EDITORIAL

Does diabetes therapy influence the risk of cancer?

U. Smith & E. A. M. Gale

# Springer-Verlag 2009

Keywords Cancer . Insulin analogues . Insulin therapy .

Metformin . Type 2 diabetes

AbbreviationsAMPK AMP-activated protein kinaseFDA Food and Drug AdministrationHMEC Human mammary epithelial cellMAPK Mitogen-activated protein kinasemTOR Mammalian target of rapamycinNPH Neutral protamine HagedornTHIN The Health Information Network

Introduction

Type 2 diabetes is associated with three of the five leadingcauses of cancer mortality in the USAcarcinoma of thecolon, pancreas and breast (postmenopausal) [1]. Theexcess risk for each cancer is ~30% (colon), ~50%(pancreas) and ~20% (breast) [24]. Type 1 diabetes carriesan excess cancer risk of ~20%, but involves a differentrange of tumours [5]. The major cancers linked with type 2diabetes are also associated with obesity or insulinresistance, suggesting that factors other than glucose play

an important role [6]. These observations, although well-attested, have attracted relatively little interest within theworld of diabetes. This is partly due to the dominant role ofcardiovascular disease, which largely accounts for thetwofold increase in mortality associated with type 2diabetes [7], and partly, perhaps, because cancer hasseemed unavoidable.

The latter assumption can no longer be consideredcorrect, for several studies have shown metformin to beassociated with a lower risk of cancer than insulin orsulfonylureas [810]. Bowker and colleagues examined therelationship between diabetes treatment and mortality in ahealth database from Saskatchewan, and found that cancermortality was almost doubled among insulin users (HR 1.9,95% CI 1.52.4, p

various cellular signals from growth factors, nutrition andenergy state to regulate protein synthesis and cell growth.Rapamycin, the inhibitor of mTOR, and its derivatives havebeen tested in several cancer trials with some success. Astudy of human prostate cancer cells demonstrated a stronganti-proliferative effect of metformin [13]. This effect wasunaffected by inhibition of the AMPK pathway, but wasassociated with cell cycle arrest in G0/G1 phase, togetherwith a major reduction in cyclin D1 levels.

Another interesting mechanism for the anti-oncogeniceffect of metformin has been postulated, based on the findingsof a study of mice with CD8+ T lymphocytes which lacktumour necrosis factor receptor-associated factor 6 (TRAF6)and are unable to generate T memory cells [14]. This failurewas associated with defective fatty acid oxidation. Metforminrestored both the metabolic defect and generation of memoryT cells. A further experiment showed that metformintreatment increased CD8+ T memory cell populations inwild-type mice, and enhanced the efficacy of anti-cancervaccination. These intriguing findings indicate a sharedmitochondrial nexus for metabolic and immune pathways,and imply that metformin may also have a direct influenceupon immune competence [14].

The welcome news, therefore, is that metformin use isassociated with a lower risk of some types of cancer, and mayeven find a role in the management of cancer in non-diabeticindividuals. This does not alter the fact the type 2 diabetes isassociated with an excess cancer risk, and that diabetestherapies that increase levels of circulating insulin mightpotentially contribute to this risk. More specifically, there isconcern that high insulin levels and associated changes in theIGF-1 axis may accelerate the progression of existing cancerfoci. Insulin treatment of type 2 diabetes was, for example,associated with a twofold increase in the risk of colorectalcancer, compared with other therapies, in an analysis thatadjusted for prior use of metformin or sulfonylureas [15]. Thesame analysis reported that cancer risk increased by anestimated 20% for each year of insulin therapy. The paper byCurrie et al. in this issue of the journal confirms an associationbetween insulin therapy and colon cancer, and suggests thatsulfonylureas may also carry an equivalent risk [10].

