 and 54 months of age reported an interactive effect of MeHg and PCB exposure as an incidental finding that emerged in the course of a study to investigate the adverse effects of PCB exposure. A finding of this nature, although interesting, should perhaps be regarded as speculative only and requiring further investigation.
Our relatively brief review of the literature highlights the current uncertainty in this field of enquiry. The two major longitudinal investigations report contradictory findings, and a number of cross-sectional studies have similarly produced inconsistent results. This data set has already been subjected to much examination [National Research Council (NRC) 2000] and further analysis in an attempt to derive appropriate environmental exposure limits for pre-natal MeHg (Office of Environmental Health 1999; Rice et al. 2003). The generally assumed superiority of longitudinal over cross-sectional designs has tended to focus most attention on the studies conducted in the Faroes and the Seychelle Islands, which are of high methodologic quality (Jacobson 2001). However, it should be noted that, given the complex and challenging nature of this type of research, the majority of the cross-sectional studies are also of relatively good quality and would normally be taken into account in a systematic review. The results of these cross-sectional studies in fact mirror the general inconsistency evident in the results of the longitudinal investigations. One is therefore drawn to the conclusion that it is not possible from currently available data to determine whether prenatal exposure to MeHg, at levels routinely experienced by populations whose diets are rich in seafood, results in adverse effects on the nervous system of the developing child.
Discussion
A primary objective of the review process is the derivation of conclusions from the available data to guide future policy. In this case, however, the development of such a policy would appear to be hindered by the existence of directly contradictory results obtained from research of equal quality. As noted above, the data from the studies contained in this review have already been the subject of extensive evaluation (NRC 2000; Rice et al. 2003) and continue to excite controversy that is so far unresolved (Davidson et al. 1999a; Grandjean and White 1999; Stern and Gochfeld 1999). A secondary objective of the review process, the identification of data gaps in the literature, appears inappropriate in circumstances where so much research has been carried out to date. Although the contribution of existing published research is unquestioned, it may be time to concede that there is little further that can be drawn from these data or, one suspects, from repeated studies of a similar type. Experience from other fields (Spurgeon 2002) suggests that further cross-sectional studies employing similar neurobehavioral outcomes will serve only to increase rather than reduce the uncertainty surrounding this issue. In the remainder of this article, therefore, I discuss some of the possible reasons for the inconsistency in the existing data and indicate some areas where alternative approaches might be required to achieve some progress in this field.
The common objective of the investigations reviewed above was to establish whether there is an association between prenatal exposure to MeHg and developmental effects. Although the various studies had many elements in common, perhaps the most noticeable feature of the studies as a group was the variation in the methods used to assess the two basic elements of the association, namely, the exposure and the effect. It is not surprising that research using different combinations of biological and psychological measures produces inconsistent results. The debate surrounding each of these elements, although undoubtedly complex, merits resolution in advance of any further research.
Exposure.
In terms of the most appropriate biological marker of prenatal exposure, opinion is divided between maternal hair and cord blood as the biological sample of choice. Studies that have attempted to define the relationship between different biological indices have produced inconsistent and somewhat wide-ranging results, and conversion from one set of values to another appears to involve a number of questionable assumptions (Office of Environmental Health 1999). Other difficulties in the interpretation of the data set arise as a result of the use of different units of measurement and a lack of clarity in some studies about whether the measure is of organic, inorganic, or total mercury concentration. Thus, there is continuing uncertainty about the association between elements of the diet and concentrations in child hair, maternal hair, cord blood, and maternal blood, as well as uncertainty about the strength of any relationship between each of these elements and the relationship between each and the actual exposure of the fetus. Elements of the debate about hair versus blood samples must be linked to a large number of other unanswered questions surrounding prenatal exposure measurement. These relate particularly to the relative importance of exposure at different periods of gestation, the relationship between these and average exposures, and the importance of peak exposures. The development of the central nervous system is time related and unidirectional. The inhibition of one stage of development tends to cause alterations to subsequent processes, with limited capacity for compensation for cell loss (Annau and Eccles 1986; Trask and Kosofsky 2000). Both the dose and timing of any environmental insult are important in terms of the specific nature of any adverse effects. How far do our current methods of prenatal exposure assessment reflect the need to take this into account?
The present enthusiasm for evidence-based policy and practice appears to offer an ideal opportunity to address these types of questions, either through the medium of an expert workshop or that of a written systematic review. The important issues in either process include a) definition of the important questions to be addressed to achieve valid and reliable assessment of prenatal exposure, b) identification of available data that could be used to answer these questions, and c) identification of new research required to fill any identified data gaps. In advance of some consensus on these issues, further research is likely to provoke more controversy rather than lead to any resolution of the current uncertainty.
Outcomes.
The outcomes used in these studies were predominantly psychological tests. Use of such tests in environmental and occupational health research, which began in the early 1980s, has always been controversial, and the apparent inconsistencies in the data produced has provoked much debate in both environmental and occupational health research (Koller et al. 2004; Levy et al. 2004). Results relating to prenatal MeHg exposure represent a particular example of a wider problem and highlight a number of questions related to the more general field of neurobehavioral toxicology.
Specifically, two main areas are of concern. The first, and perhaps the more straightforward, relates to the control of variables that either represent potential confounders or may act as modifiers of the effects under investigation (Spurgeon and Gamberale 1997). They are perhaps best considered under the broad headings of situational variables (physical testing conditions and test procedures), tester variables (reliability of the examiners), and subject variables (individual characteristics such as age, gender, and socioeconomic group). In all epidemiologic research involving psychological testing, the list of these variables is potentially very long, and researchers appear divided about which to include. In research on MeHg, the majority of studies consider important subject characteristics such as age, ethnic and socioeconomic group, and aspects of parental lifestyle. However, for a number of other variables (e.g., aspects of the caregiving environment), inclusion is patchy. For many of these variables, useful literature is available on their effects on children’s abilities or on test performance, and it may be possible to reach an evidence-based consensus on their inclusion or exclusion. For other, mainly procedural factors, data appear relatively scarce. A systematic review that encompasses other areas of psychology, for example, that pertaining to human–computer interaction, might reveal relevant information. For example, how much does the size of the screen affect performance on a computer-administered test? How much does the physical location of testing (home, laboratory, hospital) affect test performance? Existing data on the effects of time of day (Smith 1992), for example, indicate that in epidemiologic studies this factor should always be controlled. Intuitively it would seem appropriate that the physical testing situation and procedures should be standardized for all subjects as far as is practically possible, regardless of whether firm evidence exists about the influence of heating, lighting, noise control, or the arrangement of furniture. Less well-researched aspects of the test situation can be explored usefully within the researchers’ data. Is there, for example, a significant difference between test scores obtained at the beginning and at the end of the week or at different times of the year?
The effects of the tester, particularly where tests are not computer-administered, may be important, not only because of different interactions with different subjects but also because of the examiner’s variable moods, motivation, levels of fatigue, and tendency to introduce systematic errors into the testing procedure. It cannot be assumed that confining testing to one examiner or using examiners who have undergone a single period of training removes tester variation. In terms of reliability, it may be advantageous to employ more than one tester in some circumstances. Measures such as the videotaping of testing procedures, double scoring, and examination of the test data for trends related to some of these factors have all been used to account for or eliminate this potential source of variation (Harvey et al. 1988). Similarly some estimation, albeit a subjective rating, of the child’s level of co-operation with the testing procedure is important to include. Potentially this is a major source of variation in test performance rarely alluded to in published reports. Ideally, tests should also include parallel forms or practice trials to ensure that maximal performance level is recorded for each subject.
All except one of the studies in the field of research under discussion here present detailed accounts of quality control procedures in relation to MeHg assessment. It is relatively rare to find equally detailed discussion of procedures for outcome assessment. This is a situation that occurs frequently in neurobehavioral investigations. Lack of reference to quality control does not necessarily imply that control was limited but may suggest something about the attitude of researchers toward its importance. The implications for further research are 2-fold. First, systematic work is needed on the effects of factors considered likely to affect test performance, including both a review of the available data and, if necessary, further investigative work. Second, consensus must be reached on good practice such as that available in some other areas of toxicology, notably animal experimentation. Although this consensus may exist at an informal level in the field, the considerable methodologic variations between different neurobehavioral studies suggest that many aspects are currently opinion based rather than evidence based.
A second and fundamental issue in terms of outcome measures relates to the types of tests used and, by implication, the interpretation of the results they provide and the comparability of these between studies. The tests employed in the studies described above are mainly tests of intellectual functioning. However, those used in different studies, and sometimes within the same study, derive from a number of separate traditions of intellectual assessment, each of which was developed for a different purpose and different client group. Although each has some advantages, none were developed specifically for neurotoxicity research and none is entirely appropriate for this type of application.
