Homocysteine and Methylmalonic Acid: Markers to Predict and
`Supported by Eli
`Avoid Toxicity from Pemetrexed Therapy 1
`illy and Company.
`
`1L
`
`let Niyikiza, Sharyn D. Baker, David E. Seitz, et al.
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`(cid:160)C
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`Mol Cancer Ther
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` 2002;1:545-552. Published online May 1, 2002.
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`
`Vol. 1, 545–552, May 2002
`
`Molecular Cancer Therapeutics
`
`545
`
`Homocysteine and Methylmalonic Acid: Markers to Predict
`and Avoid Toxicity from Pemetrexed Therapy1
`
`Clet Niyikiza,2 Sharyn D. Baker, David E. Seitz,
`Jackie M. Walling, Katrina Nelson,
`James J. Rusthoven, Sally P. Stabler, Paolo Paoletti,
`A. Hilary Calvert, and Robert H. Allen
`Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana
`46285 [C. N., K. N., J. J. R., P. P.]; Cancer Treatment and Research
`Center (CTRC), University of Texas, San Antonio, Texas 78229
`[S. D. B.]; Indiana University School of Medicine, Indianapolis, Indiana
`46202 [D. E. S.]; Lilly Research Laboratories, Tularik Inc, South San
`Francisco, California 94080 [J. M. W.]; University of Colorado Health
`Sciences Center, Denver, Colorado 80220 [S. P. S., R. H. A.]; and
`University of Newcastle Upon Tyne, Newcastle Upon Tyne, United
`Kingdom NE4 6BE [A. H. C.]
`
`Abstract
`The purpose of this study was to identify predictive
`factors for severe toxicity caused by antifolate-
`chemotherapy using pemetrexed (ALIMTA, LY231514),
`as a model. Data on potential predictive factors for
`severe toxicity from pemetrexed were collected from
`246 patients treated between 1995 and 1999.
`Multivariate stepwise regression methods were used to
`identify markers predictive of severe toxicity. Using a
`multiple logistic regression model allowed us to
`quantify the relative risk of developing toxicities and to
`generate a validated clinical hypothesis on ways to
`improve the safety profile of pemetrexed. Pretreatment
`total plasma homocysteine (tHcy) levels significantly
`predict severe thrombocytopenia and neutropenia with
`or without associated grade 3/4 diarrhea, mucositis, or
`infection. Pretreatment methylmalonic acid (MMA)
`levels significantly and independently predict grade 3/4
`diarrhea and mucositis; however, these toxicities are
`still predicted by tHcy alone. Patients with elevated
`baseline levels of tHcy alone, or of both tHcy and
`MMA, were found to have a high risk of severe toxicity
`that led us to postulate that reducing tHcy would result
`in a reduction of severe toxicity with no harm to
`efficacy. This study points out for the first time the
`importance of pretreatment tHcy levels in predicting
`severe toxicity associated with an antifolate and sets
`the stage for a prospective clinical intervention to
`protect patients from pemetrexed-induced severe
`toxicity and possibly improve the drug’s efficacy.
`Antifolates as a class have been associated with
`sporadic severe myelosuppression with gastrointestinal
`toxicity. Although infrequent, a combination of such
`toxicities can carry a high risk of mortality. This
`phenomenon had been unpredictable until now. Our
`
`work shows that by measuring tHcy, one can identify
`patients that are at risk of toxicity before treatment.
`Most importantly, decreasing homocysteine levels via
`vitamin supplementation leads to a better safety profile
`of pemetrexed and possibly to an improved efficacy.
`
`Introduction
`In 1998, it was estimated that 90% of new anticancer agents
`designed in laboratories around the world never make it into
`routine clinical use (1). Three main reasons were put forth for
`this sobering statistic: (a) high toxicity seen with new agents
`that carry serious safety concerns; (b) lack of efficacy of
`these agents; and (c) a disconnect between the work of
`preclinical bench scientists and bedside clinicians that often
`reflected a failure to ensure that clinical trial designs for new
`agents were based on the best-known mechanism of action
`of the agent.