Insulin and cancer

The possibility of an association between insulin and cancerhas attracted intense research interest among cancerepidemiologists, since cancers of the colon, breast andpancreas have all been associated with increased circulatinglevels of endogenous insulin in the non-diabetic population[6, 16]. This might explain some of the overlap betweencancer risk in diabetes, obesity and other conditionsassociated with insulin resistance [6]. There is a possible

mechanistic basis for these epidemiological findings, in thatinsulin is a growth factor for a number of epithelial tumoursin cell culture systems, and hyperinsulinaemia also produ-ces a secondary increase in the availability of IGF-1another known tumour growth factorwhich is mediatedby a reduction in IGF binding protein-1 levels (IGFBP-1).These changes in the insulinIGF-1 axis might be expectedto favour the survival and progression of early malignantfoci [6, 17]. Tumour cells must overcome no fewer than sixroadblocks to progression before a malignant growth canbecome established. These are the acquisition of self-sufficiency in growth signals, insensitivity to growth-inhibitory signals, evasion of programmed cell death(apoptosis), limitless replication potential, sustained angio-genesis, and loss of barriers to tissue invasion [18]. Changesin the insulinIGF-1 axis have the potential to support self-sufficiency in growth signals and resistance to apoptosis, andmay thus offer an adaptive advantage to cancer foci strugglingto survive in a hostile environment [17].

The insulinIGF-1 axis

Insulin and IGF-1 are sister molecules that share a commonancestry but diverged early in vertebrate evolution, sincewhen they have co-evolved with their receptors to subservedistinct metabolic or trophic functions. The insulin and IGF-1receptors are tetrameric members of the receptor tyrosinekinase family that are composed of two extracellular domains and two intracellular domains and share consid-erable sequence homology. The metabolic consequences ofreceptor stimulation are mediated by the phosphorylation ofIRS proteins and activation of the phosphatidylinositol 3-kinaseAkt/protein kinase B pathway (the Akt pathway);activation of this pathway promotes the familiar metaboliceffects of insulin, although the Akt pathway may also transmitgrowth signals. The growth-promoting consequences ofreceptor stimulation are more generally mediated by theRasmitogen-activated protein kinase (MAPK) pathway (theRas pathway), which promotes cell growth and differentiationby regulation of gene expression [19].

Readers unfamiliar with this system might assume thatsignal specificity is intrinsic to the molecular interaction ofthese receptors and their ligands, as predicted by thetraditional lock-and-key model. The reality is more com-plicated, for in certain contexts the insulin receptor cantransmit mitogenic signals and the IGF-1 receptor cantransmit metabolic signals. This is because the insulinIGF-1 axis functions within a dense and highly flexiblesignalling network, and differences in signalling specificitymay vary with the target tissue, the density and spatiallocalisation of receptors, the kinetics of ligand binding, anddownstream modulation of post-receptor signalling, all of

Diabetologia

which contribute to this remarkable functional plasticity,quite apart from any variation in the ligand itself [20].

A whole new dimension is superimposed upon this pre-existing complexity when a cell undergoes malignanttransformation, forconsistent with the dictum that on-cology recapitulates ontogenytumour cells can re-acquire signalling capabilities that are otherwise only seenin the early stages of development. These include a variantform of the insulin receptor known as IR-A, which isabundantly expressed in both fetal and cancer tissues and isresponsive to IGF-2 as well as to insulin [21]. Cancer cellsmay thus overexpress not only the insulin and IGF-1receptors, but also IR-A and hybrid receptors formed byrecombination of their constituent proteins.

In summary, tumour cells equipped with this aberrantsignalling capacity may become dependent upon the trophiceffects of insulin and/or IGF-1, thus accelerating their owngrowth and acquiring increased resistance to apoptosis [22].Overexpression of this network of proteins is commonlyobserved in cancers of the colon, breast, pancreas andprostate. It should, however, be appreciated that cancersarise from multiple fortuitous mutations, and are thereforeheterogeneous in the extreme. Some in vitro cancer celllines are much more responsive to insulin and IGF-1signalling than others. The reported effects of insulin ontumour cell lines are highly variable, as we will shortly see,and differences between cell lines, experimental conditionsand concentrations of insulin must be taken into accountwhen interpreting results from such reports. This havingbeen said, there is abundant evidence that insulin can, insome circumstances, promote the growth of both healthyand malignant cell lines in culture systems.

Cancers take many years to develop, and it is thereforesurprising that studies such as those reported in this issue ofDiabetologia [10, 2325] can claim to detect differences incancer rates within a few years of exposure to differenttherapeutic agents. These observations, if confirmed,strongly suggest that the effects we are witnessing arisefrom differences in the rate of development of pre-existingmalignant foci rather than malignant transformation andnew cancer cell formation. This inference is consistent withthe observed effects of insulin on cells in culture. Furthersupport for this view comes from autopsy studies showingthat a high proportion of people in an ageing populationharbour early cancers. Prostate cancers, for example, arepresent in some 50% of 70-year-old men and in 80% ofthose over the age of 90 [17].