Attainment tests are attractive in the sense that they offer the opportunity to benchmark the performance of children in basic skills such as literacy and numeracy against that of their peers. However, such tests tend to reflect the use of abilities rather than the underlying abilities themselves. Given the range of social and educational factors interacting with the ability to produce attainment, this effectively introduces additional variables into the equation (Gadzella et al. 1989).
In contrast, the neuropsychological approach characterized, for example, by tests such as the Trailmaking test used in the Seychelle Islands study or the Bender Gestalt test used in the Faroes Islands study was developed to provide detailed evaluation of patients with suspected damage to the brain. Such damage might have resulted from head injury or other insult or from a degenerative disease of the nervous system. In these circumstances the purpose of assessment is to provide detailed information about the nature of the problem in functional terms and thus provide a basis for rehabilitation and progress monitoring. Assessment in a clinical setting tends to be a flexible process that draws as much on the qualitative aspects of the interaction between psychologist and client as it does on the numerical test scores. The clinician is interested in the patient as an individual and reaches a professional judgment on the basis of a number of sources of information. There is a risk that tests of this nature lose much of their value when applied in a routine fashion to large groups of people. Many who work in the field of clinical neuropsychology appear to be deeply uneasy about the transfer of these techniques to an epidemiologic setting (British Psychological Society 2001). Particular concern arises when tests designed for administration by a psychologist are adapted for computer presentation. Researchers with a neuropsychological background tend to adopt a clinical approach by administering a very large battery of tests to cover all aspects of functioning (Davidson et al. 1995a; Grandjean et al. 1997; Kjellstrom 1991). In an epidemiologic setting this can be inappropriate, resulting in multiple comparisons and the possibility of chance findings. Moreover, it often leads to confusion from a psychological point of view, where the results appear as a collection of apparently unconnected findings with no discernible meaningful pattern. Where studies use the same tests, it is common for significant associations to appear in both studies but in relation to different outcomes (Grandjean et al. 1997, 1999b). Finally, there are questions about the ability of tests designed for more severely affected patient groups to detect relatively subtle effects in community samples (Spurgeon 1996; Stollery 1985, 1990).
Tests derived from a psychometric tradition are concerned with the assessment of intelligence quotients (IQ) in the general population and were originally developed to describe normal distributions of cognitive functioning. The Wechsler Scale (Wechsler 1991) represents the most widely used test battery in this respect. Developmental scales for very young children fit within this tradition, replacing formal testing where this is impractical, although it should be noted that maternal reports of developmental milestones are subject to numerous sources of error such as inaccurate recall, differing definitions of certain behaviors, and presentational bias (Axelson and Rylander 1984).
The measurement of IQ is a reassuringly familiar concept supported by a wealth of normative data and experience built up over many years. Unfortunately, IQ tests were originally developed within a theoretical framework of cognitive functioning that prevailed more than a half-century ago. Such tests reflected a contemporary need to categorize individuals on a quantitative scale to predict future performance, an approach now considered somewhat crude and simplistic. Although such tests maintain their predictive validity in some settings (Neisser et al. 1996), they are relatively blunt instruments that combine a number of different abilities within each test (Lezak 1988). This aspect limits the information that can be derived from the assessment and makes interpretation difficult when conflicting results emerge from different studies. When placed in the context of more recent theoretical developments in cognitive psychology, established IQ tests do not provide results that can be easily linked to current models of cognitive processes.
A primary objective of neurobehavioral research is the detection of subtle effects on cognitive functioning in community samples after neurotoxicant exposure. For epidemiologic purposes, tests should be quick and easy to administer. The results should be interpretable at group level and comparable between different studies. Given these criteria, none of the tests currently in use appear to be entirely fit for this purpose. Speed and ease of administration do not represent major challenges in an age of advanced information technology. However, improvements in interpretability and comparability are more complex issues likely to require a radical change of approach. In recent years a number of authors have pointed to the overemphasis on empiricism in this field and the lack of a strong theoretical underpinning for the assessment tools employed (Stephens and Barker 1998; Stollery 1990, 1996; Williamson 1990). The development of tests grounded in well-established cognitive theory would allow results to be discussed in terms of the specific aspects of cognitive processing under investigation rather than simply by reference to broad and largely uninformative categories of effect such as “memory” or “attention.” Modern approaches to the study of memory processes, for example, have long distinguished between several elements that contribute to the final outcome (initial registration of information, encoding, transfer to long-term store, loss of information by decay or interference, and use of cues for retrieval) (Baddeley 1987). Each may be differentially susceptible to neurotoxic insult, but effects on one specific process cannot be uncovered by most current tests that provide a simple global outcome score. Moreover, overall scores may mask specific effects where subjects employ compensatory strategies among different processes to achieve maximum performance. The development of tests, for both children and adults, based on techniques currently available to separate and measure these specific processes would provide much more useful information about the nature and size of any observed effect. This type of approach would ultimately pave the way for much greater comparability between the results of different investigations and for the development of comparable assessment techniques for children at different ages during longitudinal investigations. Despite much international effort during the last 25 years, agreement on a universally approved set of tests has proved elusive (World Health Oganization 1989). At the same time, the pursuit of the goal to achieve comparability over time and between studies has tended to inhibit the development of new techniques. It seems unlikely that consensus on appropriate assessment tools will be achieved in advance of a consensus on the theoretical basis for those tools. Fortunately, much of the information required for these new developments is readily available in the existing cognitive, experimental, and developmental psychology literature.
Conclusion
Reviews of the data relating to the developmental effects of prenatal MeHg exposure have highlighted the inconsistency of the currently available evidence. The size and nature of the risk to children that is associated with seafood consumption by their mothers remains uncertain and a source of considerable controversy. It has been argued here that the present uncertainty derives from the variation between studies in the methods used to measure both the exposure and the effect. Each element would merit further consideration in advance of any future research in this field. Although consensus is required on the appropriate biological marker of exposure, there is also a particular need to address issues of both procedure and content in psychological assessment. Discussion of these issues, particularly those relating to psychological tests, may have implications that go well beyond the immediate needs of this field of inquiry. Investigation of the effects of MeHg provides one particular example of the difficulties in data interpretation that occur repeatedly in neurobehavioral studies and threaten to undermine confidence in this methodology. The increasing international anxiety about potential adverse effects of low-level neurotoxicant exposure in the environment underlines the importance of addressing these concerns, as psychological methods currently represent one of the main tools of research in this field.

methylmercury
developmental outcomes
prenatal exposure
neurobehavioral tests


Successful Internalization of a Chronic Biliary Cutaneous Fistula After Liver Transplantation: Deepithelializing the Fistula Tract

Biliary cutaneous fistulas are uncommon sequelae after biliary surgery and can be a source of significant morbidity. We describe a liver recipient who developed a biliary cutaneous fistula secondary to hepatic artery thrombosis; this subsequently drained for over 7 years. Through a novel approach, using the transabdominal fistula tract as a conduit, the fistula skin opening was deepithelialized and anastomosed to a jejunal loop, internally draining the tract. For over 7 years postoperatively, this internal drainage procedure has continued to function effectively. This approach may have value in internalizing longstanding biliary cutaneous fistulas in well-selected patients in whom there is no existing biliary ductal system or the existing system anatomically does not lend itself to restoration of functional internal drainage through conventional approaches.
Introduction
Biliary cutaneous fistulas are uncommon sequelae after biliary surgery and hepatic trauma. Persistent biliary fistulas can be associated with local skin morbidity, malabsorption of fat soluble vitamins, steatorrhea, impaired wound healing, and sepsis as a result of fistula tract obstruction.1 We describe a rare scenario in which a liver transplant recipient developed a biliary cutaneous fistula secondary to hepatic artery thrombosis. Through a novel approach, the fistula skin opening was deepithelialized and, using this transabdominal fistula tract as a conduit, anastomosed to a jejunal loop, thereby internally draining the fistula. This operative procedure, which has provided a successful long-term result, is discussed.
Materials and Methods
Case Report
A 60-year-old man of Chaldean descent underwent orthotopic liver transplant for cirrhosis secondary to chronic alcohol use. His early postoperative course was complicated by hepatic artery thrombosis with necrosis of the extrahepatic biliary system. Because of sepsis and family issues, retransplantation was not a realistic option. Surprisingly, his graft remained viable with good metabolic function despite the absence of normal extrahepatic drainage. For 7 years subsequent to transplantation, his biliary drainage was managed with a U-tube connected to a bulb drain exiting the skin in the right subcostal region (Fig. 1). As a result, he suffered from severe malnutrition, pain, and skin breakdown. The persistent external drainage markedly affected his lifestyle, as the continuously draining bile required dressing changes over the exit site two to four times per day. The U-tube, frequently becoming obstructed with biliary sludge, required bimonthly replacement. Preoperative fistulogram (Fig. 2) demonstrated the continuity of the cutaneous opening with a large channel through the mid portion of the liver, but with no evidence of a true ductal system.