`Because most anticancer agents have a narrow therapeu-
`tic window, optimizing the chance that a treatment succeeds
`without causing undue harm to the patient is of paramount
`importance. Accurate information about both the new drug
`and the patient becomes critical. Interruption of development
`of a new drug or limitation of its effectiveness or wide use
`occurs when either severe toxicity or lack of efficacy is noted.
`It is not unusual that, when a new agent shows toxicity with
`limited antitumor activity, little effort is made to persistently
`look for ways to circumvent the toxicity with the possibility of
`improving efficacy. Toxicity or lack of efficacy could be re-
`lated to a patient’s individual clinical, demographic, or ge-
`netic profile. Ideally, the goal is to devise a simple, optimal
`dosing strategy for a new agent that incorporates what is
`known about its mechanism of action and about the patient
`characteristics. This paradigm is the subject of the present
`study. We discuss how, after serious safety concerns arose,
`predictive factors for severe toxicity associated with pem-
`etrexed were identified, and how these factors led to the
`formulation of a clinical intervention to modulate the toxicity
`of this antifolate/anticancer agent while improving its effi-
`cacy. The results of this prospective clinical intervention are
`the subject of a separate upcoming publication.3
`Antifolates represent one of the most extensively investi-
`gated classes of antineoplastic agents, with aminopterin ini-
`tially demonstrating clinical activity more than 50 years ago
`(2). Methotrexate was developed shortly thereafter and, to-
`day, is a standard component of chemotherapeutic regimens
`effective for malignancies such as lymphoma, breast cancer,
`and head and neck cancer (3– 6). The cytotoxic activity and
`subsequent effectiveness of antifolates can be associated
`
`Received 3/1/02; revised 3/25/02; accepted 3/28/02.
`1 Supported by Eli Lilly and Company.
`2 To whom requests for reprints should be addressed, at Eli Lilly and
`Company, Lilly Corporate Center, Indianapolis, IN 46285.
`
`3 J. Rusthoven, C. Niyikiza, P. Bunn, et al. Reducing toxicity from pem-
`etrexed therapy with folic acid and vitamin B12 supplementation, submit-
`ted for publication.
`
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`
`546 Homocysteine and MMA Are Predictive Markers
`
`with substantial toxicity for some patients. Antifolates, as a
`class, have been associated with sporadic severe myelosup-
`pression with gastrointestinal toxicity. Although infrequent, a
`combination of such toxicities, can carry a high risk of mor-
`tality. The inability to control these toxicities has led to the
`discontinuation of clinical development of some antifolates,
`such as CB3717, and has complicated the clinical develop-
`ment of others, such as lometrexol and raltitrexed (7–9). The
`ability to predict those patients that are at greater risk of
`developing severe toxicity would represent an important ad-
`vantage in the use of these agents.
`Lometrexol
`[LY249543 (disodium form); Lilly Research
`Laboratories, Tularik Inc., South San Francisco, CA), an an-
`tifolate GARFT4 inhibitor was curtailed in its development by
`Lilly because of severe and cumulative toxicities. The onset
`of profound myelosuppression and/or mucositis, in most
`patients 6 – 8 weeks after dosing, prevented repeated admin-
`istration of this anticancer agent in most studies. This led to
`additional studies in mice (10 –11) that revealed that thera-
`peutic efficacy and toxicity of lometrexol were highly de-
`pendent on dietary folic acid intake. A subsequent Phase I
`study showed that lometrexol toxicity could be modulated by
`folic acid supplementation and that the maximum tolerated
`dose could be substantially increased (8). Yet, pharmacoki-
`netic studies conducted with 5 mg of folic acid supplemen-
`tation suggested that folic acid was not acting by enhancing
`lometrexol plasma clearance (12). Despite a 5-year effort in a
`series of preclinical and clinical investigations, researchers
`were still unable to ascertain the mechanism responsible for
`the reduction in lometrexol toxicity. The lometrexol experi-
`ence was useful when a second generation GARFT inhibitor
`(LY249543; Eli Lilly and Company), entered its clinical devel-
`opment with 5 mg of folic acid supplementation 2 days
`before, the day of, and 2 days after, as part of standard
`dosage of this anticancer agent. The development was also
`curtailed because of toxicity leaving some investigators to
`suspect that the folic acid supplementation regimen that was
`used was likely inadequate.
