Leucovorin Research Articles

LOW-RISK GESTATIONAL TROPHOBLASTIC NEOPLASIA: INSIGHTS FROM A TERTIARY CARE INSTITUTE IN INDIA

Objective: To report clinical characteristics, treatment outcomes and chemotherapy-related toxicities in patients with low-risk GTN at tertiary care centre in India. Material and Methods: This retrospective observational study was conducted at the Department of Medical Oncology of Vardhman Mahavir Medical College and Safdarjung Hospital, New Delhi over 2 years. The clinical and treatment information of every patient diagnosed with GTN between December 2021 and December 2023 were examined retrospectively in their medical records. Methotrexate (MTX) was administered with folinic acid (FA) rescue to low-risk GTN patients. EMA-CO, multiagent chemotherapy, was administered every two weeks to patients with low-risk GTN who were resistant to first-line chemotherapy. Results: Only 35 of the 40 patients with low-risk GTN were able to be evaluated since five of them were lost to follow-up throughout the course of treatment.The study found that the majority of patients (71.4%) experienced a molar pregnancy before developing gestational trophoblastic neoplasia (GTN), with 91.4% developing GTN within the first 4 months. Of these, 32 patients achieved complete responses (91.4%)with MTX therapy, while 3 experienced treatment failure (8.5%). Following multiagent chemotherapy, all three ofthe patients who had not responded to initial MTX therapy experienced complete remission (CR). All patients with GTN who were at low risk had 100% overall survival (OS) and cure rates. Conclusion: With a complete response (CR) rate of 91.4% and no serious side effects, the MTX regimen proved to be highly effective in treating women with low-risk GTN.

Assessment of anticancer drug utilization pattern and patients’ survival—A single center experience from Saudi Arabia

Background In recent years, various advancements in anticancer therapy have led to the development of multiple regimens and protocols. This study endeavors to provide an extensive evaluation of anticancer therapy prescription patterns in correlation with patient outcomes. Methods From June 2014 to April 2022, we included adult cancer patients who received anticancer therapy in our cancer center. Collected data encompassed demographic characteristics of patients and cancer, chemotherapy protocols or agents, antiemetics, drug side effects, and the patient’s last status. The prescribed drugs were assessed using the Essential Medicines List, while the prescription’s rationality was determined using the World Health Organization indicators. Results The mean age was 55.16 ± 17.04 years, with 56.4% of the patients being males. Gastrointestinal (29.7%) and breast (25.8%) cancers were the most common malignancies. The main protocols included a combination of Adriamycin and cyclophosphamide (20.1%) and folinic acid, fluorouracil, and oxaliplatin-based (FOLFOX) regimen (13.5%). The most frequently used drugs were doxorubicin (14.0%), cyclophosphamide (13.3%), and docetaxel (9.9%). The majority of patients also did not report any acute adverse events related to chemotherapy (81.1%). Antiemetics, mainly metoclopramide-based, were used in 76.07% of cases. Remarkably, 86.7% of anticancer agents were from the EML, and 90.1% were prescribed generically. Conclusion In this study, gastrointestinal cancers were the most prevalent cancers observed, with more preponderance among males. Most anticancer agents were taken from the essential drug list, with the majority being prescribed under generic names, indicating rational use.

Treatment of patients with carcinomas in advanced stages with 5-fluorouracil, folinic acid and pyridoxine in tandem

The effect of high-dose pyridoxine (PN) on activity of 5-fluorouracil (FUra) and folinic acid (FA)-containing regimens was studied in 50 patients including 14 with digestive tract, and 36 with breast carcinomas (BC) in advanced stages with poor prognostic characteristics. Patients with colorectal, and pancreas adenocarcinoma received oxaliplatin, irinotecan, FUra, FA (Folfirinox), and patients with squamous cell carcinoma of the esophagus had paclitaxel, carboplatin, FUra, FA (TCbF). Patients with BC received AVCF (doxorubicin, vinorelbine, cyclophosphamide, FUra, FA) followed by TCbF or TCbF only, and patients who overexpressed HER2 received TCbF plus trastuzumab and pertuzumab. PN (1000–3000 mg/day iv) preceded each administration of FUra and FA. 47 patients (94%) responded, including 16 (32%) with CR. Median tumor reduction was 93%. Median event-free survival (EFS) was 37.7 months. The 25 patients with tumor shrinkage ≥ 91% had EFS of 52% from 42 months onwards. Unexpected toxicity did not occur. PN enhances potency of chemotherapy regimens comprising FUra and FA.