Insulin analogues and cancer

On 9th April 2008, Pfizer (New York, NY, USA) issued aDear Healthcare Professional letter to the effect that

inhaled recombinant human insulin had been associatedwith six new cases of lung cancer in clinical trials, with onefurther post-marketing report in a patient treated withExubera. A single case had been reported in comparator-treated patients, and all cases had a history of prior cigarettesmoking [26]. The company stated that this sixfold increasein risk (0.13/1,000 cases vs 0.02) did not prove a causalconnection, but the observation may have helped to motivatethe precipitate removal of Exubera from the market on 17thOctober 2007 [27]. The effects of massive local concen-trations of insulin in the lung cannot, however, be extrapo-lated to the safety or otherwise of subcutaneous insulin.

Three potentially relevant observations provide theessential background for any discussion about insulintherapy and cancer: (1) the epidemiological finding thatinsulin concentrations within the physiological or therapeu-tic range are associated with the rate of tumour diagnosis;(2) laboratory findings that the intrinsic mitogenicity ofinsulin may vary according to the functional plasticity ofthe insulinIGF-1 signalling network, particularly in tu-mour cells; and (3) the demonstration that some tumour celllines are responsive to changes in the ambient concen-trations of either or both these ligands. Concerns that theinsulin analogues might be associated with an increasedrisk of tumour progression [28, 29] must be appreciatedagainst this complex background.

The introduction of biosynthetic human insulin openedthe way for designer insulins modified for faster or moresustained effects following injection, and it soon becameapparent that some alterations of the insulin moleculeincrease its trophic effects, as demonstrated by acceleratedDNA synthesis and cell division in cell culture systems,typically human mammary epithelial cells (HMECs). Theseeffects are mediated by prolonged binding to the insulinreceptor, or by increased cross-reactivity with the IGF-1receptor [30, 31], and all new insulins are routinelyscreened for their effects on cell growth in the course ofpreclinical evaluation. Insulin B10Asp, the first of theanalogues to be developed, was based on a single aminoacid substitution. This was, however, sufficient to produce atenfold increase in mitogenicity, compared with humaninsulin. In the light of this observation, the regulatoryauthorities required 2 year carcinogenicity studies inrodents, as against the standard 1 year toxicity testing,and the insulin was withdrawn when mammary tumoursappeared in rats [32].

Further experience revealed that the insulin and IGF-1receptors recognise the terminal part of the insulin B chainand extensions into the C chain differently. Modification ofB26B30 regions of the B chain increases IGF-1 receptorbinding, as does modification of the B10 residue andextension of the B chain by addition of arginine residues[33]. Changes at both sites have additive effects, in that

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AspB10DiArg insulin, used for experimental purposes only,produces a 90-fold increase in binding to the IGF-1 receptoron HMECs. Insulin glargine (A21Gly,B31Arg,B32Arghuman insulin) also contains arginine residues at positionsB31 and B32, together with a glycine substitution at A21;insulin aspart (B28Asp human insulin) carries a B28Aspsubstitution, and in insulin lispro (B28Lys,B29Pro humaninsulin) the sequence of proline and lysine residues at B28/B29 in human insulin are reversed. Insulin detemir, B29Lys(-tetradecanoyl),desB30 human insulin carries a fatty acylchain attached to the end of the B chain. The ability ofanalogues to stimulate HMEC growth generally correlateswith their ability to bind to the IGF-1 receptor, butprolonged interaction with either receptor also appearsnecessary for stimulation of mitotic activity [30].

Kurtzhals and colleagues used a variety of systems,including human osteosarcoma cells, to compare receptoraffinities and mitogenic potencies of the insulin analoguesin current clinical use, and found that insulin glargine has asix- to eightfold increase in receptor affinity and mitogenicpotency compared with human insulin [33]. Sanofi-aventishad previously observed a similar increase in mitogenicactivity in osteosarcoma cells [28]. In contrast, the twoshort-acting analogues were reported to resemble humaninsulin in most respects other than a slight increase in IGF-1 receptor affinity for insulin lispro. Insulin detemir hadreduced metabolic and mitotic potencies in vitro comparedwith human insulin, presumably because it carries a fattyacyl chain, which might be expected to interfere withreceptor binding, but is, for technical reasons, difficult tocompare with other insulins in such systems [33].