`It is with this background on antifolate GARFT inhibitors
`that pemetrexed (ALIMTA, LY231514; Eli Lilly and Company,
`Indianapolis, IN) clinical development was undertaken. Pem-
`etrexed is a multitargeted antifolate that has demonstrated
`broad-spectrum antitumor activity in the Phase II setting and
`is currently undergoing active clinical development (13). This
`new generation antifolate inhibits several key folate-requiring
`enzymes of the thymidine and purine biosynthetic pathways,
`in particular, thymidylate synthase, DHFR, and GARFT, by
`competing with reduced folate for binding sites (14). The
`consequent inhibition of intracellular folate metabolism leads
`to the inhibition of cell growth.
`During the course of pemetrexed clinical development,
`myelosuppression emerged as the principal drug-related
`toxicity, with 50% of all patients experiencing grade 3/4
`
`4 The abbreviations used are: GARFT, glycinamide ribonucleotide formyl-
`transferase; DHFR, dihydrofolate reductase; MMA, methylmalonic acid;
`tHcy, total plasma homocysteine; PS, performance status; AP, alkaline
`phosphatase; ALT, alanine transaminase; AST, aspartate transaminase;
`AIRCARFT, aminoimidazocarboxamide ribonucleotide formultransferase.
`
`Fig. 1. Folate and homocysteine metabolism.
`
`neutropenia (13). In particular, Grade 4 neutropenia with
`grade 3/4 infection, grade 3/4 diarrhea, or grade 3/4 mucosi-
`tis became life threatening. These toxicities, occurring typi-
`cally after two cycles of therapy, prompted a renewed ag-
`gressive clinical effort to search for ways to avoid them.
`Given the relevance of folic acid to the toxicity profile
`previously witnessed with an antifolate such as lometrexol, it
`was reasonable to postulate that functional folate status
`could be a useful predictor of toxicity from treatment with
`pemetrexed. The significant reciprocal association of hom-
`ocysteine to serum folate and RBC folate has been well
`established (15–16), and, thus, tHcy concentration may be
`used as a measure of functional folate status. Folates are
`required for the metabolism of tHcy, which is converted to
`methionine by the transfer of a methyl group from the co-
`substrate 5-methytetrahydrofolate by methionine synthase,
`an enzyme that also requires the cofactor methylcobalamin
`(Vitamin B12). Thus, under conditions of folate and/or cobal-
`amin deficiency, tHcy concentrations rise (Refs. 17–18; see
`Fig. 1). Because the enzyme L-methylmalonyl CoA mutase is
`vitamin B12 dependent, a B12 deficiency will lead to an in-
`crease in MMA (19). MMA concentrations are, therefore, a
`useful tool in differentiating folate and cobalamin deficiency
`(17–18).
`Because of previous observations on the impact of folic
`acid supplementation on the toxicity profile of lometrexol, a
`Phase I study of pemetrexed with 5 mg of folic acid 2 days
`before, the day of, and 2 days after, dosing was initiated. It
`was shown that the maximum tolerated dose could be sub-
`stantially increased (20). Still unanswered was the question
`pertaining to the precise mechanism responsible for the re-
`duction in lometrexol and pemetrexed toxicity when a patient
`receives folic acid supplementation. To find an answer, a
`programmatic change was made during the Phase II clinical
`development of pemetrexed to collect from all treated pa-
`tients a number of vitamin deficiency markers. Included in
`the panel of markers were the folic acid and/or vitamins B12
`deficiency markers, tHcy and MMA, and the vitamin B6 de-
`ficiency marker, cystathionine. This study provided initial
`answers. The objective was to identify one or more specific
`factors that might predict for pemetrexed-induced toxicity
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`Molecular Cancer Therapeutics
`
`547
`
`collected before pemetrexed treatment. These variables in-
`cluded age; gender; baseline PS; prior chemotherapy; tumor
`type; and pre-pemetrexed-treatment serum albumin, liver
`enzymes, AP, ALT, AST, platelet count, absolute neutrophil
`count, calculated area under the curve (AUC), and vitamin
`deficiency markers including tHcy, cystathionine, and MMA.