Figure 1(a) Longstanding (7-year-old) biliary cutaneous fistula draining through right upper quadrant scar. Functional U-tube in place with copious biliary drainage. (b) Close-up of U-tube exit site.Figure 2Preoperative fistulogram in which contrast was injected through the existing U-tube. The cutaneous opening at the upper left side of the frame is in continuity with the large contrast-filled channel that enters the substance of the liver. Note the complete absence of an extrahepatic biliary system.
Surgical Procedure
With the patient under general anesthesia, the abdominal cavity was entered through a right subcostal incision along the scar of the previous transplant incision. The incision was fashioned so as to preserve a 1" diameter button of full-thickness skin surrounding the opening to the fistula tract (Fig. 3). Extensive adhesions between the fascia, liver surface, and small intestine were carefully lysed. The fistula tract was dissected retrogradely from surrounding subcutaneous tissue and muscle, preserving a rectus fascial ring. This created a conduit that could be anastomosed to small intestine. Dissection of the tract was stopped proximally at the level of the liver surface to preserve its integrity and vascularity. The fistula opening was lowered into the abdominal cavity. A 60-cm long Roux-en-Y jejunal limb was constructed 50 cm distal to the ligament of Treitz using stapled technique.
Figure 3(a) At operation, the U-tube was prepped into the surgical field. (b) Dissection of the epithelialized chronic biliary cutaneous fistula tract. In entering the abdomen through the previous right subcostal incision, a 1" diameter button of full-thickness skin containing the fistula tract was preserved.
A 4-mm-wide skin edge was sharply deepithelialized circumferentially around the dissected fistula opening. This conduit was then anastomosed to the Roux limb using two layers of running 4-0 Prolene suture (Fig. 4). The inner layer approximated the deepithelialized skin edge of the fistula tract to full-thickness jejunum. The outer layer approximated the conduit’s scar tissue and the preserved rectus fascial ring to seromuscular jejunum. An omental wrap was fashioned around the anastomosis. The anastomosis was performed over an 8-French feeding tube, which was exteriorized through a previous U-tube exit site and connected to a bile bag.
Figure 4(a) Anastomosis of Roux-en-Y jejunal limb to deepithelialized fistula tract. (b) Illustration depicting the anastomosis.
The abdominal cavity was irrigated and closed in the standard fashion. The patient tolerated the 3-h procedure well. Blood loss was minimal. He was discharged on postoperative day 8.
Result
This patient has now been followed for 7 years since the described procedure and is doing well without external drainage of bile. The condition of his periincisional skin improved dramatically shortly after the procedure. At this time his liver function tests and nutritional parameters are normal and his graft function remains adequate on a low dose immunosuppressive regimen.
Discussion
Chronic biliary cutaneous fistulas are challenging to manage from many standpoints: metabolic, nutritional, hygienic, and quality of life. The patient’s being a liver transplant recipient, added further complexity. First, malignant change was reported in chronic biliary cutaneous fistulas 2; this possibility is of real concern in light of this patient’s immunosuppressed state. Second, this patient had severe malabsorption and resulting difficulty in maintaining acceptable cyclosporin blood levels. Finally, the ongoing presence of his U-tube poses the threats of ascending cholangitis, hemobilia, intrahepatic biloma, and biliary-venous fistula, which are all well-documented complications that have been associated with U-tubes.3 The decision for operative intervention was made out of these concerns and for progressive difficulty in maintaining adequate external biliary drainage. Late retransplantation was considered; however, the patient’s graft function had been excellent over the 7 years subsequent to his transplant, and the patient and his family refused to consider it.
In both the posttraumatic and postbiliary surgery settings, existing literature advocates initial nonsurgical management of biliary cutaneous fistulas, as a significant number will close either spontaneously or with nonsurgical intervention.1,4,5 In the series by Zer, four of seven biliary fistulas sealed spontaneously.5 Endoscopic approaches to reducing intrabiliary pressure and thereby encouraging drainage along a path of least resistance include endobiliary stenting, sphincterotomy, and nasobiliary drainage.1,4 Finally, selective biliary embolization, percutaneous transhepatic catheter drainage, sclerosis with tetracycline, and oral nitrates were also described to promote closure.4,6–9
Operative procedures for refractory biliary cutaneous fistula were dictated by the anatomic site of biliary tract disruption. Roux-en-Y hepaticojejunostomy, in which a jejunal loop is sutured directly to the liver capsule, was employed in the context of intrahepatic biliary injury after trauma.11,10,4 Reports in which an actual fistula tract is used as a conduit and sutured to a loop of small bowel, however, are rare. Smith described the anastomosis of a jejunal loop to a divided fistulous tract arising from the lateral surface of the liver in a patient with a penetrating injury to the upper abdomen.12 That same author described the internal drainage of a biliary cutaneous fistula secondary to blunt liver trauma into the gallbladder.12 In both cases the fistula tract was divided and then the proximal end of the divided tract was anastamosed to an intestinal loop over a tube.
Deepithelialization of the skin opening and its direct usage in an anastomosis has not, to our knowledge, been previously described. Furthermore, this procedure is unique in that the internalization was done in the case of a functioning liver after hepatic artery thrombosis. The existing anatomy that had been established over 7 years of fistulous drainage dictated the type of procedure that was performed. In contrast to fistulas that arise after biliary tract surgery (for example, after cholecystectomy with common bile duct exploration), there was no remnant of a previous biliary ductal system. The preoperative fistulogram revealed flow of contrast from the skin directly to the liver surface with no evidence of an extrahepatic ductal system. The fistula therefore provided the only source of biliary drainage for the entire liver.
An alternative surgical option would have been hepaticojejunostomy, in which the jejunum would have been sutured to a fibrous ring at the origin of the fistula tract from the liver surface. This, however, would have required more extensive dissection with possible disruption of collateral vasculature to the previously ischemic liver and to the tract itself, as well as dissection of the transverse colon, which in part bordered the tract. At operation, the superficial portion of the fistula tract was well established and appeared well vascularized. The potentially harmful deep dissection necessary for a hepaticojejunostomy was therefore avoided.
We emphasize the decision to have fashioned a tongue of omentum over the anastomosis. By nature, the tissue comprising a fistula tract is probably somewhat tenuous. This patient’s immunosuppressed and malnourished state compound the risk of anastomotic breakdown. We feel that the well-vascularized, adherent omental tongue was an important aspect of the operation in reinforcing the anastomosis.
As of 7 years of follow-up, the patient has not developed signs of biliary obstruction. His immunosuppressant levels are easier to maintain and he does not have evidence of malabsorption. It is unlikely that he will develop cicatricial narrowing of the tract, as it had remained well epithelialized for the 7 years before this operation. There is a theoretical concern about formation of squamous cell carcinoma at the anastomotic site, which we continue to keep in mind during long-term follow-up.
Conclusion
In summary, through a novel approach this patient’s biliary cutaneous fistula was internalized, which has eliminated his associated skin morbidity and malabsorption. Furthermore, his well-functioning graft was salvaged, thus saving the cost of retransplantation and sparing an additional liver for another patient. Internalization of a long-standing biliary cutaneous fistula through deepithelializing the skin opening and using the tract as a conduit is technically feasible. We believe it to be a valid approach in the patient in whom there is no existing biliary ductal system or the existing system anatomically does not lend itself to restoration of functional internal biliary drainage by conventional approaches.


Middle Segment Pancreatectomy: A Useful Tool in the Management of Pancreatic Neoplasms

Small, benign, or low-grade malignant tumors located in the neck of the pancreas are usually treated with enucleation. However, if enucleation is too risky because of possible damage of the main pancreatic duct, standard pancreatic resections are performed. Such operations can lead to impaired long-term exocrine–endocrine function. Middle segment pancreatectomy consists of a limited resection of the midportion of the pancreas and can be performed in selected patients affected by tumors of the pancreatic neck. Middle segment pancreatectomy is a safe and feasible procedure for treating tumors of the pancreatic neck; in experienced hands it is associated with no mortality but with high morbidity, even if the rate of “clinical” pancreatic fistula is about 20%. Moreover, it allows a surgeon to preserve pancreatic parenchyma and consequently long-term endocrine and exocrine pancreatic function.
Introduction
Whereas neoplastic lesion located in the pancreatic head or body-tail are usually resected by pancreaticoduodenectomy or distal pancreatectomy, tumors in the neck represent a real challenge for a surgeon. In these cases, standard or extended pancreatectomies performed for benign or borderline cases can determine the loss of a great amount of glandular tissue, significantly increasing the risk of diabetes, impaired exocrine function, and splenic loss.1–6
Enucleation would be an adequate alternative for small, benign, and low-grade malignant tumors, such as endocrine and cystic neoplasms of the pancreas. Unfortunately this conservative procedure cannot be always applicable. When the neoplastic lesion measures up to 2 cm or more, or it is encased within the pancreatic gland, enucleation is associated with a high risk of Wirsung’s duct damage; moreover in the case of tumors with uncertain biological behavior this approach should be avoided because of the risk of tumor recurrence1–5.