As indicated in the previous section, insulin analogueshave been tested in tumour cell lines with variable results.A pancreatic cancer cell line responded similarly to insulinglargine and human insulin, and survival of insulinglargine-treated patients following treatment for pancreaticcancer did not differ from that of patients on insulin orcontrols [34]. In another study, colorectal, breast andprostate cell lines showed proliferative changes andincreased resistance to apoptosis in response to exposureto pharmacological doses of insulin glargine, insulindetemir and insulin lispro, but not to human insulin [35].Another recent paper tested the effect of exposure to insulinanalogues on a panel of neoplastic and non-neoplasticmammary epithelial cell lines. Preliminary screening for theinsulin/IGF-1 receptors indicated that growth of themalignant cell line MCF7 was strongly promoted by insulinglargine, but not by other insulins, at dosage levels in thepicomolar range, comparable to those found in thecirculation of insulin-treated patients. This effect wasstrongly linked to activation of the IGF-1 receptor and theMAPK pathway. Other cell lines carrying insulin/IGF-1receptors did not respond in this way [36].

Insulin glargine is partially degraded at the injection site,yielding two bioactive products known as M1, which lacksthe diarginine residues at B31 and B32, and M2, which hasadditional deletion of the threonine at B30. Both productsretain the glycine substitution for asparagine at A21. Theseare therefore closely similar to, but not identical with,human insulin [37, 38], and their mitogenicity appears to below [38]. All three forms (unchanged insulin glargine, M1and M2) enter the circulation. Further degradation ofinsulin glargine to M1 occurs on exposure to serum,probably mediated by carboxypeptidase enzymes [38].These observations suggest that insulin glargine behavesto some extent as a prodrug, generating bioactive break-down products both at the site of injection and within thecirculation. It follows that insulin glargine may be lessmitogenic in vivo than in vitro, but the studies suggestconsiderable inter-individual variation, and a substantialproportion of the insulin injected will, on present evidence,reach the cells in the form of unaltered glargine.

In summary, it is currently impossible to extrapolatefrom the in vitro to the in vivo situation with anyconfidence. The mandatory preclinical testing proceduresto which all the insulin analogues in current clinical usehave been subjected are therefore insufficient to confirm orto exclude a possible cancer risk in humans. Preclinicaltesting has, however, identified legitimate cause for concernregarding some of the analogues.

Carcinogenicity studies in rodents

Preclinical evaluation of the analogues includes safety testsin animals, and development of insulin B10Asp was haltedwhen this was shown to promote the development ofmammary tumours in female SpragueDawley rats [32].The daily doses tested were 12.5, 50 and 200 U/kg, andmalignant tumours developed in 0, 11% and 23% of therats, respectively. Insulin glargine, by contrast, was tested atthe lower daily doses of 2, 5 and 12.5 U/kg [39], the last ofwhich is said to correspond to human daily doses ofapproximately 100 U (rats) or 50 U (mice). It is worthyof note that insulin B10Asp would have passed thecarcinogenicity testing to which insulin glargine wassubjected and would now be in clinical use. Interpretationof the insulin glargine studies was further complicated by avery high early mortality rate, which was probably due tohypoglycaemia at the higher insulin doses. This led the USFood and Drug Administration (FDA) to comment that thefindings in female mice were not conclusive due toexcessive mortality in all dose groups [40], a cautionwhich, curiously, finds no echo in the published report ofthe study [39]. Mammary tumours did indeed develop in1020% of the female rats, but were no more common in

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rats treated with insulin glargine than in those treated withneutral protamine Hagedorn (NPH) insulin or controlsolutions.

In summary, and despite the rapid increases in know-ledge that have accrued since the insulin analogues reachedthe market, we can make no firm judgement as to whetherthe insulin analogues do or do not enhance cancer riskon the basis of preclinical or laboratory testing. Sinceprospective clinical trials are evidently impracticable (whowould agree to participate?), this possibility can only beaddressed by observational studies in humans.