Methotrexate Induced Oral Mucositis and Bone Marrow Suppression in Psoriatic Patient

Methotrexate is an antimetabolite which binds to the dihydrofolate reductase enzyme and used in the treatment of malignant disorders and autoimmune diseases. Myelosuppression is a serious complication of methotrexate toxicity. The following case describes a case of 67 year old male patient diagnosed with psoriasis and wrongly taken methotrexate 7.5 mg daily for 3 days and twice daily for 4 days. He presented with oral mucositis and his lab reports showed myelosuppression. Our case emphasizes the significance of effective patient counselling to ensure the understanding of prescription and to prevent drug toxicity. The key learning points of our case report include the importance of adhering to prescribed medication regimens, awareness of MTX toxicity, early initiation of folinic acid as an antidote, vigilant monitoring of toxicity, and effective patient education on proper medication use. Adherence to established MTX therapy guidelines in dermatological conditions is crucial to minimize the risk of patient.

Real-World Evidence of FOLFIRI Combined with Anti-Angiogenesis Inhibitors or Anti-EGFR Antibodies for Patients with Early Recurrence Colorectal Cancer After Adjuvant FOLFOX/CAPOX Therapy: A Japanese Claims Database Study.

Oxaliplatin-containing adjuvant regimens (folinic acid, fluorouracil, and oxaliplatin/capecitabine and oxaliplatin [FOLFOX/CAPOX]) are used after curative resection of colorectal cancer (CRC). However, real-world evidence regarding treatment sequences and outcomes in patients with early recurrence CRC after adjuvant chemotherapy is limited. We aimed to describe the patient characteristics, treatment sequence, and overall duration of second-line (2L) therapy in patients with early recurrence CRC who received adjuvant chemotherapy (FOLFOX/CAPOX) followed by folinic acid, fluorouracil, and irinotecan (FOLFIRI) + anti-angiogenesis drugs (AA) or FOLFIRI + anti-epidermal growth factor receptor (EGFR) antibodies. This retrospective study analyzed Japanese administrative data from November 2014 to March 2023 of adult patients who underwent CRC resection surgery, started FOLFOX/CAPOX ≤3 months (mo) after surgery, and had early CRC recurrence. Early recurrence was defined as initiation of FOLFIRI+AA or FOLFIRI+anti-EGFR antibodies as 2L therapy, ≤12 mo of discontinuing adjuvant chemotherapy. Patient characteristics, treatment sequence, median time to treatment discontinuation (mTTD), i.e., duration between the start and end dates of 2L therapy (Kaplan-Meier method), and factors associated with 2L time to treatment discontinuation constituted the study outcomes (Cox regression model). Subgroup analyses were performed for timing of early CRC recurrence (≤6 mo and 6-12 mo) and tumor sidedness. Among the 832 selected patients (median age [minimum-maximum] 67 (24-86) years, 56.4% male), CAPOX (71.3%) was more commonly used than FOLFOX (28.7%) as adjuvant therapy. FOLFIRI+AA (72.5%) was used more commonly than FOLFIRI+anti-EGFR antibodies (27.5%) in 2L. AA and anti-EGFR antibodies groups had similar mTTD: 6.2 mo (95% confidence interval 5.8, 6.9) and 6.1 mo (95% confidence interval 5.2, 7.4). Age ≥70 years showed significant association with shorter 2L treatment duration (hazard ratio 1.2, 95% confidence interval 1.0, 1.4; p = 0.03). The AA cohort's mTTD was numerically shorter in the ≤6 mo recurrence subgroup compared with the 6-12 mo recurrence subgroup (6.1 mo vs 8.1 mo); the anti-EGFR antibodies cohort had similar mTTD (5.8 mo vs 6.2 mo). The AA and anti-EGFR antibodies cohorts also had similar mTTD in the left-sided CRC subgroup (6.5 mo vs 6.2 mo), but not in the right-sided subgroup (5.6 mo vs 3.9 mo). This is the first administrative data-based real-world evidence on treatment sequence and outcomes for patients with early recurrence CRC treated with FOLFIRI+AAs or FOLFIRI+ anti-EGFR antibodies after adjuvant FOLFOX/CAPOX therapy in Japan. Both regimens had similar TTD, but relapse timing and tumor sidedness may influence their efficacy.

