Temozolomide in Aggressive Pituitary Tumors and Metastatic PitNETs: A Brazilian Multicenter Real-World Cohort Study

Temozolomide in Aggressive Pituitary Tumors and Metastatic PitNETs: A Brazilian Multicenter Real-World Cohort Study | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Temozolomide in Aggressive Pituitary Tumors and Metastatic PitNETs: A Brazilian Multicenter Real-World Cohort Study Isabelle Amaro, Camila Macedo, Luciana Naves, Lidiana Bandeira, and 18 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8428238/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Mar, 2026 Read the published version in Pituitary → Version 1 posted 9 You are reading this latest preprint version Abstract Purpose To evaluate the real-world efficacy and safety of temozolomide (TMZ) in aggressive and metastatic pituitary neuroendocrine tumors (PitNETs) in a Latin American setting, addressing whether TMZ achieves meaningful radiological and biochemical disease control with acceptable toxicity. Methods We conducted a retrospective multicenter study across Brazilian reference centers including patients with aggressive/metastatic PitNETs treated with TMZ and followed for ≥ 6 months. Radiological response was assessed using RECIST 1.1. For functioning PitNETs, biochemical response was assessed using prespecified hormonal criteria. Adverse events were collected from medical records. Results Thirty patients were included (mean age 29.5 years; 53% female). All tumors were macroadenomas and 56% were giant (> 4 cm). Twenty-one PitNETs were functioning and four were metastatic. Ki-67 was > 3% in 73% of cases. Median time from diagnosis to TMZ initiation was 100 months. Radiological disease control rate (partial response or stable disease) was 96.7%. Among functioning tumors, biochemical disease control rate was 81.3%, with an objective biochemical response rate (complete + partial response) of 68.8%. Adverse events occurred in 66% of patients, most commonly nausea and myelotoxicity. Conclusion In this multicenter Brazilian real-world cohort, TMZ provided high radiological and biochemical disease control with an acceptable safety profile, supporting TMZ as preferred first-line systemic chemotherapy for aggressive/metastatic PitNETs after failure of standard therapies. aggressive pituitary adenoma pituitary carcinoma pituitary neuroendocrine tumors temozolomide aggressive PitNETs MGMT Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Pituitary neuroendocrine tumors (PitNETs) are among the most common intracranial neoplasms, accounting for approximately 15% of primary brain tumors (Melmed 2020 ). Although the majority of PitNETs follow a benign, slow-growing course, a clinically significant subset exhibits aggressive behavior, characterized by rapid growth and resistance to standard therapies (Dekkers et al. 2020 ; Kasuki & Raverot 2020 ; Raverot et al. 2025). Aggressive PitNETs (APTs) remain a major therapeutic challenge. According to the 2018 European Society of Endocrinology (ESE) guidelines (Raverot et al. 2018) which is reaffirmed by the new 2025 update, APTs are defined not solely by histopathological features, but by clinically relevant progression despite optimized multimodal therapy, including surgery, radiotherapy, and standard medical treatment (Jotanovic et al. 2024; Casar-Borota et al. 2025 ; Raverot et al. 2025). The 2025 consensus emphasizes that the current histological markers (such as Ki-67, mitotic count, and p53) have limited predictive accuracy, and no validated biomarker currently exists that can reliably predict aggressiveness or treatment response. Temozolomide (TMZ), an oral DNA-alkylating agent, is currently the only systemic therapy with established efficacy in APTs and metastatic PitNETs (Reilly et al. 1993; Raverot et al. 2012; Syro et al. 2018; Whitelaw 2019 ). It has been recommended as a first-line chemotherapeutic option for APTs and metastatic PitNETs after failure of standard treatments (Raverot et al. 2025). Its mechanism involves methylation-induced DNA damage at the O6-guanine position, ultimately leading to tumor cells apoptosis (Syro et al. 2018; Whitelaw 2019 ). However, therapeutic response remains highly variable and complete remissions are rare (Lamas et al. 2023). Although MGMT promoter methylation and low MGMT protein expression have been proposed as predictors of response, their clinical utility remains limited and inconsistent across cohorts (McCormack et al. 2009; Syro et al. 2011; Kontogeorgos & Thodou 2019 ). Most of the available data on the efficacy and safety of TMZ in APT originates from European and North American centers (Hui-Zheng & Lei 2024). To date, other populations, particularly from low- and middle-income countries, have not yet been adequately represented in the literature. This underrepresentation may be clinically relevant, as therapeutic responses could be influenced not only by genetic and epigenetic differences, but also by socioeconomic, healthcare system, and cultural factors that affect diagnosis, treatment access, and follow-up adherence (Özdemir & Dotto 2017 ; Li et al. 2024). To address this gap, the present study provides the first comprehensive multicenter analysis of TMZ treatment in APTs conducted in Brazil. By evaluating clinical outcomes, safety profiles, and therapeutic trajectories across multiple national reference centers, this study contributes real-world evidence from an underrepresented population and adds to the global effort to optimize care for these rare and challenging tumors. Methods Study design and participants This multicenter retrospective cohort study included eight Brazilian referral centers specialized in pituitary tumors. We screened patients with aggressive pituitary tumors (APTs) or metastatic PitNETs who received temozolomide (TMZ) between 2005 and 2024. The index date was the first day of TMZ cycle 1. Eligible patients had ≥ 6 months of follow-up from TMZ initiation. Aggressiveness was defined as radiological progression and/or biochemical progression despite conventional therapies (surgery, radiotherapy, and subtype-appropriate medical therapy). Metastatic PitNETs were defined by the presence of craniospinal and/or systemic metastases. Patients were excluded if they received TMZ for < 3 cycles (or < 3 months) or if severe comorbidities precluded reliable assessment of outcomes. The study was approved by the local ethics committees (with waiver of informed consent, as applicable), and data were de-identified before central analysis. Data collection Clinical, pathological, and treatment variables were extracted from medical records, including tumor subtype, baseline tumor measurements, Ki-67 labeling index, p53 immunohistochemistry, and prior therapies (surgery, radiotherapy, and pituitary-directed medications). TMZ treatment, treatment interruptions, and total number of cycles were recorded. Concomitant therapies during TMZ were documented. Response assessment Radiological response was assessed on sellar MRI (contrast-enhanced when available) using the longest tumor diameter (1D) and categorized per RECIST 1.1 as complete response (CR), partial response (PR; ≥30% decrease), stable disease (SD; <30% decrease and < 20% increase), or progressive disease (PD; ≥20% increase and/or new lesions/non-target progression). Imaging assessments followed each center’s routine schedule; when multiple scans were available and the best overall radiological response were recorded. Radiological objective response rate (ORR) was defined as CR + PR, and radiological disease control rate (DCR) as CR + PR + SD. Biochemical response was assessed only in functioning PitNETs using subtype-appropriate biochemical markers and consistent pre- and on-treatment timepoints. Responses were categorized as complete (CR; normalization of the relevant hormone measure), partial (PR; >20% reduction), stable (SD; ≤20% variation), or progressive (PD; >20% increase). Biochemical response rate was defined as CR + PR, and biochemical disease control as CR + PR + SD among functioning tumors with available biochemical data. Concordance between radiological (RECIST 1.1) and biochemical responses in functioning tumors was assessed using cross-tabulation. Data visualization was performed using heatmaps to display response pairing frequencies and alluvial diagrams to illustrate the flow of patients between response categories. Bias To mitigate bias inherent to retrospective multicenter data, we applied prespecified and standardized definitions for eligibility and outcomes (including RECIST 1D for radiological response and predefined biochemical thresholds for functioning tumors). Data were extracted using a harmonized case-report form and centrally checked for internal consistency. When multiple on-treatment assessments were available, best overall radiological response was recorded according to uniform criteria. Analyses of biochemical response were restricted to functioning tumors with available data, with no imputation for missing values. Safety Adverse events were collected from medical records and laboratory monitoring during TMZ. Events were categorized as gastrointestinal and hematologic and graded when feasible using CTCAE criteria; hematologic toxicity was defined based on standardized thresholds for neutropenia and thrombocytopenia. Outcomes Primary outcomes were radiological disease control and overall safety/tolerability. Secondary outcomes included biochemical disease control in functioning tumors and the distribution of radiological and biochemical response categories (CR/PR/SD/PD) during TMZ. Exploratory outcomes included agreement between radiological and biochemical trajectories in functioning PitNETs with available biochemical data. Statistical analysis Categorical variables are presented as n (%) and continuous variables as mean ± SD or median (IQR), as appropriate. Associations between categorical predictors and response were evaluated using Fisher’s exact test (or chi-square when applicable). Ki-67 was analyzed as a continuous variable and dichotomized at 3% and 10%. Continuous Ki-67 values across response groups were compared using the Kruskal–Wallis test with post-hoc Dunn tests when appropriate. A composite “high-proliferation” phenotype (Ki-67 ≥ 10% plus p53 positivity) was explored. Agreement between radiological and biochemical outcomes was evaluated among functioning PitNETs with available hormonal data. For the primary agreement analysis, responses were dichotomized as disease control (CR/PR/SD) versus progression (PD) in each domain, and concordance was summarized using percent agreement and Cohen’s kappa. As a descriptive secondary analysis, cross-tabulations of the original response categories (CR/PR/SD/PD) were reported to characterize discordant patterns. Analyses were two-tailed with α = 0.05 and performed in R (RStudio). This study is reported in accordance with the STROBE guidelines for cohort studies (Supp Table 1 ; Supp Fig. 2 ). Table 1 Clinical characteristics of patients treated with temozolomide (N = 30) Clinical characteristics of patients (N = 30) Age at diagnosis 29.5 years (7–56 years) Sex (female / male) 53% / 47% Tumor size at diagnosis 4.2 cm (1.2–12 cm) Giant Tumors (≥ 4 cm) 56% Metastatic PitNETs 4 / 30 patients Tumor type Prolactinoma 8 patients Non functioning 9 patients Cushing 8 patients Acromegaly 5 patients Treatment before TMZ Surgery 2.6 surgeries (1–6) Radiotherapy 19 patients Time between diagnosis and TMZ treatment 100 months Duration of TMZ treatment 10.3 months Análise Imunohistoquímica Ki-67 (27/30) 13.6% (1–30%) p53 (22/30) 59% positive Ethics approval. This study was approved by the Ethics Committee of Hospital das Clinicas da Faculdade de Medicina da Universidade de São Paulo (CAAE: 86575324.3.0000.0068). Results A total of 30 patients with aggressive or metastatic PitNETs were included (Table 1 ; suppl table 1). The mean age at diagnosis was 29.5 years (range 7–56), and 53% of the cohort were female (Table 1 , Fig. 1 ). All patients had macroadenomas, with a mean tumor diameter of 4.2 cm (range 1.2–12.0 cm); 56% were classified as giant tumors (≥ 4 cm). Four patients (13.3%) were diagnosed with metastatic PitNETs. Among the 30 patients, 70% (n = 21) had functioning tumors, while the remaining 30% (n = 9) had non-functioning PitNETs (Fig. 1 ). Among the functioning tumors, prolactinomas were the most frequent subtype (n = 8; 40%), followed by corticotroph tumors causing Cushing’s disease (n = 8; 40%), including one case of Crooke cell PitNET) and somatotroph tumors associated with acromegaly (n = 5; 25%). The Ki-67 proliferation index was assessed by immunohistochemical analysis in 27 patients (90%) and was greater than 3% in 22/30 (73%) of cases, with a mean Ki-67 of 13.6%. p53 expression was analyzed in 22 patients and was positive in 59% (Table 1 ). Before initiating TMZ, patients had undergone a median of 2.6 surgical procedures (range: 1–6), and 19 patients (63%) had received radiotherapy. The median interval between initial diagnosis and initiation of TMZ therapy was 100 months, and the median duration of TMZ treatment was 10.3 months. The main indications for initiating TMZ therapy were radiological tumor progression (53%, n = 16) and worsening biochemical markers in functioning tumors (41%, n = 13). In a smaller subset (n = 3), TMZ was introduced due to metastases. Radiological objective response rate (ORR; CR + PR) was 23.3% (7/30; 95% CI 9.9–42.3) and radiological disease control rate (DCR; PR + SD) was 96.7% (29/30; 95% CI 82.8–99.9). Progressive disease (PD) occurred in 3.3% (1/30) (Fig. 2 ). It is noteworthy that one patient (case 14) with a corticotroph functioning PitNET exhibited a 97% tumor reduction after six months of TMZ treatment. Although considered as a partial responder, the magnitude of tumor shrinkage was remarkably significant. Additionally, a Crooke cell PitNET achieved stable disease under TMZ. Progressive disease occurred in one case. Taken together, 96.7% of patients (n = 