Letton and Wilson7 reported for the first time in the English literature in 1959 two cases of traumatic midpancreatic transection followed by a reconstruction with a Roux-en-Y jejunal loop anastomized to the distal part of the gland. Dagradi and Serio,8 from our own Department of Surgery, were the first in 1984 to propose middle pancreatectomy with an “oncological” indication, treating a pancreatic insulinoma. Subsequently, other authors reported cases of resection of the middle pancreas, of varying extent, using various terms such as “central pancreatectomy,” “middle segment pancreatectomy,” “segmental pancreatectomy,” and “intermediate pancreatectomy.”9–13 The underlying indications for surgery ranged from chronic pancreatitis to benign, uncertain behavior, or low-grade malignant exocrine and endocrine neoplasms1–19. Different techniques were adopted for gastrointestinal reconstruction, including jejunal anastomosis of both the proximal and distal stump, or of only the distal stump, with pancreaticoduodenal or pancreaticogastric anastomosis.1–21
Surgical Technique
The abdomen is entered through a midline incision. The gastrocolic ligament is opened, preserving the gastroepiploic vessels, and the pancreatic gland is exposed. The posterior peritoneum along the superior and inferior margin of the pancreas is incised. The superior mesenteric vein and the portal vein must be identified and their surfaces cleared below the gland. The plane between the superior mesenteric and portal vein should be teased apart. The splenic artery and vein are dissected free and separated from the gland. Some venous tributaries to the portomesenteric axis and some minor collaterals of the splenic artery can be ligated. Then, the posterior surface of the pancreatic neck is isolated from the portomesenteric axis and a ribbon is passed behind the gland to elevate it. Sutures are placed along the superior and inferior margins to indicate where the proximal and distal transection should be performed and to ligate those vessels running along the margins. The segment of the pancreas with the tumor is subsequently transected through a knife or a stapler to the left and to the right of the lesion. The cephalic stump is sutured with interrupted stitches after elective ligation of the Wirsung’s duct or by means of a stapler. A small stent is placed in the main pancreatic duct while performing pancreojejunostomy or pancreogastrostomy; the stent can be left in place, even if in our experience we have never done it. Two closed-system suction drains are used to drain the cephalic stump of the gland and the pancreaticojejunostomy/pancreaticogastrostomy.
Discussion
It has been shown that standard pancreatic resections are nowadays associated with low mortality and morbidity if performed in high-volume centers by experienced surgeons.22–24 It is remarkable that this type of surgery can lead to long-term complications, such as diabetes, exocrine insufficiency, and late postsplenectomy infection25,26.
The incidence of postoperative exocrine and endocrine impairment is not predictable in patients with apparently “normal pancreas.” Factors such as fibrosis of the remnant, Wirsung’s duct obstruction, preexisting chronic pancreatitis, benign or malignant disease, and subclinical diabetes may play a role as “risk factors.”1–3 After standard left-sided resection there is an increased incidence of endocrine impairment and onset of diabetes reported from 17 to 85% of patients; it is reasonable that the extent of the resection is strictly related to the incidence of endocrine–exocrine long-term insufficiency27–31.
For all these reasons, more conservative surgical techniques have been advocated for small, benign, or low-grade malignant tumors located in the neck of the gland, aimed for sparing, as much as possible, pancreatic parenchyma. Whenever neoplastic lesions are not small and superficial enough to be simply enucleated, middle segment pancreatectomy should be considerated.1–6
Middle segment pancreatectomy accounts for only 3% of the pancreatic resections performed at our institution and about 100 cases have been reported in the English literature1–21,32–34: this means careful selection of patients. In fact, the small number of patients who underwent this type of operation is related to different factors: specific localizations of the neoplasm, well-selected indications (benign or low-grade malignant tumors), and a distal pancreatic stump of at least 5 cm in length.
Some authors1,3 have reported that this operation can be performed only in the case of small tumors (<5 cm in diameter); in our experience, although the mean diameter of the resected lesions was 27.4 mm, we have safely performed middle segment resection for tumor measuring more than 5 cm, harboring from the anterior face of the pancreas.
Middle segment pancreatectomy was also occasionally used for malignant disease: two islet cell carcinomas, one vipoma who subsequently developed hepatic metastases, one cystadenocarcinoma, and one carcinoma in situ2,3,10–12.
In the past we have also performed this operation for malignant tumor but we had pancreatic recurrence of the tumor in two patients (one affected by metastasis and one by intraductal papillary mucinous neoplasms [IPMN] with in situ carcinoma); moreover, two patients with adenoma and borderline main duct IPMN had a tumor recurrence in the pancreatic gland. Thus, we believe that in patients affected by primary or metastatic malignant tumor, a standard resection would be more appropriate. Moreover, middle pancreatectomy in our experience should also be avoided in patients affected by IPMN, especially main duct type because of their potential malignity and the possibility to have different degrees of dysplasia along the Wirsung’s duct in the same patients.
The surgeon must be sure to achieve tumor-free proximal and distal resection margins after performing middle segment pancreatectomy and, for this reason, frozen section examination is mandatory.
Middle segment pancreatectomy is a meticulous procedure. There is the possibility of leaks from both the closed cut edge of the head and the pancreaticojejunostomy, considering that in most patients we are dealing with a normal soft pancreatic texture with a small Wirsung’s duct. Thus, not only great care must be taken in selecting the patients who will benefit from this operation, but also an experienced pancreatic surgeon working in a high-volume center is required for performing the procedure.1–4,6,32,33
Median pancreatectomy is reported to be associated with no mortality but with a high postoperative morbidity, above all consisting of pancreatic fistula.6 In our experience the “clinical” pancreatic fistula rates after pancreaticoduodenectomy and left pancreatectomy are 10 and 20%, respectively.23,35–37 Between January 1990 and December 2005 61 patients underwent middle segment pancreatectomy at our institution. The incidence of pancreatic fistula—according to the International Study Group on Pancreatic Fistula definition22—was 51%. It is remarkable that most patients complained of Grade A fistula, which is a “biochemical” fistula without any clinical impact, whereas 13 patients (21%) developed a grade B or C fistula, which required prolonged in-hospital stay. In almost all patients the conservative management was successful; no one underwent reoperation and in four cases intraabdominal collections were treated with ultrasound-guided drainage. The mortality rate was zero.
The risk of developing a pancreatic fistula must be taken into account in the preoperative decision making; we believe that this risk is acceptable when the procedure is performed in a high-volume center and for patients with a long-life expectancy, such as young or middle-aged people affected by benign or low-grade tumors.
The most important advantage of middle segment pancreatectomy is the good endocrine and exocrine long-term function.1–6,10 Iacono et al.1 in a series of 13 patients demonstrated that postoperative oral glucose tolerance, pancreaticolauryl and fecal fat excretion were normal in all cases and they studied six patients pre- and postoperatively with oral glucose tolerance test showing no significant differences before and after surgery. Moreover, Sperti et al.3 showed, in a review of the literature, no case of impaired endocrine function in 59 evaluable patients whereas exocrine function was reported to be normal in 56 out of 59.
Another advantage of this procedure is the possibility to preserve the spleen, preventing the risk of postsplenectomy sepsis and hematologic disorders, which is low but exists in an adult.38,39
In conclusion, middle segment pancreatectomy is a safe and technically feasible surgical approach for removing pancreatic neck tumors in well-selected patients; in experienced hands it is associated with no mortality but with high morbidity. Most of the complications do no require reoperation or prolonged in-hospital stay and can be successfully managed conservatively. Moreover, it allows the surgeon to preserve pancreatic parenchyma and consequently long-term endocrine and exocrine pancreatic function.

middle segment pancreatectomy
pancreatic resection
pancreatic function
pancreatic tumors
surgical complications


Metinel Node—The First Lymph Node Draining a Metastasis—Contains Tumor-Reactive Lymphocytes

Background We previously identified tumor-reactive lymphocytes in the first lymph nodes that drain the primary tumor. In this study, we performed lymphatic mapping to investigate the possibility of finding the first lymph nodes that drain metastases, and of learning whether these lymph nodes contained tumor-reactive lymphocytes suitable for adoptive immunotherapy.
The global cancer registry Globocan shows 10.9 million new incident cases and 6.7 million deaths worldwide due to cancer in 2002.1 A total of 24.6 million persons are estimated to be alive with cancer (within 3 years of diagnosis). Leading causes of death from cancer, in order, are those of the lung (1.18 million), stomach (700,000), and liver (598,000). Most of these deaths were caused by metastatic disease, and they verify the fact that surgery and oncological therapy do not cure most patients with disseminated cancer. The incidence of cancer will continue to increase, especially in developing countries, largely due to smoking, infections, and lifestyles resembling those of people living in industrialized countries.1,2 Thus, intensified efforts are warranted, both to prevent cancer and to find new modalities of treating patients with metastatic cancer.