First observations in man

A large observational study submitted to this journal lastyear suggested that use of insulin glargine is, afteradjustment for dose, associated with a possible increase intumour risk in humans [23]. Interpretation of this analysisproved controversial, but the implications were serious. Aspecial advisory group, convened by the EASD, agreed thatit would be premature to publish these findings in isolation,and that replication was needed. The three other observa-tional analyses presented in this issue of Diabetologia weretherefore commissioned to examine the safety of thisinsulin [10, 24, 25], and the main findings will besummarised here. Coincidentally, a further paper in thisissue reports a prospective evaluation of the risk ofretinopathy progression in patients treated with insulinglargine or human NPH insulin [41]. Additional safety datarelating to cancer risk in this and other studies have beenmade available to our journal and will be published shortly.

German insurance study In this report [23], which triggeredthe remainder, Hemkens and colleagues present data from alarge insurance dataset, and compare the rate of diagnosis ofmalignant tumours in patients treated with human insulin, asagainst three of its analogues: insulin lispro, insulin aspartand insulin glargine. Insulin detemir, more recently intro-duced to the German market, was not included. The 127,031patients (39% of all those on insulin) in this large populationsample had all started insulin treatment since 2000, and wereall treated exclusively with human insulin (soluble and/orNPH) or one of the three analogues. Of these, 95,804(75.4%) were exclusively on human insulin, 23,855 (18.8%)were on insulin glargine alone, 3,269 (2.6%) were on insulinlispro and 4,103 (3.2%) were on aspart alone. It should benoted that, in Germany, patients with type 2 diabetes areoften treated with preprandial doses of rapid-acting insulinwithout a basal supplement. The insulin dose was extractedfrom the medical records. Although classification ofdiabetes is not specified in the register, those included inthis analysis will almost all have had type 2 diabetes, an

interpretation supported by the median age of ~67 years inall four groups.

The major finding of this analysis was a strong correlationbetween insulin dose and cancer risk, regardless of insulintype. The influence of dose greatly complicated the analysis,since the crude incidence of malignant neoplasms was higherin patients on human insulin than in those receiving one of thethree analogues, but patients on human insulin were alsotreated with larger doses of insulin. Insulin glargine users wereprescribed a median of ~22 U/day (95% quantile ~59 U),compared with a median of ~37 U (95% quantile ~100 U) forhuman insulin. Insulin glargine thus carried a significantlylower risk of cancer than human insulin in the unadjustedanalysis, but the risk ratio reversed itself when insulin dosewas allowed for, such that the rate of diagnosis of cancer andall-cause mortality both rose more steeply at higher doses ofinsulin glargine relative to human insulin. The adjusted HRfor diagnosis of a cancer was 1.31 (95% CI 1.201.42) forindividuals on 50 U of insulin glargine daily, as against 50 Uof human insulin. Dose for dose, insulin glargine thusappeared to carry a higher risk of cancer than human insulin.

As might be imagined from the somewhat complexnature of the analysis, this report created a dilemma for thejournal. Our referees expressed a number of majorreservations. These ranged from biological implausibility,given the short median period (1.31 years for insulinglargine) of exposure to each of the insulins, to the limitedoverlap between the dose ranges, the unexplained effect ofinsulin glargine on all-cause mortality, the lack of overalldifference in cancer risk between the four insulins in thecrude analysis, failure to correct for BMI in the doseresponse analysis, and a number of technical considera-tions. Nor was it possible to break the findings downaccording to the nature of the tumoura major limitationgiven the low probability that any one agent might producea non-specific increase in all types of cancer. Three of thesix referees initially recommended rejection on the basis ofthese limitations, some of which were inescapable. On theother hand, strengths of the study included its large size,and its main findings survived a searching and prolongedreview process. We anticipate that it will continue togenerate controversy following publication, and concludedthat it would be premature to publish these hypothesis-generating data in isolation.

Publication was therefore made conditional upon theperformance of additional studies, and these terms wereaccepted by the authors. Two national diabetes registries inSweden and Scotland were therefore invited to run theirdata against those of their respective cancer registries [24,25]. The overall null hypothesis was that patients treatedwith insulin analogues were not more likely to bediagnosed with cancer during the period of observation.

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At a later stage, a further analysis was commissioned fromPharmatelligence (Cardiff, UK), a commercial organisationwith a well-characterised diabetes database previouslyobtained from The Health Information Network (THIN) inthe UK [10].