29) achieved disease control (SD + PR) during TMZ treatment. The response profiles to treatment were remarkably similar between functioning and non-functioning PitNETs. The rates of partial response were nearly identical (22.2% vs. 23.8%, respectively; p = NS), and the proportion of patients achieving stable disease was comparable (77.8% vs. 66.7%; p = NS). The efficacy of TMZ varied significantly among functioning PitNETs (p = 0.012). Prolactinomas (n = 8) demonstrated uniform radiological stability (8/8, 100%) without measurable tumor regression; however, biochemically, partial control was achieved in 87,5% (7/8), while 12,5% (1/8) showed biochemical progression. In patients with Cushing’s disease (n = 8), one patient exhibited disease progression. Biochemically, 16% (1/6) achieved complete hormonal remission, 16% (1/6) showed partial improvement, 33% (2/6) remained stable, and 33% (2/6) experienced biochemical progression. The biochemical response rate (CR + PR) was 68.8% (11/16) and biochemical disease control (CR + PR + SD) was 81.3% (13/16), while biochemical progression occurred in 18.8% (3/16). Overall, acromegaly was associated with the highest rate of radiological response, whereas Cushing’s disease demonstrated the greatest proportion of progression events, both radiologically and biochemically, with a quarter of patients (25%) experiencing disease progression despite treatment, representing all radiological progression events within the cohort. Tumour lineage was available for 27 of 30 patients (PIT1 n = 14; TPIT n = 10; SF1 n = 3). The percentage change in maximal tumour diameter on temozolomide was broadly similar across lineages (median change: PIT1 − 13.8%, TPIT − 15.9%, SF1 − 4.8%; Fig. 3 A). Best RECIST response distributions are shown in Fig. 3 B: PIT1 (PR 4/14, SD 10/14), TPIT (PR 2/10, SD 7/10, PD 1/10), and SF1 (PR 1/3, SD 2/3). The only radiological progression event occurred in the TPIT group; all PIT1 and SF1 cases achieved radiological disease control (PR/SD). Among functioning tumors with available biochemical data (n = 16), biochemical response (CR + PR) was 68.8% (11/16; 95% CI 41.3–89.0) and biochemical disease control (CR + PR + SD) was 81.2% (13/16; 95% CI 54.4–96.0). When biochemical responses among functioning PitNETs with available hormonal data (n = 16) were dichotomized as disease control (CR/PR/SD) versus progression (PD), overall concordance between radiological and biochemical outcomes was 87.5% (14/16), with moderate agreement by Cohen’s kappa (κ = 0.44). Importantly, biochemical progression occurred in 12.5% of patients (2/16) despite radiological disease control, indicating that biochemical and radiological trajectories may diverge in a subset of cases. Although radiological and biochemical responses were generally concordant (68.7%), distinct patterns of dissociation were observed (Fig. 4 ). As shown in the heatmap (Fig. 4 - left), two patients (12.5%) presented with biochemical progression despite achieving radiologically stable disease. The alluvial diagram (Fig. 4 - right) further illustrates this divergence, highlighting that radiological stability does not necessarily guarantee hormonal control in a subset of aggressive PitNETs. Radiological outcomes differed between APTs and metastatic PitNETs. Among the four patients with metastatic disease, two patients (50%) exhibited partial response, while the remaining two (50%) experienced disease progression, with no cases of stable disease. In contrast, among the 26 patients with aggressive non-metastatic tumors, disease stabilization was the most frequent outcome (76,9%), followed by partial response (19.2%), with 1 case of progression. The potential impact of radiotherapy (RT) timing on radiological response was evaluated across the cohort. Patients were categorized into four groups based on the sequence of RT in relation to the study treatment: RT before, RT after, concurrent RT, and no prior RT. Statistical comparison between the two largest groups—'RT before' (n = 19) and 'RT after' (n = 6)—revealed no significant association between radiotherapy timing and treatment outcome (p = 0.65). A high rate of disease stabilization and partial response characterized both groups. The exploratory analysis of the proliferation marker Ki-67 did not demonstrate a statistically significant association with tumor diameter response following TMZ treatment. When evaluated categorically, the chi-square test revealed no significant difference in response proportions among Ki-67 groups (χ² = 2.47; p = 0.65). Similarly, considering the continuous Ki-67 percentage, the Kruskal–Wallis test showed no significant difference between response groups (H = 2.17; p = 0.34). The Dunn post-hoc test with Bonferroni correction confirmed the absence of relevant pairwise differences (p > 0.6 for all comparisons). Although no statistical significance was reached, there was a numerical trend toward higher Ki-67 values in tumors with poorer response: the mean Ki-67 ranged from 12.3 ± 10.0% in stable tumors to 17.8 ± 11.1% in those showing reduction, reaching 20% in progressive cases. When Ki-67 was dichotomized (< 10% vs ≥ 10%), no statistically significant difference was observed in tumor response distribution (χ² = 3.11; p = 0.21). Nevertheless, all tumors that progressed belonged to the Ki-67 ≥ 10% group. The p53 expression status, assessed by immunohistochemistry and classified as positive or negative, was analyzed with respect to TMZ response. Within the available cohort, p53 positivity was observed in a larger proportion of tumors ( n = 14; 64%) compared with p53-negative cases ( n = 8). The chi-square test did not reveal a statistically significant association between p53 expression and treatment response ( p = 0.17). However, it is noteworthy that all progressive cases (response = 4) occurred exclusively among p53-positive tumors, while none were observed in the p53-negative group. Since neither Ki-67 nor p53 expression alone reached statistical significance, we explored a composite variable integrating both proliferation and genomic instability markers. Tumors exhibiting Ki-67 ≥ 10% combined with p53 positivity were categorized as high-proliferative lesions. All cases with progressive disease (response = 4) and half of those with partial response occurred within this high-proliferation category, whereas stable disease predominated among the remaining tumors (Ki-67 < 10% and/or p53-negative). Among the 30 patients included, four (13.3%) were classified as metastatic PitNETs, comprising one case each of prolactinoma, non-functioning tumor, Cushing’s disease, and acromegaly. Treatment was generally well-tolerated, with 66.6% (20/30) of patients experiencing at least one adverse event. The majority were classified as grade 1 according to CTCAE criteria (Freites-Martinez et al. 2021). The most frequently reported events were gastrointestinal, with nausea affecting 70% of patients who reported any adverse event, followed by vomiting in 20%. Hematological toxicity (myelotoxicity) was observed in 45% of these patients. Other non-hematological events included asthenia and headache (15% each), while cutaneous reactions were infrequent (5%). Notably, 33% of the cohort reported no treatment-related adverse events. The long-term tolerability of the treatment regimen was assessed by analyzing treatment discontinuations due to adverse events. Dose reduction was required in patients with grade 3 myelotoxicity, and treatment was discontinued in four cases (13.3%), due to grade 4 myelotoxicity, one due to grade 3 myelotoxicity, and one due to grade 3 asthenia. No treatment-related deaths were reported. The discontinuation rate was 5% (1/20) in patients treated for less than 12 months, compared to 30% (3/10) in those treated for more than 12 months, suggesting that cumulative toxicity may become a more prominent factor influencing treatment feasibility over extended durations. Discussion This Brazilian multicenter cohort provides the largest Latin American dataset to date on the use of TMZ as a single therapy in aggressive and metastatic PitNETs. The radiological disease control rate of 96.7%—including 23.3% partial responses and 70% of stable disease is consistent with prior studies, which have reported control rates ranging from 60% to 85% (Hui-Zheng & Lei 2024). While no complete responses were observed, this aligns with most published cohorts, where complete radiological remission remains exceedingly rare. These findings reinforce the reproducibility of TMZ efficacy across geographic and healthcare settings, while underscoring the importance of generating data from underrepresented populations. Compared to previously published studies, our cohort from the Brazilian multicenter study demonstrated distinct demographic characteristics. With a mean age of 29.5 years, the Brazilian participants were notably younger than those in other cohorts (Losa et al. 2016; Lizzul et al. 2020; Lamas et al. 2023; Liu et al. 2025), whose mean ages ranged from 40.5 to 58.3 years. Additionally, a higher proportion of women was observed in the Brazilian cohort (53%), whereas the other studies predominantly included male participants, with rates reaching up to 87.5% (Losa et al. 2016; Lizzul et al. 2020; Lamas et al. 2023; Liu et al. 2025), These findings suggest potential differences in the epidemiological profile or inclusion criteria across studies, which should be taken into account when interpreting and comparing the results. Importantly, our results offer novel insights when stratified by tumor subtype, size, functionality, and aggressiveness. Patients with acromegaly exhibited a notably high rate of partial radiological response and no disease progression, a finding that may reflect a distinct somatotroph tumor biology or increased sensitivity to alkylating agents. In contrast, patients with prolactinomas and Cushing’s disease had higher rates of disease progression, suggesting possible lineage-related differences in TMZ responsiveness, which merit further investigation. Within the corticotroph spectrum, Crooke cell PitNETs are a high-risk variant often associated with aggressive behavior. The stable disease observed in the Crooke cell case in our cohort suggests that even histologically aggressive subtypes may retain clinically relevant chemosensitivity to temozolomide. Metastatic PitNETs exhibited a dichotomous pattern. Although the small sample precludes definitive conclusions, the data highlight the therapeutic challenge posed by metastatic PitNETs and the need for alternative strategies in this subgroup. The unpredictable clinical behavior of APT is partly explained by the absence of robust prognostic markers at diagnosis. In our series, Ki-67 and p53 expression alone did not reach statistical significance in predicting tumor response, corroborating previous evidence that conventional proliferation markers are insufficient when evaluated independently (Raverot et al. 2021; Trouillas et al. 2020). Although MGMT expression and promoter methylation have been proposed as predictors of TMZ responsiveness, their utility remains inconsistent and were not assessed in this cohort. We did not assess MGMT expression in our series, which represents a limitation given the role of this DNA repair enzyme in counteracting TMZ-induced cytotoxicity. However, the predictive value of MGMT status remains inconsistent across cohorts, and the recent ESE consensus no longer recommends routine MGMT immunohistochemistry prior to a trial of TMZ, mainly due to technical variability, temporal changes in expression, and the lack of alternative therapeutic options in this setting (Raverot et al. 2025). Thus, while MGMT remains of scientific interest, its clinical utility is limited, underscoring the need to identify more reliable biomarkers of TMZ response and resistance in APT. Biochemical responses among functioning tumors were heterogeneous. Prolactinomas exhibited the highest rate of hormonal control (60%), while acromegaly and Cushing’s disease showed variable outcomes (100% and 60%, respectively). These data, although limited by sample size, suggest that radiological and hormonal responses may not always correlate, and that endocrine control should be independently monitored in clinical practice. A hormonal response, even in the absence of a complete radiological response, may hold substantial clinical significance by contributing to morbidity reduction. Biochemical response rates to TMZ in functioning tumors have ranged widely from 19% to 100% (Zacharia et al. 2014; Losa et al. 2016; Jordan et al. 2018; McCormack et al. 2018; Elbelt et al. 2020; Lizzul et al. 2020; Zheng et al. 2020; Cooper et al. 2021; Burman et al. 2022; Du Four et al. 2022; Lamas et al. 2023). In our cohort, partial control was most frequent in prolactinomas and acromegaly, while Cushing’s disease displayed the broadest spectrum, including one case of complete remission. Taken together, our results underscore the need for harmonized definitions of biochemical response to enable comparability across studies. Consistent with previous observations, biochemical control in functioning PitNETs was variable and did not always parallel the radiological response. Notably, a recent single-center study identified early initiation of TMZ and low MGMT expression as independent predictors of a favorable outcome, particularly in functional tumors (Das et al. 2021). Nevertheless, many studies have traditionally focused on radiological outcomes as the primary endpoint, relegating hormonal response to a secondary outcome (Burman et al. 2022; Lamas et al. 2023; Raverot et al. 2025). The literature shows considerable variability in the definition of hormonal response. While some studies define response as a reduction exceeding 50% in circulating hormone levels (Luo et al. 2021 ; Lamas et al. 2023), others adopt a stratification approach analogous to the RECIST criteria, since biochemical response was defined as complete normalization of hormone