Tumor cells spread by lymphogenous and hematogenous routes into the systemic circulation to disseminate the disease. The importance of tumor lymphangiogenesis has lately been proven in studies where high levels in colorectal cancer specimens of lymphangiogenic peptides, and vascular endothelial growth factor (VEGF)-C and/or VEGF-D promote tumor lymphangiogenesis and metastasis.3 The sentinel node concept implies that the lymphatic drainage from a primary tumor first drains to a certain locoregional lymph node specific for each individual and that its tumor status is representative for the entire lymphatic field. The concept is established in staging for breast cancer4 and malignant melanoma5 and has also been evaluated in several other types of malignancies, such as colon cancer,6 ovarian cancer,7 pancreatic cancer,8 and urinary bladder cancer.9 The technique has been used as a diagnostic tool to improve staging and to tailor the extent of regional lymph node dissections. Probably most solid tumors disseminate first through the lymphatics and thereafter enter the systemic circulation via lymphovenous shunts present in the first draining lymph nodes.10–12 Approximately half of the lymphatic fluid entering a lymph node continues directly into the systemic circulation; therefore, lymphogenic spreading of the tumor cells may be mandatory for hematogenic dissemination.3,13
However, to our knowledge, only one study has previously evaluated lymphatic mapping in metastases where liver metastases were investigated.14 Because it has been proven that both metastases and primary tumors have the capacity to metastasize15–17 and induce lymphangiogenesis,18 we assume that lymphatic drainage from metastases may have an equal importance in metastases as the sentinel node concept in many solid primary tumors for correct staging. We have previously experienced surprises in lymphatic mapping within patients with colon cancer6 and urinary bladder cancer9 regarding primary tumor lymphatic drainage. We believe that a similar technique may be of use in surgery for liver metastases and other solid metastatic tumors for correct staging, and thereby for giving the patients the most appropriate therapy.
We previously showed that a sentinel node derived from a primary tumor contains specific tumor-reactive lymphocytes that have immunological reactivity toward the tumor in patients with colon cancer19 and in patients with urinary bladder cancer.20 Through the injection of Patent blue dye or radioactive tracer subserosally during surgery around the colonic tumor or transurethrally around the urinary bladder tumor, the sentinel nodes were found. A biopsy was performed, and the nodes’ immunological role and function were studied. After preparation, the lymphocytes were identified, isolated, and proliferated in vitro by stimulation of interleukins (ILs) and tumor antigen. Lymphocytes from sentinel nodes proliferated dose dependently and secreted interferon (IFN)-γ on stimulation with tumor homogenate.
The aim of the present study was to investigate lymphatic drainage from metastases and identify first draining lymph nodes. We also analyzed tumor-reactive immunological properties in lymphocytes derived from these lymph nodes.
MATERIALS AND METHODS
Patients
Nineteen patients (9 men and 10 women, average age 51.5 years) with metastases from solid tumors were included in the study. Patient characteristics are shown in Table 1. The operations were performed during the period of November 2003 to January 2007. The study was approved by the local ethical committee, and each patient provided informed consent.
Table 1.Patient characteristicsaPatientAge (y)SexPrimary tumorOrigin metinel nodeTracerNumber of metinel nodesMetinel node pos/neg for metastasesSuccessful expansionAliveFollow-up time (mo)148MColonIntra-abdominal local recurrencePB33 negYesYes38254MRectumLiver metastasisPB22 negYesYes38350MMal melanomaSubcutaneous metastasisPB22 posNoNo–477FColonLiver metastasisPB33 negNoNo–574MColonLiver metastasisPB21 posYesNo–1 neg666MColonLiver metastasisPB1MINoYes29751FOvarian cancerLiver metastasisPB21 posYesbNo–1 neg864FOvarian cancerGroin lymph nodePB/RT55 posNoNo–959FPancreatic cancerIntra-abdominal local recurrencePB4MINoNo–1039FMal melanomaGroin lymph nodePB/RT42 negNoYes111147MMal melanomaTruncal metastasisRT12 posNoNo–1245FBreast cancerCervical lymph nodePB/RT22 negYesYes51333FTongue cancerTruncal lymph nodeRT3MINoYes61464FColonIntra-abdominal local recurrencePB21 posYesYes41 neg1541FBreast cancerLiver metastasisPB22 negNoYes41639MMal melanomaCervical lymph nodePB/RT22 negNoYes21720MCholangiocarcinomaLiver metastasisPB33 negYesNo–1865MColonLiver metastasisPB31 posYesYes12 neg1942FLeiomyosarcomaLiver metastasisPB44 posYesYes1pos, positive; neg, negative; Mal, malignant; PB, Patent blue dye; MI, missing information; RT, radioactive tracer (4 × 10–15 MBq Tc-nanokolloid).a Data regarding the expansions for patients 1, 2, 5, and 6 also exist in unpublished data.b Patient died on the projected day of transfusion.
The lymph nodes that drained metastases were identified by injecting approximately 1 mL Patent blue dye (PB) (Guerbet, Paris) subserosally or subcutaneously in four places around one of the metastasis or in the tissues close to the local recurrences with a 27-gauge needle. After the injection of PB, the first draining lymph nodes turned blue within 3 to 10 minutes and were regarded as metastases-draining lymph nodes. In our previous work, a 10-minute cutoff was sufficient to identify sentinel nodes to primary tumors. We have experienced that during this period of time the first draining lymph nodes turn blue. Waiting longer means that a second echelon of draining lymph nodes may be colored blue, which does not represent the first draining lymph nodes. We arbitrarily applied the same 10-minute limit when detecting lymph nodes that drain metastases. These lymph nodes were either immediately removed or marked with sutures.
The sources of the liver metastases in our study were from colorectal cancer in five patients and in one patient each from ovarian cancer, breast cancer, cholangiocarcinoma, and leiomyosarcoma. Four patients had subcutaneous lymph node metastases from disseminated malignant melanoma, and one patient each had metastases from ovarian cancer, breast cancer, and squamous cell carcinoma of the tongue. Two patients had intra-abdominal local recurrences after surgery for colon cancer, and one patient had an intra-abdominal local recurrence after surgery for pancreatic cancer.
Because of these liver metastases, five patients underwent partial right-sided liver resections (patients 4, 5, 6, 15, and 17), and three patients underwent partial left-sided liver resection (patients 2, 18, and 19). The patient with metastases resulting from ovarian cancer (patient 7) had several large, bulky, cystic tumors, and surgeons performed an almost complete right-sided liver resection. In four cases, surgery was performed for solitary colorectal liver metastases (patients 2, 4, 5 and 6), and in the remaining five cases, tumor-reducing surgery was performed.
In six cases, a preoperative lymphoscintigraphy was performed by a subcutaneous injection of 4 × 10 to 15 MBq Tc-nanokolloid radioactive tracer in quadrants around the metastasis. The lymphoscintigraphies were performed to plan the surgical procedure by localizing the lymph node or nodes draining the metastasis. The position of the metastasis-draining lymph node or nodes was marked on the skin with a felt-tip pen. When the lymphoscintigraphy was not performed on the day of surgery, a new peritumoral injection of radioactive tracer was made at start of surgery. The draining nodes were intraoperatively identified by the use of a handheld gamma detection tube. In four of six cases, a simultaneous intraoperative injection of PB was performed.
Preparation of Specimens
Lymph nodes draining metastases and nondraining lymph nodes (for analytical purposes) were surgically removed and immediately taken to the laboratory on ice. In addition, one 5-mm slice of the whole metastasis (including the invasive margin) was also dissected and sent for analysis. Frequently, most of the lymph node or nodes were used for the expansion procedure. The remaining specimens underwent routine histopathological examinations with hematoxylin and eosin staining. At the laboratory, slices <1 mm were cut from the central and peripheral part of the lymph nodes for analysis by flow cytometry (FACS) and proliferation analyses. The metastasis was also analyzed by FACS, and a preparation of the whole tumor (homogenate) was used.
Immunological Evaluation
Single cell suspensions were made from the lymph nodes and metastases by gentle pressure with a loose-fitting glass homogenizer as previously described.19 Briefly, 1 × 106 cells were washed in phosphate-buffered saline containing 2% fetal calf serum and .05% NaN3, then stained with fluorophore conjugated antibodies toward the cell surface markers CD4, CD8, CD19, and CD56 (Becton Dickinson). Thereafter the cells were investigated with a FACSCalibur (Becton Dickinson).
