Systemic Exposure to Thiopurines and Risk of Relapse in Children With Acute Lymphoblastic Leukemia: A Children’s Oncology Group Study

Importance  Variability in prescribed doses of 6-mercaptopurine (6MP) and lack of adherence to a 6MP treatment regimen could result in intra-individual variability in systemic exposure to 6MP (measured as erythrocyte thioguanine nucleotide [TGN] levels) in children with acute lymphoblastic leukemia (ALL). The effect on relapse risk of this variability is unknown.

Objective  To determine the effect of high intra-individual variability of 6MP systemic exposure on relapse risk in children with ALL.

Design, Setting, and Participants  We used a prospective longitudinal design (Children’s Oncology Group study [COG-AALL03N1]) to monitor 6MP and disease relapse in 742 children with ALL in ambulatory care settings of 94 participating institutions from May 30, 2005, to September 9, 2011. All participants met the following eligibility criteria: (1) diagnosis of ALL at 21 years or younger; (2) first continuous remission in progress at the time of study entry; (3) receiving self-, parent-, or caregiver-administered oral 6MP during maintenance therapy; and (4) completion of at least 6 months of maintenance therapy at the time of study enrollment. The median patient age at diagnosis was 5 years; 68% were boys; and 43% had National Cancer Institute–based high-risk disease.

Main Outcomes and Measures  Daily 6MP regimen adherence was measured over 68 716 person-days using an electronic system that recorded the date and time of each 6MP bottle opening; adherence rate was defined as the ratio of days that a 6MP bottle was opened to days thata 6MP bottle was prescribed. Average monthly 6MP dose intensity was measured over 120 439 person-days by dividing the number of 6MP doses actually prescribed by the number of planned protocol doses (75 mg/m²/d). Monthly erythrocyte TGN levels (pmol/8 × 10⁸ erythrocytes) were measured over 6 consecutive months per patient (n = 3944 measurements). Using intra-individual coefficients of variation (CV%), patients were classified as having stable (CV% <85th percentile) vs varying (CV% ≥85th percentile) indices. Median follow-up time was 6.7 years from the time of diagnosis.

Results  Adjusting for clinical prognosticators, we found that patients with 6MP nonadherence (mean adherence rate <95%) were at a 2.7-fold increased risk of relapse (95% CI, 1.3-5.6; P = .01) compared with patients with a mean adherence rate of 95% or greater. Among adherers, high intra-individual variability in TGN levels contributed to increased relapse risk (hazard ratio, 4.4; 95% CI, 1.2-15.7; P = .02). Furthermore, adherers with varying TGN levels had varying 6MP dose intensity (odds ratio [OR], 4.5; 95% CI, 1.5-13.4; P = .01) and 6MP drug interruptions (OR, 10.2; 95% CI, 2.2-48.3; P = .003).

Conclusions and Relevance  These findings emphasize the need to maximize 6MP regimen adherence and maintain steady thiopurine exposure to minimize relapse in children with ALL.

Durable remissions in children with acute lymphoblastic leukemia (ALL) require about a 2-year maintenance therapy phase that includes daily self-, parent-, or caregiver-administered oral 6-mercaptopurine (6MP).¹ This drug exerts cytotoxic effects, in part, through conversion to thioguanine nucleotide metabolites (TGN) that are incorporated into DNA with resultant damage.^2-4 Systemic exposure to 6MP (as measured by erythrocyte TGN levels) is determined by the dose prescribed by the clinician, adherence by the patient or parent to the prescribed dose, absorption of 6MP from the gastrointestinal tract, and its metabolism by enzymes such as thiopurine methyltransferase (TPMT).

Previous studies have examined the impact of prescribed dose,^5-7 adherence to prescribed dose,^8-10 and erythrocyte TGN levels^11,12 on relapse risk. There is emerging evidence in non-ALL as well as ALL settings that drug interruptions result in emergence of resistance^13-15 and relapse.¹⁶ Thus, variability in dose intensity and/or adherence could contribute to variability in systemic exposure to 6MP. However, the contribution of intra-individual variability of 6MP systemic exposure to relapse in children with ALL remains unexamined.