levels; partial response corresponded to a reduction exceeding 20%, stable disease was defined as a fluctuation within ± 20%, and progressive disease as an increase of more than 20% in hormone levels (McCormack et al. 2018; Deng et al. 2025), we used the last one to our evaluations. An extensive survey demonstrated that clinically functioning tumors were more likely to respond to TMZ than non-functioning tumors, independent of MGMT status (McCormack et al. 2018). In our series, however, our radiological response rates were similar between functioning and non-functioning tumors, both partial response (23.8% vs. 22.2%, respectively) and radiological disease control, including partial response or stabilization (90.4% vs 100%). Therefore, functional status alone may not consistently discriminate responsiveness in real-world practice. This apparent discrepancy could reflect differences in patient selection, timing of TMZ initiation, or referral bias across cohorts. Beyond these differences, it is also important to note that one criterion for the definition of aggressiveness in pituitary tumors relies on resistance to standard therapies, which is more difficult to apply to non-functioning tumors. In functioning tumors, medical therapies such as cabergoline or somatostatin analogues provide a clear benchmark of resistance, whereas in non-functioning tumors, standard therapy is essentially surgical and adjuvant radiotherapy, with limited medical alternatives. Although cabergoline has been investigated in non-functioning pituitary tumors, with small series reporting disease stabilization and modest tumor shrinkage, it has not been systematically evaluated in the subset of aggressive non-functioning PitNETs (Greenman et al. 2016; Batista et al. 2019; Vargas-Ortega et al. 2022; Ayalon-Dangur et al. 2024). These conceptual differences underscore the challenges in establishing uniform criteria for aggressiveness across different tumor subtypes. Importantly, while functioning tumors allow for the additional endpoint of biochemical control, our data reinforce that radiological assessment remains the critical measure of efficacy across all subtypes. Radiological and biochemical endpoints were not fully interchangeable in functioning tumors. Although overall concordance was high, a clinically relevant discordance emerged: biochemical progression occurred in 12.5% of patients despite radiological disease control. This supports independent monitoring of imaging and biochemical markers during TMZ, particularly in functioning PitNETs where biochemical changes may directly translate into morbidity. The moderate kappa observed despite high percent agreement likely reflects the highly unbalanced distribution of radiological outcomes (predominance of PR/SD and rare radiological PD). The safety profile of TMZ in our cohort was globally acceptable, with adverse events documented in 66% of patients, predominantly grade 1. Nausea and myelotoxicity were the most frequent toxicities, and treatment discontinuation was required in four cases (13%) due to grade 3–4 events, with no deaths related to toxicity. These findings align with the broader literature synthesized in the recent international consensus on APT and metastatic PitNETs (Lamas et al. 2023; Lin et al. 2023; Raverot et al. 2025). Discontinuation rates due to adverse events ranged from 6% to 15%, most often driven by pervasive fatigue, nausea, or cytopenias, while rare but severe complications such as aplastic anemia, hepatotoxicity, or secondary hematologic malignancies have been reported (Lamas et al. 2023; Lin et al. 2023; Raverot et al. 2025). Compared with these data, our series underscores two complementary perspectives: first, that the burden of mild adverse effects is common and expected in real-world settings; second, that clinically significant hematological toxicity requiring drug withdrawal is not negligible and should be anticipated in routine practice. Importantly, while we did not observe the rare life-threatening complications highlighted in the consensus, our findings reinforce the need for vigilant hematologic monitoring, as hematologic toxicity remains the leading barrier to treatment continuity in this patient population. Our study revealed significant heterogeneity in the duration of TMZ treatment, reflecting the current lack of well-defined protocols for the management of APT and metastatic PitNETs. The mean duration of TMZ use was 10.7 months (range, 3–44 months), highlighting the variability in clinical decisions to initiate TMZ. In some cases, treatment was discontinued after six months due to disease stabilization, emphasizing the need for protocols that support both earlier initiation and longer treatment courses. Given that TMZ was most often prescribed following documented disease progression, tumor stabilization should be considered a favorable therapeutic outcome. According to the 2025 ESE Clinical Practice Guidelines (Raverot et al., 2025), for patients responding to a first course of TMZ—defined as either partial tumor regression or stabilization after rapid progression in the preceding six months—it is now recommended that therapy be continued for at least 12 months and further guided by efficacy and tolerability. Extending treatment beyond 24 months should be carefully weighed against the potential for cumulative severe toxicity. In line with these recommendations, an Italian real-life study including patients treated for more than 12 continuous cycles reported that prolonged TMZ administration was safe and associated with durable disease control and hormonal improvement, particularly among responsive functioning PitNETs (Lizzul et al. 2020). In our study, 10 patients received TMZ treatment for 12 months or longer. Among them, 81.8% experienced at least one adverse event, most of which were mild in severity. Three cases of myelotoxicity were observed, leading to treatment discontinuation in two patients. The treatment discontinuation rate due to adverse effect rate was higher in the group that used TMZ for more than 12 months (30% vs. 5%), while myelotoxicity rate was similar between the groups (30% vs. 30%). This suggests that cumulative toxicity and treatment fatigue, rather than hematologic events per se, may limit long-term tolerability of TMZ in APT. In the present cohort, despite a tumor control rate of 96.7% with TMZ, mortality was observed in 7/30 patients (23.3%); two due to direct tumor-related complications (mass effect) and five due to disease progression after TMZ withdrawal, despite having initially achieved stable disease (n = 3) or partial response (n = 2). These findings reinforce that, in the absence of severe adverse effects, maintaining TMZ in patients with a favorable response may be a safe and effective strategy, even when complete radiological remission is not achieved. Current literature and guidelines support prolonged TMZ treatment in patients with an initial response, and suggest that re-challenge may be considered, given the higher toxicity of alternative therapies. While a reduced response is typically observed during re-challenge, our study found that patients receiving a second cycle showed similar outcomes to their initial treatment response (Burman et al. 2022; Cristina Lamas, Rosa Camara, Carmen Fajardo, Pablo Remon-Ruiz, Betina Biagetti, Fernando Guerrero-Perez, Marta Araujo-Castro, Mireia Mora, Felicia Hanzu, Pedro Iglesias, Rogelio Garcıa-Centeno and Alfonso Soto 2023; Raverot et al. 2025). Among 30 patients (9 nonfunctioning and 21 functioning tumors), 26 received radiotherapy (RT) at different time points: 19 before TMZ, 6 after, and 1 concomitantly. Four patients did not receive RT. Considering treatment response, disease control (partial response + stable disease) was observed in 24/26 cases (92.3%). In the group with RT before TMZ (n = 19), there were 5 partial responses (26.3%), 12 cases of stable disease (63.2%), and 2 progressions (10.5%), totaling 89.5% disease control. When RT was delivered after TMZ (n = 6), 1 patient had a partial response (16.7%) and 5 had stable disease (83.3%), with 100% disease control. The single concomitant RT case (prolactinoma) remained stable. Among those without RT (n = 4), there was 1 partial response (25%) and 3 cases of stable disease (75%), also with no progressions (100% control). In summary, no clear advantage of RT timing on overall disease control was identified in this sample, although there was a numerical trend toward more partial responses when RT was given before TMZ (or omitted), contrasted by the fact that the only progressions occurred in that group, an observation that may reflect selection biases and the small sample size. The 2025 ESE guideline advises considering concurrent TMZ with radiotherapy for pituitary tumors showing rapid progression of a large residual that is not amenable to further surgery, particularly when high proliferative markers and/or adverse somatic mutations are present, while not otherwise specifying the optimal timing of TMZ relative to radiotherapy. It recommends that radiotherapy decisions be based on postoperative MRI findings obtained within 3 months and notes that, in aggressively growing tumors, combined TMZ–radiotherapy should be considered (Raverot et al. 2025). Retrospective series suggest higher radiological or composite response rates with combination therapy versus radiotherapy alone, though these data are constrained by heterogeneity, potential confounding, and a lack of long-term outcomes. Accordingly, the guideline emphasizes individualized, multidisciplinary decision-making when considering TMZ–radiotherapy outside the narrow indication of rapid progression (Lamas et al. 2023; Lin et al. 2023; Raverot et al. 2025). According to the recent ESE guidelines, TMZ is recommended as the first-line chemotherapeutic option for APT and metastatic PitNETs, once progression is documented despite standard therapies (Raverot et al. 2025). In our cohort, however, the median interval between diagnosis and TMZ initiation was close to 100 months, reflecting that systemic therapy is frequently reserved for late stages of disease after multiple local interventions. While this approach is consistent with the cautious positioning of current recommendations, our findings suggest that, in real-world practice, TMZ may often be introduced relatively late in the treatment course. Indeed, the benefit of early initiation on TMZ (median 15 months) was associated with higher response rates, while delays beyond 31 months predicted poorer outcomes (Das et al. 2021). However, prospective studies and the development of biological markers of aggressiveness will be crucial to refine treatment timing and harmonize practice across centers. Beyond clinical decision-making, access-related barriers also contribute to delayed initiation of TMZ. Because the drug is not formally approved for pituitary tumors in most countries, reimbursement is frequently denied by health systems, and treatment may depend on exceptional authorization. In Brazil and other Latin American settings, this challenge is particularly evident, but similar constraints are likely to affect other regions worldwide since TMZ remains off-label in many countries, not formally approved by the US Food and Drug Administration (FDA), European Medicines Agency or Australian Therapeutic Goods Administration (TGA) (McCormack 2022 ). Such regulatory and economic hurdles highlight a global gap between guideline recommendations and real-world implementation, reinforcing the need for strategies to ensure timely and equitable access to evidence-based therapies across diverse healthcare systems. Finally, the long interval between tumor diagnosis and TMZ initiation (median 100 months) raises important questions about treatment timing. Earlier initiation of systemic therapy in selected cases may improve outcomes and reduce cumulative surgical and radiotherapeutic morbidity. The heterogeneity of clinical trajectories observed in our cohort further supports the need for standardized therapeutic protocols, ideally informed by biological markers. Limitations. This study has limitations inherent to a retrospective multicenter design. Treatment timing, imaging schedules, and biochemical assays were not fully standardized across centers, which may introduce measurement variability despite the use of prespecified response definitions. Selection bias is possible because temozolomide was used in highly pretreated, clinically aggressive cases referred to tertiary centers, limiting generalisability. Key pathology variables were not available for all patients (Ki-67 and p53), and biochemical response could be evaluated only in functioning tumors with available data, which reduces power for subgroup and concordance analyses. Finally, the sample size—particularly within clinically relevant subgroups—results in imprecision around estimates and limits the feasibility of confounder-adjusted modeling; findings should therefore be interpreted as descriptive real-world evidence. Interpretation. In a real-world multicenter cohort, temozolomide was associated with high radiological disease control and frequent biochemical improvement in functioning tumors, with manageable toxicity. These findings address our objective of characterizing effectiveness and safety in Brazilian referral centers; however, inference is limited by the retrospective design, center-level heterogeneity in assessment timing, and missingness in biochemical and pathology variables, which also reduced the size of key subgroup analyses. Given the multiplicity of exploratory comparisons, subgroup signals should be interpreted as hypothesis-generating rather than definitive. Overall, our estimates align with the range of outcomes reported in prior temozolomide series in aggressive PitNETs and support standardized prospective data collection to refine predictors and monitoring strategies. Generalisability. Because this is a multicenter cohort from Brazilian tertiary referral