For cell cultures, single cell suspensions of metastasis-draining lymph node–derived cells were resuspended in RPMI 1640 proliferation media containing 10% human AB serum (Sigma), 1% penicillin-streptomycin (Sigma), and 1% glutamine (Sigma). For preparation of the antigen source, the metastasis was homogenized by an Ultra-turrax homogenizer in 5 volumes (w/v) of 2 ×  phosphate-buffered saline, followed by 5 minutes’ denaturation at 95°C. This tumor homogenate was then used for in vitro activation and clonal expansion of lymphocytes. The metastasis-draining lymph node–derived lymphocytes were finally dispersed into cell culture flasks, at 2 × 106 cells/mL of proliferation media, supplemented with 100 IU of IL-2 (Proleukin, Chiron) per milliliter of culture every 3 to 4 days. Tumor homogenate was diluted to 1:10 and 1:100 and added to the cell cultures after 3 to 4 days. After 1 to 3 weeks, the cell cultures were restimulated by irradiated peripheral blood mononuclear cells from the patient. These cells were collected by Ficoll-Hypaque centrifugation19 and used as antigen-presenting cells for restimulation together with further addition of tumor homogenate. To verify clonal expansion of T lymphocytes, Vβ repertoire analyses were performed in a few cases. To test for functionality, single cell suspensions were investigated for Th1 and Th2 cytokine production, IFN-γ, and IL-4.
After an average period of 4 weeks, expanded cells were counted, evaluated by FACS, and investigated for the exclusion of malignant cells and bacteria before they were considered ready for transfusion. Cells were washed three times in .9% saline solution (Natriumklorid Baxter Viaflo 9 mg/mL, Baxter) containing 2% human serum albumin (Baxter) and prepared in a sterile erythrocyte transfusion bag at 4 × 106 cells/mL.
The transfusions took place at the surgical ward as an intravenous transfusion over the course of 1 hour. Our criterion for adoptive transfer is a transfusion of lymphocytes to a patient, regardless of the number of transferred cells. In our previous studies (M.K., unpublished data) of adoptive transfer originating from sentinel nodes, on average, 71 million clonally expanded autologous tumor-reactive lymphocytes were transfused to each patient, and in that pilot study, the disease of all 16 patients responded to therapy. We do not know the optimal number of cells to be transfused for the best or maximum immune response, but according to our experience, 39.5 million transfused cells (the average number of transfused cells in the metinel node study) may be sufficient. The explanation may be that the CD4 cells proliferate and promote division of effector T cells and memory T cells when they are stimulated by their antigen (which is derived from the tumor). The cell expansion continues in the patient.
RESULTS
Metinel Nodes
Lymph nodes draining liver metastases, intra-abdominal recurrences, and subcutaneous metastases were identified in all 19 cases, with an average number of 2.6 nodes found per patient (range, 1–5) (Table 1). We denoted these metastasis-draining lymph nodes metinel nodes. Seventeen (40%) of the analyzed metinel nodes were positive for metastatic disease. In 4 (25%) of 16 patients where data were available, all metinel nodes were positive for metastatic disease (range, 2–5), and in 8 (50%) of 16 patients, all metinel nodes (range, 2–3) were negative for metastatic disease.
Tracer was injected around liver metastases (n = 9), intra-abdominal recurrences (n = 3), or close to subcutaneous metastases (n = 7). Draining metinel nodes appeared visually blue (Fig. 1A) within 3 to 10 minutes after the injection of PB or were found to be disease positive by lymphoscintigraphy (Fig. 1B). No complications due to the lymph node mapping procedure were observed. The method seems valid for detecting the draining metinel node regardless of tumor type because the following were identified by this method: colorectal cancer (n = 7), malignant melanoma (n = 4), ovarian cancer (n = 2), breast cancer (n = 2), pancreatic cancer (n = 1), squamous cell cancer (n = 1), cholangiocarcinoma (n = 1), and leiomyosarcoma (n = 1). Thus, we conclude that lymph nodes draining liver metastases or local recurrences can readily be identified by this simple, safe, and quick procedure.
Fig. 1.(A) After intraoperative injection of Patent blue dye around liver metastasis in patient 18, the metinel node in the hepatoduodenal ligament is blue. (B) Preoperative percutaneous injection of 4 × 15 MBq Tc-nanokolloid around ovarian cancer groin local recurrence (patient 8) demonstrates a medially and distally situated metinel node after 10 minutes.
In all cases of surgery for liver metastases, lymph nodes draining the area of the metastases were found within the liver hilum or hepatoduodenal ligament. The average number of metinel nodes from liver metastases were 2.4, and 7 (32%) of 22 of the analyzed metinel nodes were positive for metastatic disease. Two patients underwent surgery to treat intra-abdominal local recurrences from colon cancer (patients 1 and 14); resections of the recurrence and reresection of the bowel en bloc were performed. One patient with pancreatic cancer (patient 9) underwent tumor-reducing surgery to treat a local intra-abdominal recurrence after a previous Whipple operation.
Four patients underwent surgery, with lymph node biopsies performed, to treat subcutaneous metastases from disseminated malignant melanoma (patients 3, 10, 11, and 16), and one patient each underwent surgery to treat subcutaneous metastases from disseminated breast cancer (patient 12), groin lymph node metastasis due to ovarian cancer (patient 8), and subcutaneous metastases related to squamous cellular carcinoma of the tongue (patient 13) (Table 1).
Immunology
We previously demonstrated the presence of tumor-reactive T lymphocytes in primary tumor-draining sentinel nodes from patients with colon19 and urinary bladder cancers.20 Here, we investigated whether signs of immune recognition also occurred in metinel nodes draining metastases. Single cell suspensions were collected from identified metinel nodes and investigated by flow cytometry for the presence of CD4+, CD8+ T lymphocytes, CD19+ B lymphocytes, and CD56+ natural killer (NK) cells (Table 2). Most of the metinel nodes were predominated by CD4+ T helper cells, with an average CD4+/CD8+ ratio of 2.3 (range, .1–6.6). However, five patients (patients 3, 6, 11, 12, and 19) displayed an increase in the fraction of CD8+ cytotoxic T cells. Two of these patients had malignant melanoma. The metinel nodes contained an average of 9.5% (range, .1–55) CD19+ B lymphocytes and an average of 2.1% (range, 0–18.9) CD56+ NK cells.
Table 2.Immunology of single cell suspensions collected from metinel nodesPatientTime in culture (d)No. of cells at start (millions)No. of cells at end (millions)Ratio of cells start/endStart (% gated), ratio CD4/CD8 and CD19/CD56End, ratio CD4/CD8 and CD19/CD56Ratio CD4/CD8 startRatio CD4/CD8 end13110080.824/11 and 55/−24/16 and −/−1.81.52334240.9551/15 and 13/−24 and .12/−3.41.5328–––8/39 and 1/−–.2–42065.4––37/9 and −/−–4.1–543130–1.347/7.1 and 28/1.373/22 and .09/.176.63.3665––.2/1.6 and .4/.4–.125–765.7.0216.4/6.5 and 24.2/1.6–2.5–85242.6––15.9/7.6 and 9/3.4–2.1–91512––––––1074166.85.0340/7 and .5/.3–5.7–11352091409.312.8/16.3 and 1.9/0–.8–12312852.0116.4/31.8 and .1/0–.5–13243––––––1432301.05––––154836––0/.1 and 0/0–0–164520––28.25/6.95 and 1.15/0–4.1–1720894.045.8/2.6 and 4.2/.234.2/30.4 and 7.6/20.12.21.11829674.069.7/3.8 and 1.3/.1–2.6–194311151.3612.7/15.8 and 3/18.985/3 and .9/8.4.828.3
CD4+ cells are crucial for initializing both the cellular and humoral immune response toward, for example, tumors. CD4+ cells recognize antigens expressed on major histocompatibility complex II and have the capacity to catalyze the immune response in B cells, Antigen presenting cells (APCs), and CD8+ cells. CD4+ cells have, compared with CD8+ cells, a long-term capacity of acting both as effector T cells and as memory cells. CD8+ cells are mainly effector cells and will act during a few weeks, but further recruitment of CD+8 cells is maintained by CD4+ cells. The expansion procedure aims to increase the number of CD4+ cells, and the CD4+/CD8+ ratio can be regarded as a measurement of how well this can be achieved. The average ratio of CD4+/CD8+ cells at the start of the procedure was 2.6 for patients who underwent successful expansions and 2.1 for patients who underwent unsuccessful expansions. Consequently, the CD4+/CD8+ ratios were 24% higher for patients with successful expansions.