We enrolled a multiethnic and geographically diverse cohort of children with ALL receiving maintenance therapy per Children’s Oncology Group (COG) protocols. For a period of 6 consecutive months per patient, we documented all prescribed 6MP doses, monitored adherence to the prescribed dose, and measured erythrocyte 6MP metabolites monthly. We used this information to examine the contribution of intra-individual variability in adherence, dose intensity, and erythrocyte TGN levels to relapse risk.

Box Section Ref ID

Ninety-four COG member institutions (eTable 1 in Supplement) enrolled participants into this study (COG-AALL03N1) after obtaining approval from local institutional review boards. Written informed consent and/or assent was obtained from patients and/or their parents or legal guardians. Eligibility criteria were (1) diagnosis of ALL at 21 years or younger; (2) first continuous remission in progress at the time of study entry; (3) receiving self-, parent-, or caregiver-administered oral 6MP during maintenance therapy; and (4) completion of at least 6 months of maintenance therapy at the time of study enrollment. The study schema for COG-AALL03N1 is detailed in eTable 2 and eFigure 2 in Supplement. Patients enrolled into COG-AALL03N1 were comparable to patients enrolled in parent COG therapeutic protocols (eTable 3 in Supplement), with the following exceptions: (1) an overrepresentation in COG-AALL03N1 of Hispanics, Asians, and African Americans (due to purposeful overenrollment of these racial and ethnic groups in COG-AALL03N1); (2) an overrepresentation of male participants in COG-AALL03N1; and (3) a slightly younger age at diagnosis of ALL among those enrolled in COG-AALL03N1.

Leukocyte DNA was used to determine TPMT genotype status by using polymerase chain reaction–based methods specific for the TPMT *2, *3A, *3B, and *3C variant alleles.¹⁷

Participating institutions submitted monthly reports detailing prescribed 6MP doses for each day and methotrexate doses for each week of the preceding month along with dates and reasons (eg, illness, toxic effects) when 6MP or methotrexate doses were withheld. The 6MP and methotrexate doses actually prescribed were divided by the planned protocol doses (6MP, 75 mg/m²/d; methotrexate, 20 mg/m²/wk) to compute average monthly 6MP and methotrexate dose intensity.

Participating institutions submitted absolute neutrophil count (ANC) reports from blood samples obtained on days 1, 29, 57, 85, 113, and 141. These days corresponded to the days when patients returned for vincristine and steroid pulses.

Adherence to the prescribed 6MP regimen was monitored using an electronic monitoring device (TrackCap Medication Events Monitoring System [MEMS]; MWV Switzerland Ltd). This device uses microelectronic technology to record the date and time of each pill bottle opening (eFigure 1A in Supplement). At the end of the 6-month study period, data were downloaded (eFigure 1B in Supplement) from the MEMS caps. Adherence rate was defined as the number of days with a MEMS cap opening divided by the number of days prescribed for a pill bottle opening and expressed as a percentage.

Monthly erythrocyte TGN concentrations (pmol/8 × 10⁸ erythrocytes) were measured¹⁸ for 6 consecutive months per patient. The proportion of patients with evaluable samples ranged from 86.5% to 90.7% at the various time points (eTable 4 in Supplement).

Participating institutions submitted clinical status reports for up to 10 years from the time of diagnosis (every 6 months for the first 5 years, and then annually), detailing dates of last contact, relapse, second neoplasms (SNs), or non–relapse-related death during the interim period.

Patients were classified as having high systemic exposure to 6MP if their average 6MP dose intensity and erythrocyte TGN levels over the 6-month study period were at or above the median levels for the cohort. Otherwise, they were categorized as having low 6MP systemic exposure. Based on our previous observation of a high relapse risk associated with regimen adherence rates below 95%,^9,10 patients were classified as nonadherers for adherence rates below 95%; those with adherence rates of 95% or higher were classified as adherers.

The intra-patient coefficients of variation (CV%) for 6MP dose intensity, adherence, ANC, and TGN level were computed for patients with more than 1 measurement (98.8% of patients) as the ratio of the standard deviation (SD) to the mean for all measurements contributed by an individual patient and expressed as a percentage. After systematically exploring several cut points (eTable 5 in Supplement), a cut point at the 85th percentile for CV% was used to classify patients as having stable (<85th percentile) or varying (≥85th percentile) values.