centers, our findings are most generalisable to similarly selected patients with aggressive or metastatic PitNETs managed in specialized high-volume units, where temozolomide is typically used after failure of standard local and medical therapies. Extrapolation to earlier-line use, to centers with different imaging/biochemical monitoring practices, or to less aggressive tumors should be made cautiously. Nevertheless, the inclusion of multiple referral centers and routine-care assessments enhances external validity for real-world practice in comparable settings. Conclusion TMZ was effective and generally well tolerated in this Brazilian real-world multicenter cohort of APTs and metastatic PitNETs, yielding high rates of radiological disease control and a low discontinuation rate. In functioning tumors, biochemical responses were heterogeneous and could diverge from imaging outcomes, supporting parallel endocrine and radiological monitoring. When tolerated, continuation beyond the initial cycles was feasible and aligned with guideline-based systemic management when systemic therapy is indicated. These data expand the Latin American evidence base and underscore the need for collaborative prospective efforts to validate biomarkers and optimize temozolomide timing and duration within individualized therapeutic pathways for aggressive/metastatic PitNETs. Declarations Funding. RLB supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), grant 444825/2024-0. RLB is also supported by the AACR (American Association for Cancer Research), grant 23-15-01-BATI. Author Contribution I.P.A.M., C.R.P.B.M., L.A.N., and R.L.B. conceived and designed the study. I.P.A.M. and C.R.P.B.M. coordinated data collection, curated the multicenter dataset, and drafted the main manuscript. L.A.N., M.A.C., I.N.R., L.B.S., T.S.A., P.C.L.E., M.C., L.K., M.R.G., C.G.S.L.R., H.M.G., C.B., R.S.J., M.N., M.C.B.V.F., M.C.M., A.G., M.B.C.C.N., and O.F. contributed to patient inclusion and clinical/pathological data acquisition at their respective centers and critically revised the manuscript. M.P. performed/oversaw radiological assessment and contributed to figure preparation. I.P.A.M., C.R.P.B.M., and R.L.B. performed the statistical analyses and prepared the final figures/tables. R.L.B. supervised the project and finalized the manuscript. All authors reviewed and approved the final version of the manuscript. 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Cite Share Download PDF Status: Published Journal Publication published 27 Mar, 2026 Read the published version in Pituitary → Version 1 posted Editorial decision: Revision requested 13 Jan, 2026 Reviews received at journal 13 Jan, 2026 Reviews received at journal 10 Jan, 2026 Reviewers agreed at journal 28 Dec, 2025 Reviewers agreed at journal 26 Dec, 2025 Reviewers invited by journal 26 Dec, 2025 Editor assigned by journal 23 Dec, 2025 Submission checks completed at journal 23 Dec, 2025 First submitted to journal 22 Dec, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8428238","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":566469412,"identity":"2c7ad227-9cb8-43a6-ba7d-9aeaf9b00cfb","order_by":0,"name":"Isabelle Amaro","email":"","orcid":"","institution":"Universidade de São Paulo","correspondingAuthor":false,"prefix":"","firstName":"Isabelle","middleName":"","lastName":"Amaro","suffix":""},{"id":566469413,"identity":"b8545db7-5f59-4490-b436-38907bfa4723","order_by":1,"name":"Camila Macedo","email":"","orcid":"","institution":"Universidade de São 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01:18:12","extension":"xml","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":140607,"visible":true,"origin":"","legend":"","description":"","filename":"634f48f2c4a747bea9649d364b67874b1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8428238/v1/0285791187bb303ebe002ab1.xml"},{"id":99260438,"identity":"ef077164-b5d2-42cd-ac43-9eb928e6ec18","added_by":"auto","created_at":"2025-12-31 01:18:12","extension":"html","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":161256,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8428238/v1/ce778b8caeed81a468e791fa.html"},{"id":99260423,"identity":"6a6b717e-7df7-4ccb-84bf-52bc48174e73","added_by":"auto","created_at":"2025-12-31 01:18:12","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":71359,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBaseline characteristics of the cohort treated with temozolomide.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Sex distribution represented as a donut chart. (B) Clinical diagnoses at treatment initiation (prolactinoma, acromegaly, Cushing’s disease, and non-functioning PitNETs). (C) Tumor lineage classification according to PitNET lineage (PIT1, TPIT, SF1, and null cell). (D) Age distribution shown as combined violin–boxplot, illustrating median, interquartile range, and overall spread.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8428238/v1/c167a0fdcc86660abdc9e5e6.png"},{"id":99319693,"identity":"f7fae8ec-9167-49d2-af84-17226e23b299","added_by":"auto","created_at":"2025-12-31 16:37:41","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":133845,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWaterfall plot showing the individual response to temozolomide according to RECIST criteria.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEach bar represents the percentage variation in tumor diameter relative to baseline for an individual patient. Blue bars indicate partial response (≥ 30% reduction), gray bars represent stable disease (between –30% and +20%), and orange bars denote progressive disease (≥ 20% increase). Dashed lines indicate RECIST thresholds for partial response and progression.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8428238/v1/95c025d7f0e17a795b0c8307.png"},{"id":99319531,"identity":"9292134b-d947-4120-92fa-f99e9e1a4208","added_by":"auto","created_at":"2025-12-31 16:37:26","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":69416,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTemozolomide response according to pituitary tumor lineage.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) \u003cstrong\u003eTumour diameter variation (%) stratified by lineage\u003c/strong\u003e (PIT1, TPIT, SF1). Boxplots show median, interquartile range, and full distribution of radiological response; dashed lines represent RECIST cutoffs for partial response (–30%) and progressive disease (+20%). (B) \u003cstrong\u003eRECIST response distribution by lineage\u003c/strong\u003e, displayed as stacked proportions of partial response (PR), stable disease (SD), and progressive disease (PD). The number of patients per lineage is shown above each bar. PIT1-lineage tumors demonstrated the highest proportion of radiological shrinkage, whereas TPIT and SF1 tumors showed predominantly stable disease.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8428238/v1/5ee828c84584eca7ca8ebfe5.png"},{"id":99260424,"identity":"4c800db3-27a8-4d7d-ae9b-484b8f4603ed","added_by":"auto","created_at":"2025-12-31 01:18:12","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":46325,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eConcordance between radiological and biochemical responses to temozolomide in functioning PitNETs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLeft panel:\u003c/strong\u003e Heatmap cross-tabulation of radiological response by RECIST 1D (PD, PR, SD) versus biochemical response categories (PD, SD, PR, CR) among functioning tumors with available biochemical data (n=16); numbers indicate patients per cell. \u003cstrong\u003eRight panel:\u003c/strong\u003e Alluvial diagram depicting flows between radiological and biochemical response categories, with band widths proportional to the number of patients in each radiological–biochemical pair. No complete radiological responses were observed. (Abbreviations: PD, Progressive Disease; SD, Stable Disease; PR, Partial Response).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8428238/v1/33ea6b4afae3940605e48e3c.png"},{"id":105754984,"identity":"5905b944-f0af-42cb-ab30-570c190ba7e2","added_by":"auto","created_at":"2026-03-30 16:23:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1139016,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8428238/v1/17831792-50b3-4f61-bad0-8c6ff4a8d425.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Temozolomide in Aggressive Pituitary Tumors and Metastatic PitNETs: A Brazilian Multicenter Real-World Cohort Study","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePituitary neuroendocrine tumors (PitNETs) are among the most common intracranial neoplasms, accounting for approximately 15% of primary brain tumors (Melmed \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Although the majority of PitNETs follow a benign, slow-growing course, a clinically significant subset exhibits aggressive behavior, characterized by rapid growth and resistance to standard therapies (Dekkers et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Kasuki \u0026amp; Raverot \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Raverot \u003cem\u003eet al.\u003c/em\u003e 2025).\u003c/p\u003e \u003cp\u003eAggressive PitNETs (APTs) remain a major therapeutic challenge. According to the 2018 European Society of Endocrinology (ESE) guidelines (Raverot \u003cem\u003eet al.\u003c/em\u003e 2018) which is reaffirmed by the new 2025 update, APTs are defined not solely by histopathological features, but by clinically relevant progression despite optimized multimodal therapy, including surgery, radiotherapy, and standard medical treatment (Jotanovic \u003cem\u003eet al.\u003c/em\u003e 2024; Casar-Borota et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Raverot \u003cem\u003eet al.\u003c/em\u003e 2025). The 2025 consensus emphasizes that the current histological markers (such as Ki-67, mitotic count, and p53) have limited predictive accuracy, and no validated biomarker currently exists that can reliably predict aggressiveness or treatment response.\u003c/p\u003e \u003cp\u003eTemozolomide (TMZ), an oral DNA-alkylating agent, is currently the only systemic therapy with established efficacy in APTs and metastatic PitNETs (Reilly \u003cem\u003eet al.\u003c/em\u003e 1993; Raverot \u003cem\u003eet al.\u003c/em\u003e 2012; Syro \u003cem\u003eet al.\u003c/em\u003e 2018; Whitelaw \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). It has been recommended as a first-line chemotherapeutic option for APTs and metastatic PitNETs after failure of standard treatments (Raverot \u003cem\u003eet al.\u003c/em\u003e 2025). Its mechanism involves methylation-induced DNA damage at the O6-guanine position, ultimately leading to tumor cells apoptosis (Syro \u003cem\u003eet al.\u003c/em\u003e 2018; Whitelaw \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). However, therapeutic response remains highly variable and complete remissions are rare (Lamas \u003cem\u003eet al.\u003c/em\u003e 2023). Although MGMT promoter methylation and low MGMT protein expression have been proposed as predictors of response, their clinical utility remains limited and inconsistent across cohorts (McCormack \u003cem\u003eet al.\u003c/em\u003e 2009; Syro \u003cem\u003eet al.\u003c/em\u003e 2011; Kontogeorgos \u0026amp; Thodou \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMost of the available data on the efficacy and safety of TMZ in APT originates from European and North American centers (Hui-Zheng \u0026amp; Lei 2024). To date, other populations, particularly from low- and middle-income countries, have not yet been adequately represented in the literature. This underrepresentation may be clinically relevant, as therapeutic responses could be influenced not only by genetic and epigenetic differences, but also by socioeconomic, healthcare system, and cultural factors that affect diagnosis, treatment access, and follow-up adherence (\u0026Ouml;zdemir \u0026amp; Dotto \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Li \u003cem\u003eet al.\u003c/em\u003e 2024).\u003c/p\u003e \u003cp\u003eTo address this gap, the present study provides the first comprehensive multicenter analysis of TMZ treatment in APTs conducted in Brazil. By evaluating clinical outcomes, safety profiles, and therapeutic trajectories across multiple national reference centers, this study contributes real-world evidence from an underrepresented population and adds to the global effort to optimize care for these rare and challenging tumors.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e \u003cstrong\u003eStudy design and participants\u003c/strong\u003e \u003cp\u003eThis multicenter retrospective cohort study included eight Brazilian referral centers specialized in pituitary tumors. We screened patients with aggressive pituitary tumors (APTs) or metastatic PitNETs who received temozolomide (TMZ) between 2005 and 2024. The index date was the first day of TMZ cycle 1. Eligible patients had\u0026thinsp;\u0026ge;\u0026thinsp;6 months of follow-up from TMZ initiation.\u003c/p\u003e \u003c/p\u003e \u003cp\u003eAggressiveness was defined as radiological progression and/or biochemical progression despite conventional therapies (surgery, radiotherapy, and subtype-appropriate medical therapy). Metastatic PitNETs were defined by the presence of craniospinal and/or systemic metastases. Patients were excluded if they received TMZ for \u0026lt;\u0026thinsp;3 cycles (or \u0026lt;\u0026thinsp;3 months) or if severe comorbidities precluded reliable assessment of outcomes. The study was approved by the local ethics committees (with waiver of informed consent, as applicable), and data were de-identified before central analysis.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eData collection\u003c/strong\u003e \u003cp\u003eClinical, pathological, and treatment variables were extracted from medical records, including tumor subtype, baseline tumor measurements, Ki-67 labeling index, p53 immunohistochemistry, and prior therapies (surgery, radiotherapy, and pituitary-directed medications). TMZ treatment, treatment interruptions, and total number of cycles were recorded. Concomitant therapies during TMZ were documented.