To test for recognition of tumor antigen, the number of metinel node–acquired lymphocytes from nine patients (patients 1, 2, 5, 7, 12, 14, 17, 18, and 19) were increased successfully by the addition of low dose IL-2 and tumor homogenate. An average of 97 × 106 cells (range, 5–285 × 106) were collected, and the cells were expanded on average for 32 days before transfusion. At the time of transfusion, a mean of 39.5 × 106 tumor-reactive T cells (range, .7–170 × 106) were returned (Table 2). In patient 7, cells were expanded and prepared for transfusion, but the patient suddenly died without receiving any cells. No side effects from transfusion of expanded T lymphocytes were seen, and the patients were discharged from the hospital on the same day of the transfusion. The expansions were successful in five of seven patients who had colorectal primary tumor; one of two in patients with ovarian cancer and breast cancer; and one patient each with cholangiocarcinoma and leiomyosarcoma. The following expansions failed in all patients: malignant melanoma (n = 4), pancreatic cancer (n = 1), and squamous cellular cancer (n = 1). An analysis of the CD4 Vβ repertoire from patient 11 was investigated by flow cytometry of metinel node–acquired lymphocytes (Fig. 2) and revealed an increase and clonal expansion of Vβ families 7.1, 13.2, and 20. In addition, analysis of the Vβ repertoire in two independently cultured metinel nodes derived from one breast cancer metastasis (patient 12) (data not shown) demonstrated clonal expansion after 12 days’ short-term culture of the same T cell receptor families in both nodes (Vβ 4, Vβ 9, Vβ 20, and Vβ 21.3). We conclude that cultures of metinel node–acquired lymphocytes expand in the presence of autologous tumor extract and IL-2.
Fig. 2.In patient 13, the T cell receptor Vβ repertoire was investigated by flow cytometry in two sentinel (nodes at the day of operation and after in vitro cell culture (43 days). Clonal expansion of Vβ families 7.1, 13.2, and 20 were detected in the CD4+ T cell population.
Cells from patient 8, stimulated at the end of the expansion, demonstrated a high production of IFN-γ of >1000 pg/mL (Fig. 3) and no detectable IL-4 (data not shown), indicating a Th1 response. In an additional patient (patient 5), the stimulated IFN-γ secretion was 155 pg/mL while only a low IL-4 production of 30 pg/mL was found, again demonstrating a Th1-predominant response pattern.
Fig. 3.Amount of interferon gamma secreted into the supernatant measured by enzyme-linked immunosorbent assay. Data presented are for patient 7 after 35 days of in vitro cell culture.
According to more detailed analyses of some cases, we found that some T cells expressed Fox P3, indicating that these cells in fact were T regulatory cells.
DISCUSSION
We show that it is possible to find the first lymph nodes draining various types of metastases by lymphatic mapping by means of dye or radioactive tracers. We have named these metastasis-draining lymph nodes “metinel nodes” to show both the analogy and difference to the well-known term “sentinel node,” which means the first node to receive lymphatic drainage from a primary tumor. In 19 studied cases, we demonstrated three different ways of identifying these nodes by injection of tracers subserosally around liver or intra-abdominal metastases, close to subcutaneous metastases, or into local lymph node metastases.
The fact that it is possible to expand the metinel node–derived lymphocytes via in vitro expansions during several weeks of time, together with our analysis that they contain lymphocytes that show clonal expansion toward tumor antigens and produce high levels of IFN-γ in the expansions, are evidence that they contain tumor-reactive lymphocytes. These cells are mainly T-helper 1 cells that have developed an immunological response toward the metastatic cells. We suggest that these T cells may be used in future trials of adoptive cellular therapy of disseminated solid cancer.
Studies have demonstrated that tumor growth beyond a certain size required angiogenesis21 and that the amount of neovascularization peritumorally was a predictor of metastatic disease.22 Far less is known about the specific factors regulating the lymphangiogenesis around a tumor and its possible importance, much as a result of the difficulty of discriminating between lymphatics and blood vessels. However, during recent years, the molecular pathways signaling for lymphangiogenesis have been described, and these studies have revealed that lymphangiogenesis is a major component of the metastatic process.
The lymphatic capillaries consist of thin-walled, low-pressure vessels that drain the interstitium. When the surrounding interstitial fluid pressure increases, the walls of the lymphatics are stretched and open to passage of cells or fluids. The lymphatic capillaries are joined into major lymphatic collecting vessels and filtered through at least one lymph node before entering the venous circulation.23 After entering the node, approximately half of the lymphatic fluid continues directly into the blood before the rest drains into an efferent lymphatic vessel.24
A malignant tumor causes a peritumoral increase of lymphatic vessels mediated through VEGF. Studies have shown that high levels of the cytokine cascade of VEGF-C and VEGF-D promote the tumoral lymphangiogenesis and that inhibition of their joint receptor, VEGFR-3, suppresses the effects. Kawakami et al.25 showed that high levels of VEGF-C and its receptor, VEGFR-3, correlated with increased lymph node metastases and lymphatic invasion in human colorectal cancer. White et al.26 concluded that patients operated on for colon cancer had an overexpression of VEGF-D, but not of its receptor VEGFR-3, which correlated with a decrease in disease-free interval and overall survival. A study based on 110 patients with breast cancer showed that lymph vessels were present more often in metastatic axillary lymph nodes (85%) than in nonmetastatic lymph nodes (25%),18 and that intranodal and perinodal lymphatic endothelial cell proliferation fractions were higher in metastatically involved lymph nodes. Taken together, all these findings support lymphangiogenesis in metastases.
Thus, a tumor causes an upregulation of lymphangiogenic factors that leads to an increase in peritumoral lymphatic vessels in both tumors and metastases. It is likely that these newly developed lymph vessels connect to the preexisting lymphatic vessels and lymph nodes. Shed tumor cells may invade these newly formed vessels, and the dissemination of tumor cells is facilitated. The present study shows that this process can be visualized by lymphatic mapping, and the first draining lymph node from the metastasis, the metinel node, may be identified.
Animal studies showed that metastases had the capacity to metastasize. Mice inoculated with pulmonary venous blood from mice having pulmonary metastases developed disseminated cancer disease after injection into the tail vein, showing that pulmonary metastases shed viable circulating cells.16 Similarly, Hoover and Ketcham15 demonstrated that mice who had undergone amputation of their primary tumors and now only had pulmonary metastases experienced spread of the tumor cells specifically into the lungs after parabiosis.
In surgery for colorectal liver metastases, it is not standard to perform a biopsy of perihepatic lymph nodes. Nakamura et al.27 reported a 4-year survival of 40% in node-positive patients, but the study cohort was small. Most studies identify few 5-year survivors after liver resection for colorectal liver metastases involving positive perihepatic lymph nodes.28 These lymph node metastases are unlikely to be of lymphatic origin from the primary colonic tumor; instead, they may be explained by hematogenous spread to the liver followed by lymphogenic spreading from the liver metastases to the perihepatic lymph nodes.29 Correct perihepatic lymphatic staging is beyond dispute, not only because the increased morbidity in patients having perihepatic lymph node metastases, but also because of the improvements in systemic therapies. In the nine cases of resected liver metastases in our study, all metinel nodes were found among perihepatic nodes within the hepatoduodenal ligament or liver hilum. Most of the liver is drained by deep hepatic lymphatics, the upper inner part of the liver drains to nodes around the end of inferior caval vein, and the lower inner part drains to hepatic nodes through the liver hilum.30 The superficial part of the liver lymphatic drainage is divided into two main directions; the posterior surface drains into pericaval nodes, and the inferior, anterior, and superior surface drains into hepatic nodes through the liver hilum. In all cases of resected liver metastases, the injections of PB were performed subserosally on the inferior, anterior, or superior surface of the liver. Our identified perihepatic metinel nodes were located corresponding to the superficial lymphatic drainage of the liver.
Despite the prognostic importance of these perihepatic lymph nodes, we have only found one article in the literature describing lymphatic mapping originating from liver metastases. Kane et al.14 demonstrated in a study of 11 patients with colorectal liver metastases that it was possible to perform lymphatic mapping by the intraoperative injection of isosulfan blue dye in a peritumoral and subcapsular manner. The basic premise was that the occurrence of extrahepatic metastases within the normal lymphatic drainage of the liver was considered to be remetastases from the colorectal metastases. They found blue-colored nodes in seven patients (54%), all within the liver hilum, falciform ligament, or gallbladder fossa. In three of these patients, the nodes were not detected by the surgeon before the mapping procedure. Like our results, they concluded that hepatic lymphatic mapping was safe, rapid, and simple technique to perform in humans.
The importance of lymphatic mapping in liver surgery for metastases was underlined by Grobmyer et al.,31 who concluded that routine lymph node sampling had a low yield in patients without suspected perihepatic disease by computed tomographic scan or positron emission tomography, or at surgical exploration. Further, another study demonstrated that microscopic perihepatic positive lymph node disease is associated with the same poor prognosis as in macroscopic positive disease and suggests, therefore, that lymph node dissection should be systematic.32
Lymphoscintigraphy has been used to find sentinel nodes from primary tumors,33 but to our knowledge, it has not been reported as a tool to locate lymph nodes draining subcutaneous metastases. The radioactive tracer facilitated the intraoperative identification of the metinel nodes by a gamma probe used as a complement to PB. In those cases, the lymphoscintigraphy was made days before surgery, and new injections of radioactive tracers were provided on the day of surgery. One to three metinel nodes were detected in our six patients.