Evaluation of Stages, Treatment Protocols, and Outcomes of Colorectal Cancer among West Bank Patients.

Background: Colorectal cancer (CRC) is the second most widespread cancer among Palestinian patients. As cancer care improves in hospitals across the West Bank, services like palliative care, targeted therapy, bone marrow transplantation, and individualized therapy are still limited. This study aimed to assess the CRC stages, treatment protocols, and survival rates of patients in the West Bank. Methodology: This retrospective study collected data from the medical records of Al-Najah University Hospital (NUH), which specializes in the care of cancer patients. Patients with confirmed CRC (stages I-IV) undergoing surgical or medical treatment were included in the study. Data collection was standardized by using a data collection form to gather information from the medical records included in the study. All statistical analyses were performed using SPSS (version v27), and survival was assessed using a regression analysis of the number of days from the time of diagnosis to the most recent visit against the type of treatment (e.g., surgery, chemotherapy, radiotherapy). Results: A sample of 252 patients with CRC from NUH was collected, including 143 males and 109 females aged between 27 and 86 years, with the average age being 60.6 ± 11.4 years. The sample included 183 patients (72.6%) diagnosed with colon cancer only, 29 patients (11.5%) diagnosed with rectal cancer only, and 40 patients (15.9%) diagnosed with both. Diagnosis took place at CRC stage I for 3 patients (1.2%), stage II for 33 patients (13.1%), stage III for 57 patients (22.6%), and stage IV for 159 patients (63.1%). Surgery was the most prevailing mode of treatment for 230 patients (91.3%), while 227 patients (90.1%) received chemotherapy treatment, and 38 patients (15.1%) received radiotherapy. Of the 252 patients, 40 patients (15.8%) received FOLFOX (i.e., folinic acid, fluorouracil, oxaliplatin), and 25 patients (9.9%) received FOLFIRI (i.e., folinic acid, fluorouracil, irinotecan), while the 187 remaining patients (74.2%) were treated with capecitabine, oxaliplatin, bevacizumab, cetuximab, regorafenib, cisplatin, etoposide, gemcitabine, or a combination thereof. The sample was categorized into six outcomes: (1) death, (2) cure, (3) disease progression, (4) disease recurrence, (5) under-treatment, and (6) unknown. Mortality was high, with 104 patients (41.3%) dying within a short time after diagnosis, and may have been attributable to delayed diagnosis. Surgical treatment had a positive impact on increasing the survival years, and it was significant (p = 0.033). Conclusions: A high percentage of patients were diagnosed in advanced CRC stages. The treatment modes were adopted from general international guidelines; however, the cure rates were low, and mortality was high. More studies need to be undertaken to investigate the actual application of chemotherapy protocols, and survival would benefit from the involvement of clinical pharmacists in the chemotherapy protocol selection, dosing, frequency, and follow-up. The present study advocates for greater public awareness of CRC and attests to the merits of screening by primary care professionals, which can help to avoid this serious illness and to promote a better prognosis.

British Oncology Pharmacy Association Delphi consensus guidelines: Co-infusion of trometamol-containing calcium folinate (Leucovorin) with systemic anti-cancer treatments.

Drug stability and compatibility are critical factors influencing the cost and logistics of treatment delivery, therapeutic effectiveness, and patient safety. This is particularly significant in the realm of cancer chemotherapeutics, where stability and compatibility studies play a vital role in ensuring rational and safe medicine administration. Oxaliplatin, fluorouracil, and irinotecan, commonly used in various combinations for gastrointestinal cancers, are complemented by co-administration of folinic acid in certain protocols. Notably, some folinic acid preparations include trometamol as an excipient, potentially impacting the stability of the chemotherapeutic agents if infused concomitantly. This study seeks to establish guidelines for oncology multidisciplinary teams, addressing potential risks associated with the combination of trometamol-containing folinic acid and chemotherapeutics. To achieve this, a quantitative questionnaire was distributed to members of the British Oncology Pharmacy Association (BOPA) and non-BOPA members through an online survey. Nineteen healthcare professionals with oncology experience, comprising 18 pharmacists and one nurse, completed the questionnaires. Each participant rated the validity and clarity of statements on a 5-point scale. The Delphi process concluded after the fourth round, consolidating the findings and recommendations from the multidisciplinary team. Twelve recommendations for safe practice have been made.