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eResponse assessment\u003c/strong\u003e \u003cp\u003eRadiological response was assessed on sellar MRI (contrast-enhanced when available) using the longest tumor diameter (1D) and categorized per RECIST 1.1 as complete response (CR), partial response (PR; \u0026ge;30% decrease), stable disease (SD; \u0026lt;30% decrease and \u0026lt;\u0026thinsp;20% increase), or progressive disease (PD; \u0026ge;20% increase and/or new lesions/non-target progression). Imaging assessments followed each center\u0026rsquo;s routine schedule; when multiple scans were available and the best overall radiological response were recorded. Radiological objective response rate (ORR) was defined as CR\u0026thinsp;+\u0026thinsp;PR, and radiological disease control rate (DCR) as CR\u0026thinsp;+\u0026thinsp;PR\u0026thinsp;+\u0026thinsp;SD.\u003c/p\u003e \u003c/p\u003e \u003cp\u003eBiochemical response was assessed only in functioning PitNETs using subtype-appropriate biochemical markers and consistent pre- and on-treatment timepoints. Responses were categorized as complete (CR; normalization of the relevant hormone measure), partial (PR; \u0026gt;20% reduction), stable (SD; \u0026le;20% variation), or progressive (PD; \u0026gt;20% increase). Biochemical response rate was defined as CR\u0026thinsp;+\u0026thinsp;PR, and biochemical disease control as CR\u0026thinsp;+\u0026thinsp;PR\u0026thinsp;+\u0026thinsp;SD among functioning tumors with available biochemical data. Concordance between radiological (RECIST 1.1) and biochemical responses in functioning tumors was assessed using cross-tabulation. Data visualization was performed using heatmaps to display response pairing frequencies and alluvial diagrams to illustrate the flow of patients between response categories.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eBias\u003c/strong\u003e \u003cp\u003eTo mitigate bias inherent to retrospective multicenter data, we applied prespecified and standardized definitions for eligibility and outcomes (including RECIST 1D for radiological response and predefined biochemical thresholds for functioning tumors). Data were extracted using a harmonized case-report form and centrally checked for internal consistency. When multiple on-treatment assessments were available, best overall radiological response was recorded according to uniform criteria. Analyses of biochemical response were restricted to functioning tumors with available data, with no imputation for missing values.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eSafety\u003c/strong\u003e \u003cp\u003eAdverse events were collected from medical records and laboratory monitoring during TMZ. Events were categorized as gastrointestinal and hematologic and graded when feasible using CTCAE criteria; hematologic toxicity was defined based on standardized thresholds for neutropenia and thrombocytopenia.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eOutcomes\u003c/strong\u003e \u003cp\u003ePrimary outcomes were radiological disease control and overall safety/tolerability. Secondary outcomes included biochemical disease control in functioning tumors and the distribution of radiological and biochemical response categories (CR/PR/SD/PD) during TMZ. Exploratory outcomes included agreement between radiological and biochemical trajectories in functioning PitNETs with available biochemical data.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eStatistical analysis\u003c/strong\u003e \u003cp\u003eCategorical variables are presented as n (%) and continuous variables as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD or median (IQR), as appropriate. Associations between categorical predictors and response were evaluated using Fisher\u0026rsquo;s exact test (or chi-square when applicable). Ki-67 was analyzed as a continuous variable and dichotomized at 3% and 10%. Continuous Ki-67 values across response groups were compared using the Kruskal\u0026ndash;Wallis test with post-hoc Dunn tests when appropriate. A composite \u0026ldquo;high-proliferation\u0026rdquo; phenotype (Ki-67\u0026thinsp;\u0026ge;\u0026thinsp;10% plus p53 positivity) was explored. Agreement between radiological and biochemical outcomes was evaluated among functioning PitNETs with available hormonal data. For the primary agreement analysis, responses were dichotomized as disease control (CR/PR/SD) versus progression (PD) in each domain, and concordance was summarized using percent agreement and Cohen\u0026rsquo;s kappa. As a descriptive secondary analysis, cross-tabulations of the original response categories (CR/PR/SD/PD) were reported to characterize discordant patterns. Analyses were two-tailed with α\u0026thinsp;=\u0026thinsp;0.05 and performed in R (RStudio).\u003c/p\u003e \u003c/p\u003e \u003cp\u003eThis study is reported in accordance with the STROBE guidelines for cohort studies (Supp Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; Supp Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eClinical characteristics of patients treated with temozolomide (N\u0026thinsp;=\u0026thinsp;30)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eClinical characteristics of patients (N\u0026thinsp;=\u0026thinsp;30)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge at diagnosis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e29.5 years (7\u0026ndash;56 years)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSex (female / male)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e53% / 47%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTumor size at diagnosis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.2 cm (1.2\u0026ndash;12 cm)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGiant Tumors (\u0026ge;\u0026thinsp;4 cm)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e56%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMetastatic PitNETs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4 / 30 patients\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eTumor type\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProlactinoma\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8 patients\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNon functioning\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9 patients\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCushing\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8 patients\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAcromegaly\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5 patients\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eTreatment before TMZ\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSurgery\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.6 surgeries (1\u0026ndash;6)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRadiotherapy\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e19 patients\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTime between diagnosis and TMZ treatment\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100 months\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDuration of TMZ treatment\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10.3 months\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eAn\u0026aacute;lise Imunohistoqu\u0026iacute;mica\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKi-67 (27/30)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13.6% (1\u0026ndash;30%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ep53 (22/30)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e59% positive\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eEthics approval.\u003c/strong\u003e \u003cp\u003e This study was approved by the Ethics Committee of Hospital das Clinicas da Faculdade de Medicina da Universidade de S\u0026atilde;o Paulo (CAAE: 86575324.3.0000.0068).\u003c/p\u003e \u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eA total of 30 patients with aggressive or metastatic PitNETs were included (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; suppl table 1). The mean age at diagnosis was 29.5 years (range 7\u0026ndash;56), and 53% of the cohort were female (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e). All patients had macroadenomas, with a mean tumor diameter of 4.2 cm (range 1.2\u0026ndash;12.0 cm); 56% were classified as giant tumors (\u0026ge;\u0026thinsp;4 cm). Four patients (13.3%) were diagnosed with metastatic PitNETs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAmong the 30 patients, 70% (n\u0026thinsp;=\u0026thinsp;21) had functioning tumors, while the remaining 30% (n\u0026thinsp;=\u0026thinsp;9) had non-functioning PitNETs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Among the functioning tumors, prolactinomas were the most frequent subtype (n\u0026thinsp;=\u0026thinsp;8; 40%), followed by corticotroph tumors causing Cushing\u0026rsquo;s disease (n\u0026thinsp;=\u0026thinsp;8; 40%), including one case of Crooke cell PitNET) and somatotroph tumors associated with acromegaly (n\u0026thinsp;=\u0026thinsp;5; 25%).\u003c/p\u003e \u003cp\u003eThe Ki-67 proliferation index was assessed by immunohistochemical analysis in 27 patients (90%) and was greater than 3% in 22/30 (73%) of cases, with a mean Ki-67 of 13.6%. p53 expression was analyzed in 22 patients and was positive in 59% (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBefore initiating TMZ, patients had undergone a median of 2.6 surgical procedures (range: 1\u0026ndash;6), and 19 patients (63%) had received radiotherapy. The median interval between initial diagnosis and initiation of TMZ therapy was 100 months, and the median duration of TMZ treatment was 10.3 months.\u003c/p\u003e \u003cp\u003eThe main indications for initiating TMZ therapy were radiological tumor progression (53%, n\u0026thinsp;=\u0026thinsp;16) and worsening biochemical markers in functioning tumors (41%, n\u0026thinsp;=\u0026thinsp;13). In a smaller subset (n\u0026thinsp;=\u0026thinsp;3), TMZ was introduced due to metastases.\u003c/p\u003e \u003cp\u003eRadiological objective response rate (ORR; CR\u0026thinsp;+\u0026thinsp;PR) was 23.3% (7/30; 95% CI 9.9\u0026ndash;42.3) and radiological disease control rate (DCR; PR\u0026thinsp;+\u0026thinsp;SD) was 96.7% (29/30; 95% CI 82.8\u0026ndash;99.9). Progressive disease (PD) occurred in 3.3% (1/30) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e). It is noteworthy that one patient (case 14) with a corticotroph functioning PitNET exhibited a 97% tumor reduction after six months of TMZ treatment. Although considered as a partial responder, the magnitude of tumor shrinkage was remarkably significant. Additionally, a Crooke cell PitNET achieved stable disease under TMZ. Progressive disease occurred in one case. Taken together, 96.7% of patients (n\u0026thinsp;=\u0026thinsp;29) achieved disease control (SD\u0026thinsp;+\u0026thinsp;PR) during TMZ treatment.\u003c/p\u003e \u003cp\u003eThe response profiles to treatment were remarkably similar between functioning and non-functioning PitNETs. The rates of partial response were nearly identical (22.2% vs. 23.8%, respectively; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;NS), and the proportion of patients achieving stable disease was comparable (77.8% vs. 66.7%; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;NS).\u003c/p\u003e \u003cp\u003eThe efficacy of TMZ varied significantly among functioning PitNETs (p\u0026thinsp;=\u0026thinsp;0.012). Prolactinomas (n\u0026thinsp;=\u0026thinsp;8) demonstrated uniform radiological stability (8/8, 100%) without measurable tumor regression; however, biochemically, partial control was achieved in 87,5% (7/8), while 12,5% (1/8) showed biochemical progression. In patients with Cushing\u0026rsquo;s disease (n\u0026thinsp;=\u0026thinsp;8), one patient exhibited disease progression. Biochemically, 16% (1/6) achieved complete hormonal remission, 16% (1/6) showed partial improvement, 33% (2/6) remained stable, and 33% (2/6) experienced biochemical progression. The biochemical response rate (CR\u0026thinsp;+\u0026thinsp;PR) was 68.8% (11/16) and biochemical disease control (CR\u0026thinsp;+\u0026thinsp;PR\u0026thinsp;+\u0026thinsp;SD) was 81.3% (13/16), while biochemical progression occurred in 18.8% (3/16). Overall, acromegaly was associated with the highest rate of radiological response, whereas Cushing\u0026rsquo;s disease demonstrated the greatest proportion of progression events, both radiologically and biochemically, with a quarter of patients (25%) experiencing disease progression despite treatment, representing all radiological progression events within the cohort.\u003c/p\u003e \u003cp\u003eTumour lineage was available for 27 of 30 patients (PIT1 \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;14; TPIT \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10; SF1 \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3). The percentage change in maximal tumour diameter on temozolomide was broadly similar across lineages (median change: PIT1 \u0026minus;\u0026thinsp;13.8%, TPIT \u0026minus;\u0026thinsp;15.9%, SF1 \u0026minus;\u0026thinsp;4.8%; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Best RECIST response distributions are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB: PIT1 (PR 4/14, SD 10/14), TPIT (PR 2/10, SD 7/10, PD 1/10), and SF1 (PR 1/3, SD 2/3). The only radiological progression event occurred in the TPIT group; all PIT1 and SF1 cases achieved radiological disease control (PR/SD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAmong functioning tumors with available biochemical data (n\u0026thinsp;=\u0026thinsp;16), biochemical response (CR\u0026thinsp;+\u0026thinsp;PR) was 68.8% (11/16; 95% CI 41.3\u0026ndash;89.0) and biochemical disease control (CR\u0026thinsp;+\u0026thinsp;PR\u0026thinsp;+\u0026thinsp;SD) was 81.2% (13/16; 95% CI 54.4\u0026ndash;96.0). When biochemical responses among functioning PitNETs with available hormonal data (n\u0026thinsp;=\u0026thinsp;16) were dichotomized as disease control (CR/PR/SD) versus progression (PD), overall concordance between radiological and biochemical outcomes was 87.5% (14/16), with moderate agreement by Cohen\u0026rsquo;s kappa (κ\u0026thinsp;=\u0026thinsp;0.44). Importantly, biochemical progression occurred in 12.5% of patients (2/16) despite radiological disease control, indicating that biochemical and radiological trajectories may diverge in a subset of cases.