In the late 1950s, the immunosurveillance hypothesis was introduced34 supporting the fact that tumor cells possessed new antigenic potentials and provoked an immunological reaction that could eradicate a tumor completely. In the last decade, the hypothesis has been proven in mice and humans. The role of the immune system in cancer diseases is demonstrated by immunodeficient patients having both an overrepresentation of virus-induced malignancies and an increased probability of developing cancer of nonviral cause. Cardiac transplant recipients have, for example, been shown to have a 25-fold higher prevalence of lung tumors than the general population.35 Much data also supports the fact that both the cellular and the humoral immune systems develop immune responses against tumors. Bevacizumab, a monoclonal antibody against VEGF, functions as an inhibitor of angiogenesis, inhibits growth of human tumor cells in mice, and prolongs the overall median survival in human metastatic colorectal cancer.36 The presence of tumor-reactive lymphocytes in sentinel nodes19,20 is an example of the function of the cellular immune system and a positive prognostic factor for survival in colorectal and ovarian cancer.37–39
In our earlier study, we made a single cell suspension of the lymphocytes from sentinel nodes. They were expanded in vitro, stimulated by tumor homogenate, IL-2, and antigen-presenting cells. In the present expansions, we proved it was possible to find tumor-reactive lymphocytes in metinel nodes and to perform adoptive immunotherapy based on them. The preliminary results seems promising, with total or partial regression of some liver and lung metastases (M.K., unpublished data). In this study, the expansions were performed in exactly the same way as in our previous studies, except that the source of the lymphocytes was derived from lymph nodes draining metastases—that is, metinel nodes. The fact that we succeeded in nine cases in proliferating the lymphocytes via in vitro expansions over more than a month is evidence for the presence of tumor-reactive lymphocytes. Because the expansions were continuously stimulated with autologous tumor homogenate and antigen-presenting cells together with IL, only lymphocytes that were a priori stimulated by antigens derived from the tumor could proliferate and survive for such a long time. Figure 2 demonstrates the Vβ repertoire in CD4-positive cells in two metinel nodes in patient 11, thus showing which types of T-helper cell receptors were found at the end of expansion. The figure shows clonal expansion of Vβ 7.1, 13.2, and 20, demonstrating the presence of tumor-reactive lymphocytes toward three different types of tumor antigens.
These collective facts point out the presence of tumor-reactive type 1 T-helper lymphocytes in metinel nodes. T helper lymphocytes play a central role in initiating the adoptive immune defense. In 9 of 19 patients, the cells survived until the time for transfusion, and 8 patients underwent intravenous adoptive autologous T cell transfusion. Approximately half of all patients who underwent the expansion procedure died as a result of a combination of too few cells at the start and the initial presence of immunosuppressive factors that not could be overcome. Six of eight patients who received transfusions are still alive, although the follow-up time varies (Table 1). Our results are encouraging because the prognosis in these type of cases is usually poor.
In this study, we have shown that it is possible to find the draining lymph nodes from different types of metastases. We named these nodes “metinel nodes,” and they contain tumor-reactive lymphocytes, preferentially T-helper cells with Th1 response. We have also demonstrated that it is possible to proliferate these cells in vitro and, if the expansion procedure is successful, to use the cells for adoptive immunotherapy.

metastases
lymphatic mapping
immunotherapy
sentinel node
solid tumors


Tonsillar metastasis of oesophageal adenocarcinoma

In the literature less than 100 cases of metastatic carcinoma of the palatine tonsil have been reported. Tonsillar metastasis of adenocarcinoma of the oesophagus has not been reported previously. We report a case of a 57-year-old male with a primary adenocarcinoma of the distal esophagus with a metastasis in the right palatine tonsil. Metastatic tumour involving the palatine tonsil is rare. The route of dissemination remains unclear. Hypothetically the dissemination of tumour cells could be lymphogenic or secondary by transportation due to vomiting or at the time of endoscopy, but most likely represents haematogenous spread.
Introduction
The vast majority of cancers in the head and neck region are of epithelial origin. Lymphoma is the next most common malignancy in this area. Metastases in the head and neck other than lymph node metastases are rare.
The occurrence of tonsillar metastases from non-haematological malignant neoplasms is very rare [10]. We present a patient with metastasis in the right tonsil of a primary adenocarcinoma of the distal oesophagus.
Case report
A 57-year-old man was referred to our department of otolaryngology and head and neck surgery, because of dysphagia, pain and swelling of the right palatine tonsil. He also complained of frequent vomiting. Five months previously, an adenocarcinoma of the distal oesophagus with lymph node and liver metastases had been diagnosed. He received chemotherapy with combined capecitabine and oxaliplatin, which was stopped after two cycles because of disease progression. On physical examination an enlarged and ulcerating right palatine tonsil was seen (Fig. 1). A suspicious ipsilateral cervical lymph node of 2 × 4 cm was found at level II. A biopsy of the tonsillar tumour was performed and revealed intestinal-type adenocarcinoma. The histopathological picture corresponded with that of the oesophageal tumour (Fig. 2). Tonsillectomy was not performed because of severe clotting disorders attributed to liver metastases. The patient died 4 weeks after his first visit to our department.
Fig. 1Enlarged right palatine tonsil with ulcerationFig. 2Biopsy right tonsil: adenocarcinoma (intestinal type)
Discussion
Second primary tumours relatively often involve the head and neck region. These tumours develop in head and neck cancer patients due to their common factors in carcinogenesis, e.g., tobacco, alcohol and, probably, genetic susceptibility [4]. However, this common aetiology applies for squamous cell carcinoma and not for adenocarcinoma. Simultaneous primary adenocarcinomas are not frequently found. In our patient the corresponding histopathology indicates a common origin, suggesting metastatic disease.
Primary oesophageal adenocarcinoma is a relatively uncommon tumour in western countries [9]. The pattern of metastasis of oesophageal adenocarcinoma can be lymphogenic, haematogenic and by transportation of tumour cells by regurgitation or endoscopes [2, 10]. Lymphogenic spread of distal oesophageal adenocarcinoma generally involves the regional nodes in the lower posterior mediastinum, the paracardiac region, along the lesser curvature and the left gastric artery [6]. In case of extensive lymphogenic spread, lymph-node metastases can be found in the tracheal bifurcation region, the proximal mediastinum, coeliac axis and supra-clavicular region [6]. Lymphogenic spread of adenocarcinoma of the distal oesophagus and the gastro-oesophageal junction is present in 70% of the patients at the time of diagnosis of the primary tumour [9].
Haematogenous dissemination occurs at a later stage and is less frequent. Liver and lung are the most common organs involved [6].
Metastatic tumour involving the palatine tonsil is distinctly rare. These metastases represent only 0.8% of all tonsillar tumours [5]. Bilateral metastases of gastric carcinoma to the tonsils have been described [7]. Less than 100 cases of metastatic carcinoma of the palatine tonsil have been reported [3, 7]. The most frequent tumours metastasizing to the tonsil are melanoma, lung carcinoma, breast carcinoma and renal cell carcinoma [3]. Eleven cases were associated with gastric adenocarcinoma [2, 7, 8]. Tonsillar metastasis of adenocarcinoma of the esophagus has not been reported previously. The occurrence of a tonsillar metastasis as the first manifestation of an occult neoplasm is very unusual [8]. In the majority of cases the occurrence of a tonsillar metastasis becomes manifest after the diagnosis of the primary tumour and is generally part of a widespread systemic disease [2]. Therefore, mean survival time for patients with tonsillar metastases is short, with a mean of 9 months, irrespective of the histological type of primary tumour [3].
In this case there are four hypothetical pathways for metastatic spread to the tonsils: Haematogenous dissemination of tumour from an esophageal carcinoma may occur via the portal circulation, the liver, the pulmonary circulation and systemic arterial vessels, thus reaching the palatine tonsil. A second haematogenous route may be through the paravertebral plexus of Batson, bypassing the lungs [1]. Retrograde flow via this valveless plexus occurs during elevated intra-abdominal and intrathoracic pressure, as is brought about by straining, coughing or as in this case, vomiting [1]. A third route of metastasis may be through the thoracic duct followed by retrograde cervical lymphatic spread to the tonsil [3]. This route of dissemination is very rare and is mostly seen in cases of seminomas [3]. Finally, some authors believe the possibility of metastasis by direct translumenal implantation, which might be favoured by a previous lesion in the mucosa of the tonsil [2]. The tumour cells would be transported secondary to regurgitation from the oesophagus [8], or as in this case possibly because of frequent vomiting, or at the time of endoscopy [2].
In conclusion, when finding a tumour of the tonsil a metastasis from a distant primary malignancy should be considered, although this is very rare. The route of dissemination eventually remains unclear.

tonsil
metastasis
adenocarcinoma
oesophagus
head and neck