All patients were in complete continuous remission at the time of study entry. Cumulative incidence of first relapse (at any site) was calculated, treating SNs and death due to noncancer causes as competing risks.¹⁹ The time at risk was calculated from the date when the maintenance phase was initiated (start of maintenance therapy) to the date of last contact, date of relapse, date of SNs, or date of noncancer death, whichever came first. Proportional subdistribution hazard (PSH) regression models (with SNs and death as competing risk,^20,21 and adjusting for National Cancer Institute [NCI] risk classification [based on age at diagnosis and presenting white blood cell count],²² race or ethnicity, methotrexate dose intensity, and ALL chromosomal abnormalities²³) were used to evaluate the impact of the following variables on relapse risk individually: TPMT genotype (wild type vs others); 6MP dose intensity, TGN level (as high vs low), and ANC (as high vs low values); and 6MP adherence (as adherers vs nonadherers and stable vs varying values) and 6MP treatment interruptions (yes vs no). Variables identified to have a significant association with relapse (P < .05) were entered into a multivariable model, adjusting for NCI risk classification, race or ethnicity, methotrexate dose intensity, and ALL chromosomal abnormalities. Time from start of maintenance therapy to study entry was included in all analyses to adjust for the varying intervals between start of maintenance therapy and study entry. The effects of the predictor variables on the competing risks (SMNs and noncancer deaths) were examined, and no association was identified (eTable 6 in Supplement).

Relationships between TGN level, 6MP regimen adherence, 6MP dose intensity, and ANC (treated as continuous variables) were assessed using the Pearson correlation coefficient.²⁴ Logistic regression was used to determine the characteristics of individuals with high intra-individual variability in TGN levels.

The PROCs PHREG and LOGISTIC packages of SAS software, version 9.3 (SAS Institute Inc) and SAS macro PSHREG were used for analysis. Two-sided tests showing P < .05 were considered to indicate statistical significance.

Table 1 summarizes the demographic and clinical characteristics of the 742 children with ALL enrolled in the study and the indices of systemic exposure to 6MP. Of these 742 patients, 464 were included in previous studies describing the racial or ethnic differences in adherence to oral 6MP regimens.^8-10 Median age at the time of diagnosis was 5 years (range, 1-19 years) and at the time of study entry, 6 years (range, 2-21 years). Sixty-eight percent of the participants were male; 43% presented with high-risk disease (using the NCI classification); and 6% presented with unfavorable ALL chromosomal abnormalities. Of the 742 patients, 470 (63%) provided MEMS-based 6MP adherence data for 68 716 person-days. The 470 patients with adherence data were comparable to the 272 who lacked adherence data with respect to clinical and demographic variables, indices of thiopurine exposure, length of follow-up, and disease recurrence (eTable 7 in Supplement).

After a median follow-up of 6.7 years from the time of diagnosis (range, 1.2-12.0 years), 62 patients experienced relapse of their primary disease; 10 patients developed SNs; and 3 died from other causes. The mean (SD) cumulative incidence of relapse was 9.0% (1.1%) at 6 years from the start of maintenance therapy.

The impact of indices of systemic thiopurine exposure on relapse risk was examined univariately (cumulative incidence of relapse). The association between each index and relapse risk was also examined after adjusting for NCI risk classification, ALL chromosomal abnormalities, race or ethnicity, methotrexate dose intensity, and time from start of maintenance therapy (adjusted univariate analysis; Table 2, model 1).

Relapse risk did not differ by low (<0.86) vs high (≥0.86) 6MP dose intensity. The mean (SD) cumulative incidence of relapse at 6 years for the low-intensity group was 10.1% (1.6%), while for the high-intensity group it was 7.9% (1.5%) (P = .20) (Figure 1A); hazard ratio (HR), 0.8; 95% CI, 0.4 to 1.5 (P = .50) (Table 2). Relapse risk also did not differ by varying (CV% ≥35.5) vs stable (CV% <35.5) 6MP dose intensity. The mean (SD) cumulative incidence of relapse at 6 years for the varying group was 13.8% (3.5%), while for the stable group it was 9.0% (3.5%) (P = .07) (Figure 1B); HR, 1.3; 95% CI, 0.7 to 2.7 (P = .40) (Table 2).