\u003c/p\u003e \u003cp\u003eAlthough radiological and biochemical responses were generally concordant (68.7%), distinct patterns of dissociation were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). As shown in the heatmap (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e - left), two patients (12.5%) presented with biochemical progression despite achieving radiologically stable disease. The alluvial diagram (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e - right) further illustrates this divergence, highlighting that radiological stability does not necessarily guarantee hormonal control in a subset of aggressive PitNETs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eRadiological outcomes differed between APTs and metastatic PitNETs. Among the four patients with metastatic disease, two patients (50%) exhibited partial response, while the remaining two (50%) experienced disease progression, with no cases of stable disease. In contrast, among the 26 patients with aggressive non-metastatic tumors, disease stabilization was the most frequent outcome (76,9%), followed by partial response (19.2%), with 1 case of progression.\u003c/p\u003e \u003cp\u003eThe potential impact of radiotherapy (RT) timing on radiological response was evaluated across the cohort. Patients were categorized into four groups based on the sequence of RT in relation to the study treatment: RT before, RT after, concurrent RT, and no prior RT. Statistical comparison between the two largest groups\u0026mdash;'RT before' (n\u0026thinsp;=\u0026thinsp;19) and 'RT after' (n\u0026thinsp;=\u0026thinsp;6)\u0026mdash;revealed no significant association between radiotherapy timing and treatment outcome (p\u0026thinsp;=\u0026thinsp;0.65). A high rate of disease stabilization and partial response characterized both groups.\u003c/p\u003e \u003cp\u003eThe exploratory analysis of the proliferation marker Ki-67 did not demonstrate a statistically significant association with tumor diameter response following TMZ treatment. When evaluated categorically, the chi-square test revealed no significant difference in response proportions among Ki-67 groups (χ\u0026sup2; = 2.47; p\u0026thinsp;=\u0026thinsp;0.65). Similarly, considering the continuous Ki-67 percentage, the Kruskal\u0026ndash;Wallis test showed no significant difference between response groups (H\u0026thinsp;=\u0026thinsp;2.17; p\u0026thinsp;=\u0026thinsp;0.34). The Dunn post-hoc test with Bonferroni correction confirmed the absence of relevant pairwise differences (p\u0026thinsp;\u0026gt;\u0026thinsp;0.6 for all comparisons). Although no statistical significance was reached, there was a numerical trend toward higher Ki-67 values in tumors with poorer response: the mean Ki-67 ranged from 12.3\u0026thinsp;\u0026plusmn;\u0026thinsp;10.0% in stable tumors to 17.8\u0026thinsp;\u0026plusmn;\u0026thinsp;11.1% in those showing reduction, reaching 20% in progressive cases. When Ki-67 was dichotomized (\u0026lt;\u0026thinsp;10% vs\u0026thinsp;\u0026ge;\u0026thinsp;10%), no statistically significant difference was observed in tumor response distribution (χ\u0026sup2; = 3.11; p\u0026thinsp;=\u0026thinsp;0.21). Nevertheless, all tumors that progressed belonged to the Ki-67\u0026thinsp;\u0026ge;\u0026thinsp;10% group.\u003c/p\u003e \u003cp\u003eThe p53 expression status, assessed by immunohistochemistry and classified as positive or negative, was analyzed with respect to TMZ response. Within the available cohort, p53 positivity was observed in a larger proportion of tumors (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;14; 64%) compared with p53-negative cases (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8). The chi-square test did not reveal a statistically significant association between p53 expression and treatment response (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.17). However, it is noteworthy that all progressive cases (response\u0026thinsp;=\u0026thinsp;4) occurred exclusively among p53-positive tumors, while none were observed in the p53-negative group.\u003c/p\u003e \u003cp\u003eSince neither Ki-67 nor p53 expression alone reached statistical significance, we explored a composite variable integrating both proliferation and genomic instability markers. Tumors exhibiting Ki-67\u0026thinsp;\u0026ge;\u0026thinsp;10% combined with p53 positivity were categorized as high-proliferative lesions. All cases with progressive disease (response\u0026thinsp;=\u0026thinsp;4) and half of those with partial response occurred within this high-proliferation category, whereas stable disease predominated among the remaining tumors (Ki-67\u0026thinsp;\u0026lt;\u0026thinsp;10% and/or p53-negative).\u003c/p\u003e \u003cp\u003eAmong the 30 patients included, four (13.3%) were classified as metastatic PitNETs, comprising one case each of prolactinoma, non-functioning tumor, Cushing\u0026rsquo;s disease, and acromegaly.\u003c/p\u003e \u003cp\u003eTreatment was generally well-tolerated, with 66.6% (20/30) of patients experiencing at least one adverse event. The majority were classified as grade 1 according to CTCAE criteria (Freites-Martinez \u003cem\u003eet al.\u003c/em\u003e 2021). The most frequently reported events were gastrointestinal, with nausea affecting 70% of patients who reported any adverse event, followed by vomiting in 20%. Hematological toxicity (myelotoxicity) was observed in 45% of these patients. Other non-hematological events included asthenia and headache (15% each), while cutaneous reactions were infrequent (5%). Notably, 33% of the cohort reported no treatment-related adverse events.\u003c/p\u003e \u003cp\u003eThe long-term tolerability of the treatment regimen was assessed by analyzing treatment discontinuations due to adverse events. Dose reduction was required in patients with grade 3 myelotoxicity, and treatment was discontinued in four cases (13.3%), due to grade 4 myelotoxicity, one due to grade 3 myelotoxicity, and one due to grade 3 asthenia. No treatment-related deaths were reported.\u003c/p\u003e \u003cp\u003eThe discontinuation rate was 5% (1/20) in patients treated for less than 12 months, compared to 30% (3/10) in those treated for more than 12 months, suggesting that cumulative toxicity may become a more prominent factor influencing treatment feasibility over extended durations.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis Brazilian multicenter cohort provides the largest Latin American dataset to date on the use of TMZ as a single therapy in aggressive and metastatic PitNETs. The radiological disease control rate of 96.7%\u0026mdash;including 23.3% partial responses and 70% of stable disease is consistent with prior studies, which have reported control rates ranging from 60% to 85% (Hui-Zheng \u0026amp; Lei 2024). While no complete responses were observed, this aligns with most published cohorts, where complete radiological remission remains exceedingly rare. These findings reinforce the reproducibility of TMZ efficacy across geographic and healthcare settings, while underscoring the importance of generating data from underrepresented populations.\u003c/p\u003e \u003cp\u003eCompared to previously published studies, our cohort from the Brazilian multicenter study demonstrated distinct demographic characteristics. With a mean age of 29.5 years, the Brazilian participants were notably younger than those in other cohorts (Losa \u003cem\u003eet al.\u003c/em\u003e 2016; Lizzul \u003cem\u003eet al.\u003c/em\u003e 2020; Lamas \u003cem\u003eet al.\u003c/em\u003e 2023; Liu \u003cem\u003eet al.\u003c/em\u003e 2025), whose mean ages ranged from 40.5 to 58.3 years. Additionally, a higher proportion of women was observed in the Brazilian cohort (53%), whereas the other studies predominantly included male participants, with rates reaching up to 87.5% (Losa \u003cem\u003eet al.\u003c/em\u003e 2016; Lizzul \u003cem\u003eet al.\u003c/em\u003e 2020; Lamas \u003cem\u003eet al.\u003c/em\u003e 2023; Liu \u003cem\u003eet al.\u003c/em\u003e 2025), These findings suggest potential differences in the epidemiological profile or inclusion criteria across studies, which should be taken into account when interpreting and comparing the results.\u003c/p\u003e \u003cp\u003eImportantly, our results offer novel insights when stratified by tumor subtype, size, functionality, and aggressiveness. Patients with acromegaly exhibited a notably high rate of partial radiological response and no disease progression, a finding that may reflect a distinct somatotroph tumor biology or increased sensitivity to alkylating agents. In contrast, patients with prolactinomas and Cushing\u0026rsquo;s disease had higher rates of disease progression, suggesting possible lineage-related differences in TMZ responsiveness, which merit further investigation. Within the corticotroph spectrum, Crooke cell PitNETs are a high-risk variant often associated with aggressive behavior. The stable disease observed in the Crooke cell case in our cohort suggests that even histologically aggressive subtypes may retain clinically relevant chemosensitivity to temozolomide. Metastatic PitNETs exhibited a dichotomous pattern. Although the small sample precludes definitive conclusions, the data highlight the therapeutic challenge posed by metastatic PitNETs and the need for alternative strategies in this subgroup.\u003c/p\u003e \u003cp\u003eThe unpredictable clinical behavior of APT is partly explained by the absence of robust prognostic markers at diagnosis. In our series, Ki-67 and p53 expression alone did not reach statistical significance in predicting tumor response, corroborating previous evidence that conventional proliferation markers are insufficient when evaluated independently (Raverot et al. 2021; Trouillas et al. 2020).\u003c/p\u003e \u003cp\u003eAlthough MGMT expression and promoter methylation have been proposed as predictors of TMZ responsiveness, their utility remains inconsistent and were not assessed in this cohort. We did not assess MGMT expression in our series, which represents a limitation given the role of this DNA repair enzyme in counteracting TMZ-induced cytotoxicity. However, the predictive value of MGMT status remains inconsistent across cohorts, and the recent ESE consensus no longer recommends routine MGMT immunohistochemistry prior to a trial of TMZ, mainly due to technical variability, temporal changes in expression, and the lack of alternative therapeutic options in this setting (Raverot \u003cem\u003eet al.\u003c/em\u003e 2025). Thus, while MGMT remains of scientific interest, its clinical utility is limited, underscoring the need to identify more reliable biomarkers of TMZ response and resistance in APT.\u003c/p\u003e \u003cp\u003eBiochemical responses among functioning tumors were heterogeneous. Prolactinomas exhibited the highest rate of hormonal control (60%), while acromegaly and Cushing\u0026rsquo;s disease showed variable outcomes (100% and 60%, respectively). These data, although limited by sample size, suggest that radiological and hormonal responses may not always correlate, and that endocrine control should be independently monitored in clinical practice. A hormonal response, even in the absence of a complete radiological response, may hold substantial clinical significance by contributing to morbidity reduction. Biochemical response rates to TMZ in functioning tumors have ranged widely from 19% to 100% (Zacharia \u003cem\u003eet al.\u003c/em\u003e 2014; Losa \u003cem\u003eet al.\u003c/em\u003e 2016; Jordan \u003cem\u003eet al.\u003c/em\u003e 2018; McCormack \u003cem\u003eet al.\u003c/em\u003e 2018; Elbelt \u003cem\u003eet al.\u003c/em\u003e 2020; Lizzul \u003cem\u003eet al.\u003c/em\u003e 2020; Zheng \u003cem\u003eet al.\u003c/em\u003e 2020; Cooper \u003cem\u003eet al.\u003c/em\u003e 2021; Burman \u003cem\u003eet al.\u003c/em\u003e 2022; Du Four \u003cem\u003eet al.\u003c/em\u003e 2022; Lamas \u003cem\u003eet al.\u003c/em\u003e 2023). In our cohort, partial control was most frequent in prolactinomas and acromegaly, while Cushing\u0026rsquo;s disease displayed the broadest spectrum, including one case of complete remission. Taken together, our results underscore the need for harmonized definitions of biochemical response to enable comparability across studies. Consistent with previous observations, biochemical control in functioning PitNETs was variable and did not always parallel the radiological response. Notably, a recent single-center study identified early initiation of TMZ and low MGMT expression as independent predictors of a favorable outcome, particularly in functional tumors (Das \u003cem\u003eet al.\u003c/em\u003e 2021). Nevertheless, many studies have traditionally focused on radiological outcomes as the primary endpoint, relegating hormonal response to a secondary outcome (Burman \u003cem\u003eet al.\u003c/em\u003e 2022; Lamas \u003cem\u003eet al.\u003c/em\u003e 2023; Raverot \u003cem\u003eet al.\u003c/em\u003e 2025). The literature shows considerable variability in the definition of hormonal response. While some studies define response as a reduction exceeding 50% in circulating hormone levels (Luo et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Lamas \u003cem\u003eet al.