FOLFIRI Plus Durvalumab With or Without Tremelimumab in Second-Line Treatment of Advanced Gastric or Gastroesophageal Junction Adenocarcinoma

Efficacy of second-line chemotherapy in advanced gastric or gastrooesphageal junction (GEJ) adenocarcinoma remains limited. To determine the efficacy of 1 or 2 immune checkpoint inhibitors combined with FOLFIRI (leucovorin [folinic acid], fluorouracil, and irinotecan) in the treatment of advanced gastric/GEJ adenocarcinoma. The PRODIGE 59-FFCD 1707-DURIGAST trial is a randomized, multicenter, noncomparative, phase 2 trial, conducted from August 27, 2020, and June 4, 2021, at 37 centers in France that included patients with advanced gastric/GEJ adenocarcinoma who had disease progression after platinum-based first-line chemotherapy. Patients were randomized to receive FOLFIRI plus durvalumab (anti-programmed cell death 1 [PD-L1]) (FD arm) or FOLFIRI plus durvalumab and tremelimumab (anti-cytotoxic T-lymphocyte associated protein 4 [CTLA-4]) (FDT arm). The efficacy analyses used a clinical cutoff date of January 9, 2023. The primary end point was progression-free survival (PFS) at 4 months according to RECIST 1.1 criteria evaluated by investigators. Overall, between August 27, 2020, and June 4, 2021, 96 patients were randomized (48 in each arm). The median age was 59.7 years, 28 patients (30.4%) were women and 49 (53.3%) had GEJ tumors. Four month PFS was 44.7% (90% CI, 32.3-57.7) and 55.6% (90% CI, 42.3-68.3) in the FD and FDT arms, respectively. The primary end point was not met. Median PFS was 3.8 and 5.4 months, objective response rates were 34.7% and 37.7%, and median overall survival was 13.2 and 9.5 months in the FD and FDT arms, respectively. Disease control beyond 1 year was 14.9% in the FD arm and 24.4% in the FDT arm. Grade 3 to 4 treatment-related adverse events were observed in 22 (47.8%) patients in each arm. A combined positive score (CPS) PD-L1 of 5 or higher was observed in 18 tumors (34.0%) and a tumor proportion score (TPS) PD-L1 of 1% or higher in 13 tumors (24.5%). Median PFS according to CPS PD-L1 was similar (3.6 months for PD-L1 CPS ≥5 vs 5.4 months for PD-L1 CPS <5) by contrast for TPS PD-L1 (6.0 months for PD-L1 TPS ≥1% vs 3.8 months for PD-L1 TPS <1%). Combination of immune checkpoint inhibitors with FOLFIRI in second-line treatment for advanced gastric/GEJ adenocarcinoma showed an acceptable safety profile but antitumor activity only in a subgroup of patients. ClinicalTrials.gov Identifier: NCT03959293.

Engineering 5-flourouracil and leucovorin-loaded vesicular systems for possible colon specific delivery: In vitro evaluation and real time cell assay against HCT-116 colon cell lines

5-flourouracil (5-FU) is typically modulated with leucovorin (LEU) in clinical practice to improve clinical efficacy and patient survival rates. However, this combination has undesirable side effects and makes 5-FU more toxic. Hence, integrating a vesicular system (proniosomes) with another delivery vehicle may improve drug targeting, while resolving the aforementioned drawbacks. This study aimed to engineer 5-FU/LEU proniosomes for possible delivery to the colon. The modified slurry approach was used to create drug-loaded proniosomes (150 mg/9 g of carrier) using both water-soluble (dextrin (DEX) and lactose (LAC)) and insoluble (Neusilin FH2 (NEU)) carriers. The powdered formulations were filled into Eudragit S100 (10 %)-coated capsules or Eudragit FS 30D capsules for enteric- or colon-specific delivery. In vitro evaluations (flow properties, powder X-ray diffractometry (XRD) analysis, particle size analysis, entrapment efficiency, drug release, scanning electron microscopy (SEM), polydispersity index, Fourier transform infrared spectroscopy (FTIR), and stability studies) were performed on the formulations. An in vitro cytotoxicity test [real-time cell assay (RTCA)] against HCT-116 colon cancer cell lines was performed using the optimized formulation. In vitro evaluations showed that the nanoparticles had good physicochemical properties. RTCA studies showed sustained cell death with the formulations compared to the pure drug and placebo. The sequential drug release of the colon-targeted capsules containing 5-FU and LEU- loaded proniosomes showed negligible drug release in SGF (pH 1.2) and phosphate buffer solution (pH 6.8) (approximately 11 %) but profound drug release (>80 %) at pH 7.4. Drug-loaded proniosomes engineered for colon targeting (Eudragit S100 (10 %) capsules or Eudragit FS 30D capsules) showed good colon-specific targeting and favorable in vitro cytotoxicity profiles.