Sweden The Swedish study linked data from a number ofregistries to identify 114,841 patients who received pre-scriptions for insulin in 2005 [24]. These records were thenlinked to data from the cancer registry for the twosubsequent calendar years. The specific focus was oninsulin glargine, as noted above, and some limitationsshould be noted. For example, duration of insulin therapyand exposure to other insulins prior to 2005 could not beconsidered. Patients were then divided into three groups:insulin glargine only (5,970 individuals), insulin glargineplus other insulins (20,316 individuals) and insulin usersnot on insulin glargine (88,555 individuals). Classificationof diabetes was based on age at diagnosis, and thosediagnosed after 30 years of age, including almost all thoseon insulin glargine alone, were considered to have type 2diabetes. Insulin dose could only be estimated in terms ofthe number of insulin prescriptions filled, which meant thata doseresponse relationship could not be examined in thesame way as in the German study [23], which was based onrecorded insulin doses. The endpoints were diagnosis ofany neoplasm, and a diagnosis of a cancer of the breast,prostate or gastrointestinal tract. Joint consideration of allgastrointestinal tumours might be considered a furtherlimitation of this study, since colon cancer is a muchstronger candidate for an insulin effect.

The analysis found no statistically significant differencein cancer incidence between the two largest groups, those oninsulins other than insulin glargine, and those on insulinglargine plus other insulins. Those on insulin glargine alonedid, however, have a higher risk of breast cancer than thoseon insulins other than insulin glargine, with an RR of 1.99(95% CI 1.313.03), all other cancer risks being equal. Thisobservation was robust in statistical terms, in that it was littleaffected by any of the subsequent adjustments that weremade, and metformin use did not emerge as a confounder.

As in any observational study, this finding must beinterpreted with caution. To begin with, the number ofbreast cancers was relatively low: 25 cases on insulinglargine vs 183 on insulins other than insulin glargine.Furthermore, it is puzzling that the reported effect should belimited to users of insulin glargine alone, rather than allinsulin glargine users regardless of other insulins. Theinsulin glargine plus other insulin group did, however,contain a much higher proportion of younger patients(presumably on basal bolus therapy) than the other twogroups. This indicates the strong possibility of an allocationbias, sometimes termed confounding by indication, i.e.

differences between exclusive insulin glargine users and thecomparator groups that might also influence their relativerisk of breast cancer. Statistical corrections can limit thispossibility, but cannot rule it out. Conversely, the observa-tion has biological plausibility, for breast cancer would be aprime candidate for an insulin glargine effect in any a priorihypothesis based on laboratory data.

Scotland The Scottish analysis [25] is based on a nationalclinical database which covers almost everyone withdiagnosed diabetes in Scotland. The analysis included allpatients exposed to insulin therapy for the calendar years2002, 2003 and 2004. An open cohort analysis included49,197 patients on insulin, divided, as in the Swedishanalysis, into insulin glargine alone, insulin glargine plusother insulins, and non-glargine insulins. These groupswere then matched with cancer registry data validated up tothe end of 2005. The analysis considered overall cancerincidence, and the frequency with which cancer of thebreast, colon, prostate and pancreas were diagnosed. As inthe Swedish study, there were clear differences between thepatient groups; for example, those on insulin glargine alonewere older than those on insulin glargine plus other insulins(68 vs 41 years) and users of other insulins (60 years). Notsurprisingly, those on insulin glargine alone were also moreoverweight, more hypertensive, and more likely to be onoral glucose-lowering agents; 97% had a diagnosis of type2 diabetes, as against 37% of those on insulin glargine plusother insulins. Relatively few Scottish patients were oninsulin glargine at the time of study, with 3,512 on insulinglargine plus other insulins and only 447 on glargine alone.Those on insulin glargine with rapid-acting insulin had aslightly lower rate of cancer progression than did the humaninsulin group (HR 0.8, 95% CI 0.551.17, p

both, or on insulin (subdivided into insulin glargine only,NPH insulin only, human biphasic and analogue biphasicinsulin only). An additional group of diet-treated diabetes,plus patients in the 3 year period prior to their diagnosisof diabetes, allowed cancer risk to be examined inmedication-naive individuals. The analysis was limited topatients who entered a given treatment category later thanthe year 2000, although insulin users might, for example,have previously taken oral agents. This dataset wastherefore more sharply defined in terms of diabetes therapythan the two national registries, but was also smaller.