`Vitamin deficiency markers were measured over time before
`each cycle of treatment as long as the patient remained on
`study. Weekly laboratory studies included complete blood
`cell and differential WBC counts, serum creatinine, total bil-
`irubin, ALT, AST, and AP. Vitamin deficiency markers were
`quantified using previously published methods (21). Normal
`ranges were determined previously using 50 blood donors
`(25 male, 25 female; ages, 18 – 65) at the Belle Bonfils Blood
`in Denver, Colorado. Whole blood was allowed to clot for 1 h
`at room temperature before serum was collected. Values
`were calculated as the mean ⫾ 2 SDs after log normalization.
`In the multivariate statistical search for predictive factors
`for toxicity, dependent outcome variables included the fol-
`lowing worst-grade toxicities: (a) grade 4 neutropenia; (b)
`grade 4 thrombocytopenia; (c) grade 3 or 4 mucositis; (d)
`grade 3 or 4 diarrhea; (e) grade 4 neutropenia and grade 3 or
`4 infection; and (f) grade 4 hematological toxicity or grade 3
`or 4 nonhematological toxicity, where a patient experienced
`any or a combination of the above-listed toxicities. Toxicity
`was graded according to the National Cancer Institute com-
`mon toxicity criteria (22).
`To identify the most statistically significant predictive fac-
`tor(s) for a given toxicity, multivariate stepwise regression
`methods were used whereby variables significant at the 0.25
`level were entered into the model and those not significant at
`the 0.10 level were removed from the model. At each step, a
`test was performed to verify that the factors included in the
`model significantly impacted the toxicity of interest (23).
`To assess the risk of developing severe hematological or
`nonhematological toxicities associated with the vitamin de-
`ficiency marker of tHcy, alone or with MMA, at study entry, a
`multiple logistic regression analysis was performed sepa-
`rately for tHcy and MMA while adjusting for the other inde-
`pendent factors (23). Quartiles were determined for each
`marker using baseline distribution of
`the marker levels.
`Ranges were defined using these quartiles to calculate the
`risk of toxicity for a given patient falling within a specific
`range. Odds ratios were also calculated as a measure of the
`extent to which the risk of severe toxicity was affected as
`baseline tHcy and MMA levels fell above or below the se-
`lected reference range.
`
`Results
`A total of 1063 courses of pemetrexed were administered.
`The number of courses per patient ranged from 1 to 17
`cycles with a mean of 4 cycles. There were an equal number
`of males and females (Table 1). Age ranged from 25 to 90
`years (mean, 57.8 years), and 25% of the patients were 65
`years or older. Ninety percent of patients had a PS of 0 or 1.
`Myelosuppression was the major toxicity encountered (Ta-
`ble 2). Grade 4 neutropenia was seen in ⬃32% of the pa-
`tients, whereas the presence of grade 4 hematological or
`grade 3 or 4 nonhematological toxicity was observed in 37%
`
`Fig. 2. Patient population description.
`
`such as baseline patient characteristics, cumulative dose,
`and baseline levels of the vitamin deficiency markers tHcy,
`MMA, and cystathionine.
`
`Patients and Methods
`Patient Population. A total of 305 of the 1300 patients,
`treated in Phases I and II between September 1995 and
`November 1995, had the vitamin deficiency markers of tHcy,
`MMA, and cystathionine collected before and during pem-
`etrexed therapy. Pemetrexed was developed with doses of
`500 mg or 600 mg/m2 every 21 days. Other dosing schedules
`were explored early in the Phase 1 program but were found
`not to be feasible for further development. To eliminate the
`complicating factor of folic acid supplementation on toxicity
`and the impact of doses not pursued, any patient who re-
`ceived folic acid supplementation at any point during therapy
`or who received any dosing regimen other than pemetrexed
`500 – 600 mg/m2, was removed from the analysis. This left a
`final sample size of 246 patients with data on vitamin defi-
`ciency markers (see Fig. 2).
`Protocol and informed consent documents were approved
`by each site’s Ethical Review Board before the enrollment of
`any patient. All of the patients were informed of the nature of
`the study, and all of the patients signed a written informed
`consent document before enrollment.