\u003c/em\u003e 2023), others adopt a stratification approach analogous to the RECIST criteria, since biochemical response was defined as complete normalization of hormone levels; partial response corresponded to a reduction exceeding 20%, stable disease was defined as a fluctuation within \u0026plusmn;\u0026thinsp;20%, and progressive disease as an increase of more than 20% in hormone levels (McCormack \u003cem\u003eet al.\u003c/em\u003e 2018; Deng \u003cem\u003eet al.\u003c/em\u003e 2025), we used the last one to our evaluations.\u003c/p\u003e \u003cp\u003eAn extensive survey demonstrated that clinically functioning tumors were more likely to respond to TMZ than non-functioning tumors, independent of MGMT status (McCormack \u003cem\u003eet al.\u003c/em\u003e 2018). In our series, however, our radiological response rates were similar between functioning and non-functioning tumors, both partial response (23.8% vs. 22.2%, respectively) and radiological disease control, including partial response or stabilization (90.4% vs 100%). Therefore, functional status alone may not consistently discriminate responsiveness in real-world practice. This apparent discrepancy could reflect differences in patient selection, timing of TMZ initiation, or referral bias across cohorts. Beyond these differences, it is also important to note that one criterion for the definition of aggressiveness in pituitary tumors relies on resistance to standard therapies, which is more difficult to apply to non-functioning tumors. In functioning tumors, medical therapies such as cabergoline or somatostatin analogues provide a clear benchmark of resistance, whereas in non-functioning tumors, standard therapy is essentially surgical and adjuvant radiotherapy, with limited medical alternatives. Although cabergoline has been investigated in non-functioning pituitary tumors, with small series reporting disease stabilization and modest tumor shrinkage, it has not been systematically evaluated in the subset of aggressive non-functioning PitNETs (Greenman \u003cem\u003eet al.\u003c/em\u003e 2016; Batista \u003cem\u003eet al.\u003c/em\u003e 2019; Vargas-Ortega \u003cem\u003eet al.\u003c/em\u003e 2022; Ayalon-Dangur \u003cem\u003eet al.\u003c/em\u003e 2024). These conceptual differences underscore the challenges in establishing uniform criteria for aggressiveness across different tumor subtypes. Importantly, while functioning tumors allow for the additional endpoint of biochemical control, our data reinforce that radiological assessment remains the critical measure of efficacy across all subtypes.\u003c/p\u003e \u003cp\u003eRadiological and biochemical endpoints were not fully interchangeable in functioning tumors. Although overall concordance was high, a clinically relevant discordance emerged: biochemical progression occurred in 12.5% of patients despite radiological disease control. This supports independent monitoring of imaging and biochemical markers during TMZ, particularly in functioning PitNETs where biochemical changes may directly translate into morbidity. The moderate kappa observed despite high percent agreement likely reflects the highly unbalanced distribution of radiological outcomes (predominance of PR/SD and rare radiological PD).\u003c/p\u003e \u003cp\u003eThe safety profile of TMZ in our cohort was globally acceptable, with adverse events documented in 66% of patients, predominantly grade 1. Nausea and myelotoxicity were the most frequent toxicities, and treatment discontinuation was required in four cases (13%) due to grade 3\u0026ndash;4 events, with no deaths related to toxicity. These findings align with the broader literature synthesized in the recent international consensus on APT and metastatic PitNETs (Lamas \u003cem\u003eet al.\u003c/em\u003e 2023; Lin \u003cem\u003eet al.\u003c/em\u003e 2023; Raverot \u003cem\u003eet al.\u003c/em\u003e 2025). Discontinuation rates due to adverse events ranged from 6% to 15%, most often driven by pervasive fatigue, nausea, or cytopenias, while rare but severe complications such as aplastic anemia, hepatotoxicity, or secondary hematologic malignancies have been reported (Lamas \u003cem\u003eet al.\u003c/em\u003e 2023; Lin \u003cem\u003eet al.\u003c/em\u003e 2023; Raverot \u003cem\u003eet al.\u003c/em\u003e 2025). Compared with these data, our series underscores two complementary perspectives: first, that the burden of mild adverse effects is common and expected in real-world settings; second, that clinically significant hematological toxicity requiring drug withdrawal is not negligible and should be anticipated in routine practice. Importantly, while we did not observe the rare life-threatening complications highlighted in the consensus, our findings reinforce the need for vigilant hematologic monitoring, as hematologic toxicity remains the leading barrier to treatment continuity in this patient population.\u003c/p\u003e \u003cp\u003eOur study revealed significant heterogeneity in the duration of TMZ treatment, reflecting the current lack of well-defined protocols for the management of APT and metastatic PitNETs. The mean duration of TMZ use was 10.7 months (range, 3\u0026ndash;44 months), highlighting the variability in clinical decisions to initiate TMZ. In some cases, treatment was discontinued after six months due to disease stabilization, emphasizing the need for protocols that support both earlier initiation and longer treatment courses. Given that TMZ was most often prescribed following documented disease progression, tumor stabilization should be considered a favorable therapeutic outcome.\u003c/p\u003e \u003cp\u003e According to the 2025 ESE Clinical Practice Guidelines (Raverot et al., 2025), for patients responding to a first course of TMZ\u0026mdash;defined as either partial tumor regression or stabilization after rapid progression in the preceding six months\u0026mdash;it is now recommended that therapy be continued for at least 12 months and further guided by efficacy and tolerability. Extending treatment beyond 24 months should be carefully weighed against the potential for cumulative severe toxicity. In line with these recommendations, an Italian real-life study including patients treated for more than 12 continuous cycles reported that prolonged TMZ administration was safe and associated with durable disease control and hormonal improvement, particularly among responsive functioning PitNETs (Lizzul \u003cem\u003eet al.\u003c/em\u003e 2020).\u003c/p\u003e \u003cp\u003eIn our study, 10 patients received TMZ treatment for 12 months or longer. Among them, 81.8% experienced at least one adverse event, most of which were mild in severity. Three cases of myelotoxicity were observed, leading to treatment discontinuation in two patients. The treatment discontinuation rate due to adverse effect rate was higher in the group that used TMZ for more than 12 months (30% vs. 5%), while myelotoxicity rate was similar between the groups (30% vs. 30%). This suggests that cumulative toxicity and treatment fatigue, rather than hematologic events per se, may limit long-term tolerability of TMZ in APT.\u003c/p\u003e \u003cp\u003eIn the present cohort, despite a tumor control rate of 96.7% with TMZ, mortality was observed in 7/30 patients (23.3%); two due to direct tumor-related complications (mass effect) and five due to disease progression after TMZ withdrawal, despite having initially achieved stable disease (n\u0026thinsp;=\u0026thinsp;3) or partial response (n\u0026thinsp;=\u0026thinsp;2). These findings reinforce that, in the absence of severe adverse effects, maintaining TMZ in patients with a favorable response may be a safe and effective strategy, even when complete radiological remission is not achieved.\u003c/p\u003e \u003cp\u003e Current literature and guidelines support prolonged TMZ treatment in patients with an initial response, and suggest that re-challenge may be considered, given the higher toxicity of alternative therapies. While a reduced response is typically observed during re-challenge, our study found that patients receiving a second cycle showed similar outcomes to their initial treatment response (Burman \u003cem\u003eet al.\u003c/em\u003e 2022; Cristina Lamas, Rosa Camara, Carmen Fajardo, Pablo Remon-Ruiz, Betina Biagetti, Fernando Guerrero-Perez, Marta Araujo-Castro, Mireia Mora, Felicia Hanzu, Pedro Iglesias, Rogelio Garcıa-Centeno and Alfonso Soto 2023; Raverot \u003cem\u003eet al.\u003c/em\u003e 2025).\u003c/p\u003e \u003cp\u003eAmong 30 patients (9 nonfunctioning and 21 functioning tumors), 26 received radiotherapy (RT) at different time points: 19 before TMZ, 6 after, and 1 concomitantly. Four patients did not receive RT. Considering treatment response, disease control (partial response\u0026thinsp;+\u0026thinsp;stable disease) was observed in 24/26 cases (92.3%). In the group with RT before TMZ (n\u0026thinsp;=\u0026thinsp;19), there were 5 partial responses (26.3%), 12 cases of stable disease (63.2%), and 2 progressions (10.5%), totaling 89.5% disease control. When RT was delivered after TMZ (n\u0026thinsp;=\u0026thinsp;6), 1 patient had a partial response (16.7%) and 5 had stable disease (83.3%), with 100% disease control. The single concomitant RT case (prolactinoma) remained stable. Among those without RT (n\u0026thinsp;=\u0026thinsp;4), there was 1 partial response (25%) and 3 cases of stable disease (75%), also with no progressions (100% control). In summary, no clear advantage of RT timing on overall disease control was identified in this sample, although there was a numerical trend toward more partial responses when RT was given before TMZ (or omitted), contrasted by the fact that the only progressions occurred in that group, an observation that may reflect selection biases and the small sample size.\u003c/p\u003e \u003cp\u003e The 2025 ESE guideline advises considering concurrent TMZ with radiotherapy for pituitary tumors showing rapid progression of a large residual that is not amenable to further surgery, particularly when high proliferative markers and/or adverse somatic mutations are present, while not otherwise specifying the optimal timing of TMZ relative to radiotherapy. It recommends that radiotherapy decisions be based on postoperative MRI findings obtained within 3 months and notes that, in aggressively growing tumors, combined TMZ\u0026ndash;radiotherapy should be considered (Raverot \u003cem\u003eet al.\u003c/em\u003e 2025). Retrospective series suggest higher radiological or composite response rates with combination therapy versus radiotherapy alone, though these data are constrained by heterogeneity, potential confounding, and a lack of long-term outcomes. Accordingly, the guideline emphasizes individualized, multidisciplinary decision-making when considering TMZ\u0026ndash;radiotherapy outside the narrow indication of rapid progression (Lamas \u003cem\u003eet al.\u003c/em\u003e 2023; Lin \u003cem\u003eet al.\u003c/em\u003e 2023; Raverot \u003cem\u003eet al.\u003c/em\u003e 2025).\u003c/p\u003e \u003cp\u003eAccording to the recent ESE guidelines, TMZ is recommended as the first-line chemotherapeutic option for APT and metastatic PitNETs, once progression is documented despite standard therapies (Raverot \u003cem\u003eet al.\u003c/em\u003e 2025). In our cohort, however, the median interval between diagnosis and TMZ initiation was close to 100 months, reflecting that systemic therapy is frequently reserved for late stages of disease after multiple local interventions. While this approach is consistent with the cautious positioning of current recommendations, our findings suggest that, in real-world practice, TMZ may often be introduced relatively late in the treatment course. Indeed, the benefit of early initiation on TMZ (median 15 months) was associated with higher response rates, while delays beyond 31 months predicted poorer outcomes (Das \u003cem\u003eet al.\u003c/em\u003e 2021). However, prospective studies and the development of biological markers of aggressiveness will be crucial to refine treatment timing and harmonize practice across centers.\u003c/p\u003e \u003cp\u003eBeyond clinical decision-making, access-related barriers also contribute to delayed initiation of TMZ. Because the drug is not formally approved for pituitary tumors in most countries, reimbursement is frequently denied by health systems, and treatment may depend on exceptional authorization. In Brazil and other Latin American settings, this challenge is particularly evident, but similar constraints are likely to affect other regions worldwide since TMZ remains off-label in many countries, not formally approved by the US Food and Drug Administration (FDA), European Medicines Agency or Australian Therapeutic Goods Administration (TGA) (McCormack \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Such regulatory and economic hurdles highlight a global gap between guideline recommendations and real-world implementation, reinforcing the need for strategies to ensure timely and equitable access to evidence-based therapies across diverse healthcare systems.\u003c/p\u003e \u003cp\u003eFinally, the long interval between tumor diagnosis and TMZ initiation (median 100 months) raises important questions about treatment timing. Earlier initiation of systemic therapy in selected cases may improve outcomes and reduce cumulative surgical and radiotherapeutic morbidity. The heterogeneity of clinical trajectories observed in our cohort further supports the need for standardized therapeutic protocols, ideally informed by biological markers.\u003c/p\u003e \u003cp\u003e \u003cb\u003eLimitations.\u003c/b\u003e This study has limitations inherent to a retrospective multicenter design. Treatment timing, imaging schedules, and biochemical assays were not fully standardized across centers, which may introduce measurement variability despite the use of prespecified response definitions. Selection bias is possible because temozolomide was used in highly pretreated, clinically aggressive cases referred to tertiary centers, limiting generalisability. Key pathology variables were not available for all patients (Ki-67 and p53), and biochemical response could be evaluated only in functioning tumors with available data, which reduces power for subgroup and concordance analyses. Finally, the sample size\u0026mdash;particularly within clinically relevant subgroups\u0026mdash;results in imprecision around estimates and limits the feasibility of confounder-adjusted modeling; findings should therefore be interpreted as descriptive real-world evidence.