The most striking finding to emerge from this analysiswas the protective effect of metformin. This has been notedpreviously [9], but the present analysis has shown that therisk of cancer in metformin-treated patients is equivalent tothat in treatment-naive individuals prior to diabetes therapy,and that the rate of cancer development associated withmonotherapy with sulfonylureas or insulin is lower whenthese therapies are combined with metformin. Furthermore,metformin was associated with a reduced rate of cancer ofthe colon or pancreas, but no reduction was seen for cancerof the breast or prostate. The difference in risk of pancreaticcancer was striking, yet is consistent with experimentalstudies in hamsters [42]. It has also recently been shownthat metformin abrogates sitagliptin-induced pancreaticductal metaplasia, a precursor of carcinoma, in a rat modelof type 2 diabetes [43]. These observations suggest thatmetformin may come to play a major role in cancerprevention in diabetes. For present purposes, however, thepoints to note are that concomitant metformin use ispotentially a major confounder when it comes to estimatingthe risks of insulin therapy. Furthermore, the lack ofeffect of metformin on breast cancer, if confirmed, mighthelp to explain why this particular cancer has tended toemerge from the analyses conducted in the previous twostudies [10].

This study was essentially negative when it came tocomparison of the four insulin-treated groups, whether interms of all cancers or cancer of the breast, pancreas, orcolorectal cancer, or a basket of all three cancer types. Itwill also be noted that the four insulin-treated groups wereless evidently heterogeneous than patients in the otheranalyses we have considered. Numbers were, however,relatively small, with 2,286 on insulin glargine alone,compared with 1,262 on NPH insulin, and once again adosage-based comparison, as performed in the Germanstudy [23], did not prove feasible.

Insulin glargine and retinopathy

A further safety concern requiring human studies arosewhen one of the early clinical trials [44] was reported to

have observed a threefold increase in retinopathy progres-sion with insulin glargine compared with human insulin[41]. Curiously, this fact is not mentioned in the originalreport [44], which concludes with the statement that insulinglargine has a safety profile that, apart from reducednocturnal hypoglycaemia, is otherwise similar to NPHinsulin. Equally curious, a later review also cites the samepaper as documenting a three step or greater retinopathyprogression in 7.5% of those on insulin glargine vs 2.7% ofthose on NPH (p

A further point to consider is that differences in cancerrisk between treatments have emerged within a remarkablyshort period of exposure to the putative agent. This isalmost without precedent in the field of cancer [24], and theobservation, if confirmed, can only be interpreted in termsof activation or accelerated progression of latent malignantfoci. The potential importance of factors that promotecancer progression is suggested by the observation that therate of clinical prostate cancer is about tenfold greater in theUSA than in Japan, whereas the prevalence of latentprostate cancer in autopsy studies is much the same in thetwo populations [17] (Fig. 1). Breast cancer screening hasbeen implemented in both Sweden and Scotland, and mighthave contributed to earlier diagnosis. There is no reason tobelieve that insulin therapy causes cancer, but there is,nonetheless, reason to suspect that high concentrations ofinsulin may promote its development.

When it comes to interpretation of the studies them-selves, it is important to note that the model upon which theGerman analysis is based depends on a number of

assumptions that may or may not prove to be justified.This study does, however, introduce the issue of dose,which may prove central to future consideration of thisissue. The Swedish and Scottish studies [24, 25], whichwere especially commissioned to consider the safety ofinsulin glargine, are reasonably consistent. Both show aclear difference between the cancer risk of the group oninsulin glargine plus other insulin and that of the group oninsulin glargine alone. The demographic characteristics ofboth groups also differ, in that the former groups werecomposed largely of younger patients on basal bolustherapy. The baseline risk of cancer is much lower in thisage group, together with the likelihood of pre-existingcancer foci. Furthermore, a proportion had type 1diabetes, which is associated with a different range ofcancers [5]. Statistical adjustment cannot fully compensatefor biological differences between groups. The insulinglargine only groups also differed, although to a lesserextent, from the comparator groups on human insulin. TheGerman study indicated an increased cancer risk in bothsexes, suggesting that it would be premature to focusfurther attention on one specific tumour type to theexclusion of all others. This having been said, both studiesindependently indicate a signal for breast cancer, abiologically plausible tumour, and this observation cannotsimply be ignored.