`Data Collection and Statistical Analysis. Data from mul-
`tiple, potentially predictive (or independent) variables were
`
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`
`548 Homocysteine and MMA Are Predictive Markers
`
`Table 1 Patient demographics and baseline folic acid, B6, and B12 vitamin deficiency markers
`
`Study
`
`Age (no. of
`patients)
`
`Gender (no. of
`patients)
`
`⬍65 ⱖ65 Male
`
`Female
`
`BSAa (m2)
`mean, range
`
`PS
`(no. of
`patients)
`
`tHcy (␮mol/liter)
`mean, range
`
`Cyst (␮mol/liter)
`mean, range
`
`MMA (nmol/liter)
`mean, range
`
`Phase I combination,b n ⫽ 79
`Phase II
`Colorectal cancer, n ⫽ 35
`Pancreas cancer, n ⫽ 41
`Esophageal and gastric
`cancer, n ⫽ 23
`Breast cancer, n ⫽ 46
`Cervical cancer, n ⫽ 9
`NSCLC cancer, n ⫽ 13
`
`61
`
`24
`26
`16
`
`42
`8
`9
`
`18
`
`11
`15
`7
`
`4
`1
`4
`
`51
`
`22
`23
`17
`
`0
`0
`10
`
`28
`
`13
`18
`6
`
`46
`9
`3
`
`1.89, 1.27–2.47
`
`1.90, 1.45–2.23
`1.85, 1.22–2.61
`1.69, 1.26–2.22
`
`1.77, 1.48–2.22
`1.61, 1.25–1.97
`1.84, 1.30–2.16
`
`0–1
`
`79
`
`31
`34
`21
`
`42
`9
`11
`
`2
`
`7
`
`4
`7
`2
`
`4
`0
`2
`
`9.06, 3.6–27.4
`
`376, 98–2481
`
`206, 67–1065
`
`11.7, 6–22.5
`12.8, 4.7–132.4
`12.6, 6.3–31.9
`
`224, 50–1303
`235, 52–869
`250, 87–718
`
`9.0, 4.3–20.5
`7.7, 5.4–10.5
`9.1, 3.7–15.4
`
`396, 80–2234
`155, 89–282
`533, 198–1921
`
`280, 68–2170
`341, 29–8507
`262, 96–1192
`
`173, 79–734
`223, 78–482
`179, 97–303
`
`Total patients
`
`Mean values
`
`186
`
`60
`
`123
`
`123
`
`224 22
`
`1.84, 1.22–2.61
`
`10.3, 3.5–132.4
`
`309, 50–2481
`
`237, 29–8507
`
`a BSA, body surface area; Cyst, cystathionine; NSCLC, non-small cell lung cancer.
`b Patients with different primary tumor types.
`
`Table 2 Prevalence of selected toxicities in patients treated with
`pemetrexed (n ⫽ 246)
`
`Toxicity
`
`Grade 4 neutropenia
`Grade 4 thrombocytopenia
`Grade 3/4 mucositis
`Grade 3/4 diarrhea
`Any grade 4 hematological toxicity or grade 3/4
`nonhematological toxicity
`Grade 4 neutropenia ⫹ grade 3/4 mucositis
`Grade 4 neutropenia ⫹ grade 3/4 diarrhea
`Grade 4 neutropenia ⫹ grade 3/4 infection
`
`No. of
`patients %
`79
`32
`20
`8
`12
`5
`15
`6
`92
`37
`
`8
`8
`6
`
`3
`3
`2
`
`of the patients. Grade 4 neutropenia coupled with grade 3 or
`4 diarrhea was observed in 3% of patients.
`Baseline tHcy and MMA were found to be highly correlated
`(R2 ⫽ 0.8870). The analysis performed to further assess the
`impact of this correlation (both with and without MMA, as an
`independent variable) revealed an important interaction be-
`tween these two vitamin deficiency markers with respect to
`pemetrexed induced toxicity. tHcy correlated significantly
`with severe hematological toxicity, as well as with severe
`diarrhea and severe neutropenia, whether or not MMA was
`included in the model (significance levels are shown in Table
`3). Interestingly, when MMA was included as an independent
`variable in the model, it was significantly correlated with
`diarrhea and mucositis, whereas tHcy was not. However,
`when MMA was excluded from the analysis, the model se-
`lected tHcy as the main predictor for diarrhea and mucositis.