Relapse risk did not differ by low (<145.7) vs high (≥145.7) TGN level. The mean (SD) cumulative incidence of relapse at 6 years for the low-TGN group was 9.9% (1.6%), while for the high-TGN group it was 8.0% (1.5%) (P = .30) (Figure 1C); HR, 1.3; 95% CI, 0.8 to 2.3 (P = .30) (Table 2). Furthermore, relapse risk did not differ when red cell TGN level was treated as a continuous variable, dichotomized (deciles, quartiles), or categorized using absolute values (eTable 8 in Supplement).

However, patients with varying TGN levels (CV% ≥ 55.7) had a significantly higher risk of relapse (mean [SD] cumulative incidence, 20.0% [4.3%]) than patients with stable TGN levels (4.3% [1.1%]) (P < .001) (Figure 1D); HR, 2.3; 95% CI, 1.2 to 4.3 (P = .01) (Table 2).

Mean (SD) cumulative risk of relapse was higher among 6MP regimen nonadherers (13.9% [2.6%]) than among adherers (4.7% [1.3%]) (P = .001) (Figure 2); HR, 2.7, 95% CI, 1.3 to 5.6 (P = .01) (Table 2). Risk of relapse among patients with missing adherence data (n = 272) was 1.8-fold higher than among adherers (HR, 1.8; 95% CI, 0.9-3.8; P = .10). Nonadherers demonstrated high variability in adherence (mean CV%, 17.7), whereas adherers had stable adherence rates (mean CV% 2.5) (P < .001). Patients with varying adherence had a significantly higher risk of relapse than patients with stable adherence (HR, 2.6; 95% CI, 1.2-5.4; P = .01).

Nonadherence to oral 6MP regimens and varying TGN levels were examined in a multivariable regression model. As summarized by model 2 in Table 2, nonadherence emerged as the only predictor of relapse risk (HR, 2.5; 95% CI, 1.1-5.6; P = .03). The interaction between adherence and varying TGN level was not statistically significant (HR, 0.2; 95% CI, 0.04-1.4; P = .10).

Separate models were constructed for adherers and nonadherers by adjusting each predictor individually for NCI risk classification, ALL chromosomal abnormalities, race or ethnicity, methotrexate dose intensity, and time since start of maintenance therapy. Among adherers, varying TGN level emerged as the only variable associated with relapse risk (HR, 4.4; 95% CI, 1.2-15.7; P = .02) (Table 2, model 3). On the other hand, among nonadherers, varying TGN level was not associated with relapse (HR, 0.9; 95% CI, 0.3-2.6; P = .80) (Table 2, model 4). However, varying adherence was associated with an increased risk of relapse (HR, 2.7; 95% CI, 1.1-6.7; P = .03).

Correlations between absolute values of the indices of thiopurine exposure are summarized in eTable 9 in Supplement; correlations between varying indices are summarized in eTable 10 in Supplement. A positive correlation was observed between 6MP regimen adherence and erythrocyte TGN level (P = .002); a negative correlation was observed between adherence and 6MP dose intensity (P < .001) and between adherence and ANC (P < .001). A positive correlation was observed between varying adherence and varying TGN level (P < .001); a negative correlation was observed between varying adherence and varying ANC (P = .003). A positive correlation was observed between 6MP dose intensity and ANC (P < .001). A positive correlation was observed between 6MP dose intensity CV% and TGN level CV% (P < .001) and between 6MP dose intensity CV% and ANC CV% (P < .001).

As summarized in Table 3, we used multivariable logistic regression analysis to identify characteristics of patients with varying TGN levels. We examined the simultaneous impact of the following variables: stable vs varying 6MP dose intensity, low vs high 6MP dose intensity, drug treatment interruptions, and nonadherence to oral 6MP regimens. This multivariable analysis was adjusted for NCI risk groups, chromosomal abnormalities, race and ethnicity, and TMPT genotype. In the model for the entire cohort, 3 variables characterized individuals with varying TGN levels: varying 6MP dose intensity (odds ratio [OR], 2.4; 95% CI, 1.2-5.2; P = .02), drug treatment interruptions (OR, 2.2; 95% CI, 1.1-4.3; P = .03), and nonadherence to oral 6MP regimen (OR, 2.0; 95% CI, 1.1-3.6; P = .03). A similar analysis restricted to adherers revealed that varying 6MP dose intensity (OR, 4.5; 95% CI, 1.5-13.4; P = .01) and drug treatment interruptions (OR, 10.2; 95% CI, 2.2-48.3; P = .003) predicted for individuals with varying TGN levels. On the other hand, varying adherence was the only variable that characterized individuals with varying TGN level among nonadherers (OR, 1.06 per unit increase in CV% adherence; 95% CI, 1.03-1.09; P < .001).