The THIN study managed to match the study groupsmore closely [10]; it also allowed a time-matched compar-ison to be made between monotherapy with human insulinand insulin glargine, which was not possible in the previoustwo studies [24, 25]. In the event, no difference emergedbetween the therapies [10]. The number of patients studiedwas, however, relatively low, and interpretation of all threestudies must allow for the relatively low frequency of breastcancer: 25 cases in patients on insulin glargine alone inSweden, six cases in Scotland and ten cases in THIN. Thelatter study therefore provides some reassurance in relationto the two previous studies, but does not resolve thequestion at issue.

Where next?

The difficulties we encountered during the course of thisanalysis provide an excellent demonstration of the problemsand pitfalls of observational studies [47, 48]. Prospectiveclinical trials set out to compare like with like, whereaspharmacoepidemiological studies such as those reportedhere almost inevitably need to make statistical adjustmentsfor unmatched comparisons. You can only correct forconfounders when you know what the confounders are. Aprospective clinical trial would be the best way of resolvingthe issue, but would be unfeasible, arguably unethical, and

Fig. 1 Scanning electron micrograph showing prostate cancer.Prostate cancer appears to have a weak negative association with type2 diabetes, possibly because a common polymorphism in the HNF1Bgene, which predisposes to type 2 diabetes, also protects againstprostate cancer [54]. Image from Steve Gschmeissner/Science PhotoLibrary

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far too slow to perform. Clinical trial data held on file bysanofi-aventis may allow some of these questions to beanswered, but independent review would be an essentialelement of any such analysis. An additional approach,supplementary to the foregoing, would be a much largerpharmacoepidemiological analysis, jointly designed andconducted by industry and representatives of the scientificassociations, and independently analysed. We believe thatsuch a study would achieve as close an approximation tothe truth as is likely to be possible.

Summary and conclusions

The focus of this series of investigations has been oninsulin glargine. It has not proved possible to placeinsulin detemir under similar scrutiny, but it would beprudent for this insulin analogue to be investigated inmore detail. On current evidence, the short-actinganalogues do not appear to present a potential problem.With respect to insulin glargine, it is in no ones interestto mount a witch-hunt against this popular and widelyused insulinmany will reflect upon the fate ofrosiglitazonebut it is in everyones interest for thetruth to be known. The evidence presented in this set ofpapers is sufficient to establish that there is a case toanswer, but is entirely insufficient to bring in a verdict.Certain reassurances do, however, seem justified. Thereis no evidence that insulin, however formulated, causescancer. There is no evidence of an overall increase in therate of cancer development in patients on insulinglargine, and some suggestion that the risk may actuallybe reduced. There is no evidence of harm in type 1diabetes, or in premenopausal breast cancer. On the otherhand, it has to be said that insulin glargine has not beenshown to be more effective than human insulin inachieving glycaemic control in type 2 diabetes; its mainbenefit (if any) is in relation to symptomatic episodes ofnocturnal hypoglycaemia [4952]. We have safe and effec-tive alternatives to offer our patients with type 2 diabetes.

The observations presented here require further anal-ysis and evaluation, and are likely to open a much widerdebate. Once the safety of the analogues has, as wewould all wish, been confirmed, the wider debate willcentre around the relationship between insulin andcancer, and the possibility of reducing this risk bylifestyle measures and metformin. One point has becomeabundantly clear, and this is that cancer must now benumbered among the complications of diabetes. Further-more, and as with ischaemic heart disease, cancer isassociated with a higher mortality in those with diabetesthan in those without [53]. Cancer risk and preventionmay become increasingly important considerations in

diabetes therapy, and the implications of the studiesreported here are likely to be very far-reaching.

Acknowledgements This article is dedicated to the memory ofMichael Berger. We think he might have enjoyed reading it. Ourgrateful thanks are due to the teams of investigators in differentcountries who worked under high pressure to generate the datareported in the accompanying papers, and managed to do so in timefor our joint publication deadline.

Duality of interest The authors declare that there is no duality ofinterest associated with this manuscript.

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Does diabetes therapy influence the risk of cancer?IntroductionInsulin and cancerThe insulinIGF-1 axisInsulin analogues and cancerCarcinogenicity studies in rodentsFirst observations in manInsulin glargine and retinopathyWhat does it all mean?Where next?Summary and conclusionsReferences

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