`The prevalence of selected severe toxicities was found to
`increase as pretreatment tHcy and MMA levels increased
`(see Fig. 3). A ␹2 test for trend (24) indicated significantly
`increased prevalence of severe toxicities with increased pre-
`treatment levels of tHcy (grade 4 neutropenia, P ⫽ 0.0185;
`grade 4 thrombocytopenia, P ⫽ 0.0002; and grade 4 neu-
`tropenia ⫹ grade 3/4 infection, P ⫽ 0.0064), and MMA (grade
`4 neutropenia, P ⬍ 0.0001 and grade 4 neutropenia ⫹ grade
`3/4 diarrhea, P ⫽ 0.0005). This trend was seen also with
`selected hematological and nonhematological toxicity (see
`
`Fig. 4). Statistically significant increases in prevalence of
`severe hematological or nonhematological toxicity with in-
`creasing pretreatment levels were observed for MMA (P ⫽
`0.0001), homocysteine (P ⫽ 0.0011), and for tHcy and MMA
`quartile intersections (P ⫽ 0.0014). The most dramatic in-
`crease in such toxicities was observed in patients with si-
`multaneous elevations of both markers, in which 15 of 19
`patients experienced severe toxicity.
`Using quartile-defined ranges, we performed multiple lo-
`gistic regression analyses, including the same independent
`variables reported in Table 3. These analyses were run sep-
`arately for tHcy and MMA and are reported in Fig. 5. In
`addition, the relative risk for a patient whose pretreatment
`levels fell above the third quartile for both homocysteine
`(⬎11.5 ␮mol/liter) and MMA (⬎219.3 nmol/liter) was re-
`ported. The tHcy interquartile range of 7.4 –11.5 ␮mol/liter in
`this study is similar to that considered a normal range in the
`cardiovascular literature (25–28). As a result, the interquartile
`range of 7.4 –11.5 ␮mol/liter was used as the normal range
`for the purpose of relative risk assessment for toxicity (see
`Fig. 5C). Patients with pretreatment tHcy levels below 7.5
`␮mol/liter had an odds ratio of 0.7, a 30% reduction in the
`risk of developing a severe toxicity when compared with
`patients with normal baseline tHcy. This risk reduction was
`not found to be statistically significant (P ⫽ 0.3672). Patients
`with baseline tHcy levels above 11.5 ␮mol/liter had an odds
`ratio of 3.1, a 300% increase in the risk of developing a
`severe hematological or nonhematological
`toxicity when
`compared again to those patients with normal baseline tHcy.
`This increase in the risk was found to be highly statistically
`significant (P ⫽ 0.0040).
`Using MMA interquartile range as reference in the analysis,
`we showed that patients with baseline MMA ⬍119.0 nmol/
`liter had a statistically significant decrease in risk of severe
`toxicity, with an odds ratio of 0.3 when compared with that of
`patients with MMA in the reference range of 119.0 –219.3
`nmol/liter. Patients whose MMA was ⬎219.3 nmol/liter had a
`borderline significant increase in risk, with an odds ratio of
`2.2 when compared with the same reference range (Fig. 5C).
`
`
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`
`Molecular Cancer Therapeutics
`
`549
`
`Table 3 Factors selected as significantly correlated to severe toxicity (n ⫽ 246)a
`
`Dependent variable
`
`G4b neutropenia
`
`G4 neutropenia ⫹
`G3/4 infection
`
`G4
`thrombocytopenia
`
`G3/4
`diarrhea
`
`Correlated independent
`variable (P)
`
`Homocysteine
`(P ⫽ 0.003)
`PS (P ⫽ 0.03)
`
`Combination
`therapy
`(P ⫽ 0.01)
`
`Homocysteine
`(P ⬍ 0.0001)
`
`Homocysteine
`(P ⬍ 0.0001)
`Baseline ANC
`(P ⫽ 0.02)
`
`MMA
`(P ⬍ 0.0001)
`
`a Total homocysteine and MMA are correlated at R2 ⫽ 0.8830.
`b G4, grade 4; G3/4, grade 3/4; ANC, absolute neutrophil count.
`
`G4 neutropenia
`⫹ G3/4 diarrhea
`
`Homocysteine
`(P ⬍ 0.0001)
`
`G3/4 mucositis
`
`MMA
`(P ⬍ 0.0001)
`Baseline ANC
`(P ⫽ 0.0005)
`
`Fig. 3. Prevalence of selected
`toxicities by homocysteine and
`MMA quartile.