We evaluated the impact of intra-individual variability in 6MP systemic exposure on relapse risk in children with ALL. We found that in addition to the previously reported association between nonadherence to oral 6MP regimen and relapse risk, high intra-individual variability in TGN level (due to varying 6MP dose intensity and drug treatment interruptions) contributed to an increased risk of relapse among those who adhered to their therapy regimens.

Unlike previous reports,^11,12,25 the current study identified no clear threshold for erythrocyte TGN levels that was associated with relapse risk. A previous study from St Jude Children’s Research Hospital also had not demonstrated such an association⁵; this lack of association was believed to be owing to high TGN levels that likely exceeded a minimum threshold for efficacy, coupled with the weekly clinic visits for parenteral methotrexate administration that likely reinforced adherence to the 6MP regimen (although adherence was not measured directly). Unlike the single-institution St Jude study,⁵ the current study enrolled patients from 94 geographically diverse institutions, and patients returned to the clinic every 28 days for vincristine and steroid pulses (and not weekly), since the weekly methotrexate dose was taken orally at home. Observed TGN levels in the current study were lower than those in the previous single-institution study,⁵ yet no association was identified between TGN levels and relapse risk, even after accounting for objectively measured 6MP regimen adherence. Furthermore, no association was identified between relapse risk and 6MP dose intensity in the current study—a finding that differed from previous studies.^5,16 The impact of TGN levels and 6MP dose intensity on relapse risk is likely dependent on other elements of ALL therapeutic regimens.

In the current report, we reaffirmed the increased risk of relapse associated with adherence rates lower than 95% reported in the previous studies using this cohort.^9,10 However, in the current study, we extended our findings beyond our previous reports to show that nonadherent patients were more likely to have higher 6MP dose intensity as well as high and stable ANC compared with adherent patients. These findings may be explained by the fact that high, nonfluctuating ANC (due to nonadherence) may trigger dose escalation (high 6MP dose intensity) by the clinician.

In addition, our findings suggest that among adherers, high intra-individual variability in TGN level was associated with an increased risk of relapse. The association between high variability in TGN level and relapse could possibly be explained by the ability of residual malignant stem cells to escape control when the minimum threshold of cytotoxic metabolites needed for efficacy was not maintained. Patients with varying TGN levels were more likely to have varying 6MP dose intensity and 6MP drug treatment interruptions. The variability in 6MP dose intensity correlated with variability in ANC. Thus, taken together, the findings suggest that among adherers, variability in drug dosing and drug treatment interruptions (possibly to maintain ANC within a certain range) are associated with varying TGN levels, and this variability in TGN levels is associated with an increased risk of relapse.

A previous study had shown that a significantly larger proportion of maintenance therapy was omitted among patients in the lowest quartile for 6MP dose intensity.⁵ These findings suggest that an aggressive approach to titrating the dose of 6MP to maintain ANC within a certain window could result in drug treatment interruptions and in high variability in 6MP dose intensity, which in turn could then result in varying TGN levels and could increase the risk of relapse.

Treatment interruptions are a predictor of drug resistance in HIV-positive individuals¹³ and in ovarian¹⁴ and lung¹⁵ cancer. In the pediatric ALL setting, studies suggest that excessive toxic effects may compromise outcome due to interruptions of therapy.^6,26 In fact, patients with discontinuations of 6MP treatment that spanned greater than 10% of the maintenance therapy period had an increased risk of hematologic relapse.¹⁶ Despite these observations, controversy exists whether outcome can be improved by advancing 6MP dose to the point of toxicity (at the risk of dose omission due to neutropenia) or whether it is better to proceed with smaller dose increments, thereby causing less severe neutropenia.^6,7,16,25-29 There exists a lack of standardized guidelines in responding to variation in ANC, as illustrated by the varying recommendations for ANC-based titration of 6MP on the parent therapeutic protocols (eTable 11 in Supplement). However, whether a dosing approach with a goal of minimizing variation in daily 6MP dose will improve outcomes compared with an approach that attempts to maintain full protocol dose at the expense of drug treatment interruptions has not been tested.