Efficacy and safety of neoadjuvant sintilimab in combination with FLOT chemotherapy in patients with HER2-negative locally advanced gastric or gastroesophageal junction adenocarcinoma: an investigator-initiated, single-arm, open-label, phase II study.

The addition of immune checkpoint inhibitors to neoadjuvant chemotherapy in operable advanced gastric or gastroesophageal junction (G/GEJ) cancer aroused wide interest. This study was designed to assess the efficacy and safety of neoadjuvant sintilimab, a programmed cell death protein-1 (PD-1) inhibitor, in combination with fluorouracil, leucovorin, oxaliplatin, and docetaxel (FLOT) chemotherapy for HER2-negative locally advanced G/GEJ cancer. Eligible patients with clinical stage cT4 and/or cN+M0 G/GEJ cancer were enroled in this phase II study. Patients received neoadjuvant sintilimab (200mg every 3 weeks) for three cycles plus FLOT (50mg/m 2 docetaxel, 80mg/m 2 oxaliplatin, 200mg/m 2 calcium levofolinate, 2600mg/m 2 5-fluorouracil every 2 weeks) for four cycles before surgery, followed by four cycles of adjuvant FLOT with same dosages after resection. The primary endpoint was the pathological complete response (pCR) rate. Thirty-two patients were enroled between August 2019 and September 2021, with a median follow-up of 34.8 (95% CI, 32.8-42.9) months. Thirty-two (100%) patients received neoadjuvant therapy, and 29 underwent surgery with an R0 resection rate of 93.1%. The pCR (TRG0) was achieved in 5 (17.2%; 95% CI, 5.8-35.8%) patients, and the major pathological response was 55.2%. Twenty-three (79.3%) patients had T downstaging, 21 (72.4%) had N downstaging, and 19 (65.5%) had overall TNM downstaging. Six (20.7%) patients experienced recurrence. Patients achieving pCR showed better event-free survival (EFS), disease-free survival (DFS), and overall survival (OS) than non-pCR. The estimated 3-year EFS rate, 3-year DFS rate, and 3-year OS rate were 71.4% (95% CI, 57.2-89.2%), 78.8% (95% CI, 65.1-95.5%), and 70.9% (95% CI, 54.8-91.6%), respectively. The objective response rate and disease control rate were 84.4% (95% CI, 68.3-93.1%) and 96.9% (95% CI, 84.3-99.5%), respectively. Twenty-five (86.2%) received adjuvant therapy. The main grade ≥3 treatment-related adverse events (TRAEs) were lymphopenia (34.4%), neutropenia (28.1%), and leukopenia (15.6%). no patients died from TRAE. The LDH level exhibited a better predictive value to pathological responses than PD-L1 and MSI status. The study demonstrated an encouraging efficacy and manageable safety profile of neoadjuvant sintilimab plus FLOT in HER2-negative locally advanced G/GEJ cancer, which suggested a potential therapeutic option for this population.

Post-therapy emergence of an NBN reversion mutation in a patient with pancreatic acinar cell carcinoma.

Pancreatic acinar cell carcinoma (PACC) is a rare form of pancreatic cancer that commonly harbors targetable alterations, including activating fusions in the MAPK pathway and loss-of-function (LOF) alterations in DNA damage response/homologous recombination DNA repair-related genes. Here, we describe a patient with PACC harboring both somatic biallelic LOF of NBN and an activating NTRK1 fusion. Upon disease progression following 13 months of treatment with folinic acid, fluorouracil, irinotecan, and oxaliplatin (FOLFIRINOX), genomic analysis of a metastatic liver biopsy revealed the emergence of a novel reversion mutation restoring the reading frame of NBN. To our knowledge, genomic reversion of NBN has not been previously reported as a resistance mechanism in any tumor type. The patient was treated with, but did not respond to, targeted treatment with a selective NTRK inhibitor. This case highlights the complex but highly actionable genomic landscape of PACC and underlines the value of genomic profiling of rare tumor types such as PACC.