`(homocysteine
`quartiles in ␮M: ⬍7.4, 7.4 –9.2,
`9.3–11.5, and ⬎11.5; MMA quar-
`tiles in nM: ⬍119, 119 –156.5,
`156.6 –219.3, and ⬎219.3). The
`␹2 test for trend indicated a sta-
`tistically significantly different in-
`cidence of certain toxicities in
`different quartiles for both hom-
`ocysteine (G4 neutropenia, P ⫽
`0.0185; G4 thrombocytopenia,
`P ⫽ 0.0002; and G4 neutropenia
`⫹ G3/4 infection, P ⫽ 0.0064)
`and MMA (G4 neutropenia, P ⬍
`0.0001; and G4 neutropenia ⫹
`G3/4 diarrhea, P ⫽ 0.0005).
`
`Patients with baseline tHcy (⬎11.5 ␮mol/liter) and MMA
`(⬎219.3 nmol/liter) levels above the third quartile had an
`odds ratio of 15.6 when compared with those with pretreat-
`ment tHcy levels in the normal range (see Fig. 5C, white bar).
`An similar increase in risk was seen when toxicity prevalence
`in this patient group was evaluated relative to MMA reference
`ranges of 119.0 –219.3 nmol/liter with an odd ratios 6.0 (see
`Fig. 5C, white bar).
`
`Discussion
`Potentially life-threatening toxicity remains a major limitation
`to the optimal administration of commonly used chemother-
`apeutic agents. In some cases, a supportive intervention has
`been clinically indicated in an attempt to counter undesired
`side effects and, hence, to permit safe, maximal dosing of a
`chemotherapeutic agent. Such is indeed the case with the
`use of corticosteroids and antihistamines to prevent anaphy-
`lactic reactions to taxanes, or the use of hydration to reduce
`nephrotoxicity from cisplatin. The ability to predict which
`patients are more likely to experience drug-associated
`toxicity from pretreatment characteristics represents an im-
`portant improvement in the management of this problem.
`Antifolates have been associated with sporadic severe
`
`Fig. 4. Prevalence of any grade 4 hematological or grade 3/4 nonhema-
`tological toxicity in each homocysteine and MMA quartile, as well as in
`each quartile intersection. (Q1 intersection: n ⫽ 24; Q2 intersection: n ⫽
`15; Q3 intersection: n ⫽ 11; Q4 intersection: n ⫽ 19). Statistically signif-
`icant increases in the prevalence of severe hematological or nonhemato-
`logical toxicity by increasing quartile were observed for MMA (P ⫽
`0.0001), homocysteine (P ⫽ 0.0011), and for total homocysteine and MMA
`quartile intersections (P ⫽ 0.0014).
`
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`
`550 Homocysteine and MMA Are Predictive Markers
`
`Fig. 5. Risk of developing any severe hematological or nonhematological toxicity in relation to a total homocysteine (tHcy): reference range, 7.4 –11.5 (A);
`total homocysteine (tHcy) reference range, ⬍7.4 (B); MMA reference range, ⬍119.0 (C); and MMA reference range, 119.0 –219.3 (D). A, a: odds ratio, 0.7;
`95% confidence interval (CI), 0.3–1.6; P ⫽ 0.3672; b: odds ratio, 3.1; 95% CI, 1.4 – 6.7; P ⫽ 0.0040; c: odds ratio, 15.6; 95% CI, 3.6 –96.9; P ⫽ 0.0008. B,
`a: odds ratio, 1.5; 95% CI, 0.6 –3.4; P ⫽ 0.36; b: odds ratio, 8.8; 95% CI, 2.7–32.8; P ⫽ 0.0006; c: odds ratio, 50.5; 95% CI, 4.5–1222.8; P ⫽ 0.0049. C,
`a: odds ratio, 0.3; 95% CI, 0.1– 0.7; P ⫽ 0.0058; b: odds ratio, 2.2; 95% CI, 1.0 – 4.7; P ⫽ 0.0365; c: odds ratio, 6.0; 95% CI, 1.6 –30.0; P ⫽ 0.0131. D, a:
`odds ratio, 3.3; 95% CI, 1.5–7.9; P ⫽ 0.0058; b: odds ratio, 12.5; 95% CI, 3.6 –54.7; P ⫽ 0.0002; c: odds ratio, 185.0; 95% CI, 15.4 – 8468.0; P ⫽ 0.0007.