The current study found that nonadherers to the 6MP treatment regimen demonstrate high variability in adherence, which was associated with an increased risk of relapse. Patients with varying adherence were more likely to have varying TGN levels. Thus, the association between relapse and high intra-individual variability in adherence could be explained in part through increased variability in TGN levels. However, the lack of a direct association between varying TGN level and relapse suggests that lack of adherence to other medication regimens (not measured in the current study) could possibly contribute to relapse.

The present study had certain limitations. It is difficult to draw cause-and-effect relationships from observational studies such as this one, especially since it did not capture adherence to the regimens of other oral medications such as methotrexate and corticosteroids. Thus, the association between high variability in TGN levels and relapse risk among adherers, as well as the attribution of this high variability in TGN levels to varying dose intensity and drug treatment interruptions needs to be tested and confirmed in the setting of a prospective clinical trial. Such a trial would explore 6MP dosing strategies that examine the potential benefit derived from minimizing variation in daily 6MP dose compared with standard approaches that maintain full protocol dose at the expense of drug treatment interruptions.

Another limitation is the relatively small number of events in certain subgroups that precluded the emergence of a statistically significant association. The small sample in these subgroups was evidenced by the wide CIs surrounding the estimates. An example of such an association includes the 4.4-fold increased risk of relapse among individuals with the TPMT wild-type genotype compared with those with TPMT heterozygous or mutant genotype.

These limitations notwithstanding, the current study has several strengths, including the prospective design, the large geographically and ethnically diverse study population, and the evaluation of simultaneously captured regimen adherence, dose intensity, and red cell TGN levels on relapse risk.

Taken together, the findings from the present study emphasize the need to maximize adherence to 6MP maintenance therapy regimens and to maintain steady thiopurine exposure to minimize relapse in children with ALL.

Accepted for Publication: February 6, 2015.

Corresponding Author: Smita Bhatia, MD, MPH, University of Alabama at Birmingham, 1600 Seventh Ave S, Lowder 500, Birmingham, AL 35233 (sbhatia@peds.uab.edu).

Published Online: March 26, 2015. doi:10.1001/jamaoncol.2015.0245.

Author Contributions: Dr Bhatia had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study concept and design: Bhatia, Landier, Bostrom, Lew.

Acquisition, analysis, or interpretation of data: Bhatia, Landier, Hageman, Chen, Kim, Sun, Kornegay, Evans, Angiolillo, Casillas, Lew, Maloney, Mascarenhas, Ritchey, Termuhlen, Carroll, Wong, Relling.

Drafting of the manuscript: Bhatia, Landier, Kim, Sun, Wong.

Critical revision of the manuscript for important intellectual content: Bhatia, Landier, Hageman, Chen, Kornegay, Evans, Angiolillo, Bostrom, Casillas, Lew, Maloney, Mascarenhas, Ritchey, Termuhlen, Carroll, Wong, Relling.

Statistical analysis: Bhatia, Chen, Kim, Sun, Wong.

Obtained funding: Bhatia, Relling.

Administrative, technical, or material support: Bhatia, Landier, Hageman, Kornegay, Evans, Angiolillo, Casillas, Termuhlen, Carroll, Relling.

Study supervision: Bhatia, Evans, Lew, Carroll, Wong, Relling.

Conflict of Interest Disclosures: St Jude Children’s Research Hospital allocates a portion of the income it receives from licensing inventions and tangible research materials to those researchers responsible for creating the intellectual property; Drs Evans and Relling receive a portion of the income St Jude receives from licensing patent rights related to testing for TPMT genetic polymorphisms. No other disclosures are reported.

Funding/Support: This study was supported in part by grants R01 CA096670, U10 CA098543, U10 CA095861, R37 CA36401, CA21765 GM92666, CA156449, and M01-RR00043 from the National Institutes of Health and by the American Lebanese Syrian Associated Charities.

Role of the Funder/Sponsor: The funding institutions had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; or preparation, review, or approval of the manuscript.

Previous Presentation: This study was presented as a podium presentation at the American Society of Hematology Conference; December 6-10, 2013; New Orleans, Louisiana.