\u003c/p\u003e \u003cp\u003e \u003cb\u003eInterpretation.\u003c/b\u003e In a real-world multicenter cohort, temozolomide was associated with high radiological disease control and frequent biochemical improvement in functioning tumors, with manageable toxicity. These findings address our objective of characterizing effectiveness and safety in Brazilian referral centers; however, inference is limited by the retrospective design, center-level heterogeneity in assessment timing, and missingness in biochemical and pathology variables, which also reduced the size of key subgroup analyses. Given the multiplicity of exploratory comparisons, subgroup signals should be interpreted as hypothesis-generating rather than definitive. Overall, our estimates align with the range of outcomes reported in prior temozolomide series in aggressive PitNETs and support standardized prospective data collection to refine predictors and monitoring strategies.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGeneralisability.\u003c/b\u003e Because this is a multicenter cohort from Brazilian tertiary referral centers, our findings are most generalisable to similarly selected patients with aggressive or metastatic PitNETs managed in specialized high-volume units, where temozolomide is typically used after failure of standard local and medical therapies. Extrapolation to earlier-line use, to centers with different imaging/biochemical monitoring practices, or to less aggressive tumors should be made cautiously. Nevertheless, the inclusion of multiple referral centers and routine-care assessments enhances external validity for real-world practice in comparable settings.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eTMZ was effective and generally well tolerated in this Brazilian real-world multicenter cohort of APTs and metastatic PitNETs, yielding high rates of radiological disease control and a low discontinuation rate. In functioning tumors, biochemical responses were heterogeneous and could diverge from imaging outcomes, supporting parallel endocrine and radiological monitoring. When tolerated, continuation beyond the initial cycles was feasible and aligned with guideline-based systemic management when systemic therapy is indicated. These data expand the Latin American evidence base and underscore the need for collaborative prospective efforts to validate biomarkers and optimize temozolomide timing and duration within individualized therapeutic pathways for aggressive/metastatic PitNETs.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding.\u003c/h2\u003e \u003cp\u003eRLB supported by the Conselho Nacional de Desenvolvimento Cient\u0026iacute;fico e Tecnol\u0026oacute;gico (CNPq), grant 444825/2024-0. RLB is also supported by the AACR (American Association for Cancer Research), grant 23-15-01-BATI.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eI.P.A.M., C.R.P.B.M., L.A.N., and R.L.B. conceived and designed the study. I.P.A.M. and C.R.P.B.M. coordinated data collection, curated the multicenter dataset, and drafted the main manuscript. L.A.N., M.A.C., I.N.R., L.B.S., T.S.A., P.C.L.E., M.C., L.K., M.R.G., C.G.S.L.R., H.M.G., C.B., R.S.J., M.N., M.C.B.V.F., M.C.M., A.G., M.B.C.C.N., and O.F. contributed to patient inclusion and clinical/pathological data acquisition at their respective centers and critically revised the manuscript. M.P. performed/oversaw radiological assessment and contributed to figure preparation. I.P.A.M., C.R.P.B.M., and R.L.B. performed the statistical analyses and prepared the final figures/tables. R.L.B. supervised the project and finalized the manuscript. All authors reviewed and approved the final version of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAyalon-Dangur I, Turjeman A, Hirsch D, Robenshtok E, Tsvetov G, Gorshtein A, Masri H, Shraga-Slutzky I, Manisterski Y, Akirov A et al 2024 Cabergoline treatment for surgery-na\u0026iuml;ve non-functioning pituitary macroadenomas. 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Pituitary 23 359\u0026ndash;366\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLosa M, Bogazzi F, Cannavo S, Ceccato F, Curt\u0026ograve; L, De Marinis L, Iacovazzo D, Lombardi G, Mantovani G, Mazza E et al 2016 Temozolomide therapy in patients with aggressive pituitary adenomas or carcinomas. J Neurooncol 126 519\u0026ndash;525\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLuo M, Tan Y, Chen W, Hu B, Wang Z, Zhu D, Jiao H, Duan C, Zhu Y, Wang H (2021) Clinical Efficacy of Temozolomide and Its Predictors in Aggressive Pituitary Tumors and Pituitary Carcinomas: A Systematic Review and Meta-Analysis. Front Neurol 12:700007\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMcCormack A (2022) Temozolomide in aggressive pituitary tumours and pituitary carcinomas. Best Pract Res Clin Endocrinol Metab 36:101713\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMcCormack AI, McDonald KL, Gill AJ, Clark SJ, Burt MG, Campbell KA, Braund WJ, Little NS, Cook RJ, Grossman AB et al 2009 Low O6-methylguanine-DNA methyltransferase (MGMT) expression and response to temozolomide in aggressive pituitary tumours. Clin Endocrinol 71 226\u0026ndash;233\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMcCormack A, Dekkers OM, Petersenn S, Popovic V, Trouillas J, Raverot G, Burman P \u0026amp; ESE survey collaborators 2018 Treatment of aggressive pituitary tumours and carcinomas: results of a European Society of Endocrinology (ESE) survey 2016. Eur J Endocrinol 178 265\u0026ndash;276\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMelmed S (2020) Pituitary-tumor endocrinopathies. N Engl J Med 382:937\u0026ndash;950\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u0026Ouml;zdemir BC, Dotto G-P (2017) Racial differences in cancer susceptibility and survival: More than the color of the skin? Trends Cancer 3:181\u0026ndash;197\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRaverot G, Castinetti F, Jouanneau E, Morange I, Figarella-Branger D, Dufour H, Trouillas J \u0026amp; Brue T 2012 Pituitary carcinomas and aggressive pituitary tumours: merits and pitfalls of temozolomide treatment. Clin Endocrinol 76 769\u0026ndash;775\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRaverot G, Burman P, McCormack A, Heaney A, Petersenn S, Popovic V, Trouillas J, Dekkers OM \u0026amp; European Society of Endocrinology 2018 European Society of Endocrinology Clinical Practice Guidelines for the management of aggressive pituitary tumours and carcinomas. Eur J Endocrinol 178 G1\u0026ndash;G24\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRaverot G, Burman P, Abreu AP, Heaney AP, van Hulsteijn L, Lin AL, Marcus H, McCormack A, Minniti G, Petersenn S et al 2025 Revised European Society of Endocrinology Clinical Practice Guideline for the management of aggressive pituitary tumours and pituitary carcinomas. Eur J Endocrinol 192 R45\u0026ndash;R78\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eReilly -O\u0026rsquo;, Newlands SM, Glaser ES, Brampton MG, Rice-Edwards M, Illingworth JM, Richards RD, Kennard PG, Colquhoun C, Lewis IR et al 1993 Temozolomide: a new oral cytotoxic chemotherapeutic agent with promising activity against primary brain tumours. Eur J Cancer 29 940\u0026ndash;942\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSyro LV, Ortiz LD, Scheithauer BW, Lloyd R, Lau Q, Gonzalez R, Uribe H, Cusimano M, Kovacs K \u0026amp; Horvath E 2011 Treatment of pituitary neoplasms with temozolomide. Cancer 117 454\u0026ndash;462\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSyro LV, Rotondo F, Camargo M, Ortiz LD, Serna CA, Kovacs K 2018 Temozolomide and pituitary tumors: Current understanding, unresolved issues, and future directions. Front Endocrinol 9. (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fendo.2018.00318\u003c/span\u003e\u003cspan address=\"10.3389/fendo.2018.00318\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVargas-Ortega G, Gonz\u0026aacute;lez-Virla B, Balc\u0026aacute;zar-Hern\u0026aacute;ndez L, Arreola-Rosales R, Benitez-Rodr\u0026iacute;guez FJ, L\u0026oacute;pez F\u0026eacute;lix B \u0026amp; Mercado M 2022 Efficacy of cabergoline therapy in patients with non-functioning pituitary adenomas: A single center clinical experience. Archives Endocrinol Metabolism 66 506\u0026ndash;511\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWhitelaw BC (2019) How and when to use temozolomide to treat aggressive pituitary tumours. Endocrine-related Cancer 26:R545\u0026ndash;R552\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZacharia BE, Gulati AP, Bruce JN, Carminucci AS, Wardlaw SL, Siegelin M, Remotti H, Lignelli A, Fine RL 2014 High response rates and prolonged survival in patients with corticotroph pituitary tumors and refractory Cushing disease from capecitabine and temozolomide (CAPTEM): a case series. Neurosurgery 74 E447\u0026ndash;E455; discussion E455.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZheng G, Lu L, Zhu H, You H, Feng M, Liu X, Dai C, Yao Y, Wang R, Zhang H et al 2020 Clinical, Laboratory, and Treatment Profiles of Silent Corticotroph Adenomas That Have Transformed to the Functional Type: A Case Series With a Literature Review. Front Endocrinol 11 558593\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"pituitary","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pitu","sideBox":"Learn more about [Pituitary]()","snPcode":"11102","submissionUrl":"https://submission.nature.com/new-submission/11102/3","title":"Pituitary","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"aggressive pituitary adenoma, pituitary carcinoma, pituitary neuroendocrine tumors, temozolomide, aggressive PitNETs, MGMT","lastPublishedDoi":"10.21203/rs.3.rs-8428238/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8428238/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eTo evaluate the real-world efficacy and safety of temozolomide (TMZ) in aggressive and metastatic pituitary neuroendocrine tumors (PitNETs) in a Latin American setting, addressing whether TMZ achieves meaningful radiological and biochemical disease control with acceptable toxicity.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eWe conducted a retrospective multicenter study across Brazilian reference centers including patients with aggressive/metastatic PitNETs treated with TMZ and followed for \u0026ge;\u0026thinsp;6 months. Radiological response was assessed using RECIST 1.1. For functioning PitNETs, biochemical response was assessed using prespecified hormonal criteria. Adverse events were collected from medical records.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThirty patients were included (mean age 29.5 years; 53% female). All tumors were macroadenomas and 56% were giant (\u0026gt;\u0026thinsp;4 cm). Twenty-one PitNETs were functioning and four were metastatic. Ki-67 was \u0026gt;\u0026thinsp;3% in 73% of cases. Median time from diagnosis to TMZ initiation was 100 months. Radiological disease control rate (partial response or stable disease) was 96.7%. Among functioning tumors, biochemical disease control rate was 81.3%, with an objective biochemical response rate (complete\u0026thinsp;+\u0026thinsp;partial response) of 68.8%. Adverse events occurred in 66% of patients, most commonly nausea and myelotoxicity.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eIn this multicenter Brazilian real-world cohort, TMZ provided high radiological and biochemical disease control with an acceptable safety profile, supporting TMZ as preferred first-line systemic chemotherapy for aggressive/metastatic PitNETs after failure of standard therapies.\u003c/p\u003e","manuscriptTitle":"Temozolomide in Aggressive Pituitary Tumors and Metastatic PitNETs: A Brazilian Multicenter Real-World Cohort Study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-31 01:18:07","doi":"10.21203/rs.3.rs-8428238/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-13T22:36:00+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-13T22:27:19+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-10T11:58:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"31460319461035269574349368372886303523","date":"2025-12-28T07:50:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"91426584089253981697513154785663978380","date":"2025-12-26T10:55:52+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-26T07:15:43+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-23T14:39:28+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-23T14:38:26+00:00","index":"","fulltext":""},{"type":"submitted","content":"Pituitary","date":"2025-12-22T20:19:58+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"pituitary","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pitu","sideBox":"Learn more about [Pituitary]()","snPcode":"11102","submissionUrl":"https://submission.nature.com/new-submission/11102/3","title":"Pituitary","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"49c34dc7-6fc7-4bf1-9b0c-b8e562464259","owner":[],"postedDate":"December 31st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-30T16:18:41+00:00","versionOfRecord":{"articleIdentity":"rs-8428238","link":"https://doi.org/10.1007/s11102-026-01663-z","journal":{"identity":"pituitary","isVorOnly":false,"title":"Pituitary"},"publishedOn":"2026-03-27 16:11:06","publishedOnDateReadable":"March 27th, 2026"},"versionCreatedAt":"2025-12-31 01:18:07","video":"","vorDoi":"10.1007/s11102-026-01663-z","vorDoiUrl":"https://doi.org/10.1007/s11102-026-01663-z","workflowStages":[]},"version":"v1","identity":"rs-8428238","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8428238","identity":"rs-8428238","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}