`
`myelosuppression with gastrointestinal toxicity. Pemetrexed
`was no exception to this rule. Although infrequent, a combi-
`nation of such toxicities can actually carry a high risk of
`mortality. The inability to control these toxicities has led to
`the discontinuation of clinical development of some anti-
`folates.
`The dependence of therapeutic efficacy and toxicity of
`antifolates such as lometrexol on dietary folic acid intake,
`observed early in the 1990s both in animal models and in
`patients, together with the observation that folic acid was
`not acting by enhancing plasma clearance of these com-
`pounds, had left researchers unable to pin down the
`mechanism responsible for the observed reduction in tox-
`icity. Our work provides the missing link. Using statistical
`approaches, we have uncovered the importance of base-
`line tHcy and MMA as biomarkers for predicting severe
`hematological or nonhematological
`toxicity associated
`with pemetrexed therapy. The statistical investigation re-
`vealed that independent, and/or simultaneous, elevations
`in pretreatment tHcy and MMA levels are more closely
`associated with increased risk of toxicity from pemetrexed
`than from other routine biochemical, hematological, or
`clinical parameters. It also showed that in the absence of
`MMA levels,
`tHcy predicts those toxicities otherwise
`linked to MMA. Because tHcy is a surrogate marker for
`functional folate, with an increase in tHcy concentration
`indicating folate and/or vitamin B12 deficiency, it might be
`expected that treatment with pemetrexed would increase
`plasma tHcy level. However, the folate product of the
`methionine synthase reaction is tetrahydrofolate, which is
`
`converted to 5,10-methylenetetrahydrofolate and then to
`5-methyltetrahydrofolate (the substrate for methionine
`synthase) by the enzymes serine-hydroxymethyl transfer-
`ase and 5,10-methylenetetrahydrofolate reductase,
`re-
`spectively (29 –30). Because there are currently no data to
`suggest that either of these enzymes is inhibited by peme-
`trexed, one can hypothesize that this cycle will continue
`even in the presence of pemetrexed. Indeed, tHcy levels
`were not increased by pemetrexed therapy in the patients
`studied here.
`MMA elevation (a marker for vitamin B12 deficiency) was
`found to be the most significant predictor of severe diarrhea
`and mucositis. Vitamin B12 deficiency is usually attributable
`to malabsorption of the vitamin, which suggests that gastro-
`intestinal pathology is present in the majority of patients
`experiencing B12 deficiency (31). These patients, may, there-
`fore, experience greater toxicity from antifolates, agents with
`known gastrointestinal side effects.
`Simultaneous elevations in both tHcy and MMA with con-
`centrations above the respective third quartiles resulted in a
`striking increase in the prevalence of severe hematological or
`nonhematological toxicity. As such, the relative risk of an
`individual patient with values in these quartiles developing
`severe toxicity during treatment with pemetrexed was dra-
`matically increased. Although the confidence interval for this
`observation is quite large because of the small number of
`patients involved (n ⫽ 19), the magnitude of the increase in
`risk points to a likely relationship between elevations of both
`markers and a substantial risk of toxicity.
`
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`
`Molecular Cancer Therapeutics
`
`551
`
`Despite the high degree of predictability provided by base-
`line tHcy and MMA levels, we were unable to identify all of
`the patients at risk for severe toxicity using these markers.
`This may have been attributable to interindividual differences
`or other untested pharmacological and biological variables
`not characterized in the present study, such as cell mem-
`brane transport, the formation of polyglutamates, or levels of
`the pemetrexed targets thymidylate synthase, DHFR, and
`GARFT/AICARFT.
`Standard medical therapy in response to severe toxicity
`after treatment with antifolate drugs has involved the admin-
`istration of reduced folates (e.g., leucovorin) to rescue pa-
`tients, rather than prophylactically administering folate to
`those who may be at increased risk for toxicity if given
`antifolate therapy. This study has demonstrated that tHcy
`