Analysis of 9 patients with adolescence-onset methylenetetrahydrofolate reductase deficiency

Objective: To explore the diagnosis and treatment of adolescence-onset methylenetetrahydrofolate reductase (MTHFR) deficiency. Methods: This was a retrospective case study. Nine patients with adolescence-onset MTHFR deficiency were diagnosed at Peking University First Hospital from January 2016 to December 2022, and followed up for more than 1 year. Their general information, clinical manifestations, laboratory tests, cranial images, MTHFR gene variants, diagnosis, treatment, and outcome were analyzed retrospectively. Results: The 9 patients came from 8 families. They had symptoms at age of 8.0 years to 17.0 years and diagnosed at 9.0 years to 17.5 years. Eight were male and 1 was female. Two patients were brothers, the elder brother developed abnormal gait at 17.0 years; and the younger brother was then diagnosed at 15.0 years of age and treated at the asymptomatic stage, who was 18.0 years old with normal condition during this study. The main manifestations of the 8 symptomatic patients included progressive dyskinesia and spastic paralysis of the lower limbs, with or without intellectual decline, cognitive impairment and behavioral abnormalities. Totally, 15 variants of MTHFR gene were identified in the 9 patients, including 8 novel variants. Five patients had brain image abnormalities. Increased plasma total homocysteine level (65-221 μmol/L) was found in all patients, and decreased to 20-70 μmol/L after treatment with betaine and calcium folinate. Besides, the 8 symptomatic patients had their behavior and cognitive problems significantly improved, with a legacy of lower limb motor disorders. Conclusions: Late-onset MTHFR deficiency can occur in adolescence. The diagnosis is usually delayed because of non-specific clinical symptoms. The test of blood total homocysteine could be used as a selective screening test. Eight novel varients of MTHFR gene were identified. Timely treatment can improve clinical condition significantly, and pre-symptomatic treatment may prevent brain damage.

Abstract 2000: Role of O-GlcNAc conjugation and TS translational repression in 5-FU/LV resistance in CRC and CF10 response

Abstract Rationale: Fluoropyrimidine drugs (FPs; e.g., 5-FU) are widely used to treat colorectal cancer (CRC), the 3rd leading cause of cancer-related mortality. However, acquired resistance to 5-FU is a principal cause of treatment failure in the metastatic setting. Further, previous studies selected for cells resistant to single agent 5-FU however 5-FU is invariably used clinically in combination with the reduced folate leucovorin (LV). Our studies focus on resistance mechanisms upon treatment with 5-FU+LV in folate-restricted media and test if CF10, a novel polymeric FP being developed in our laboratory displays activity in CRC cells resistant to 5-FU+LV. Experimental: We selected CRC cells (HCT-116, LS174T, HCT15, MC38) resistant to 5-FU+LV by selecting for survival upon culture in folate-restricted media with stepwise increased concentrations of 5-FU+LV over 5 months. Since elevated thymidylate synthase (TS) levels were observed in multiple studies selecting CRC cells for 5-FU resistance, we performed Western blot on cell lysates from parental and (5-FU+LV)-R cells. Since Myc and p53 expression are regulated in part via translational inhibition by TS binding to these mRNAs we also compared Myc and p53 levels in parental and (5-FU+LV)-R cells by Western blot. Further, since TS, Myc, and p53 protein stability are all regulated in part via O-GlcNAc conjugation we tested if OGT levels were changed in (5-FU+LV)-R cells. Resistance factors for CF10 were also determined in (5-FU+LV)-R cells. Results: A surprising result was that basal TS levels were reduced in (5-FU+LV)-R cells relative to the parental cells. We also observed that TS levels were increased in both parental and (5-FU+LV)-R cells by 5-FU+LV treatment however the effect was much greater in (5-FU+LV)-R cells. We observed strongly decreased p53 and Myc levels in the (5-FU+LV)-R cells. CF10 treatment further decreased p53 and Myc levels in parental cells, but increased p53 and further decreased Myc in (5-FU+LV)-R cells. In contrast, 5-FU alone treatment in (5-FU+LV)-R cells increased Myc and decreased p53 levels and further decreased in combination with LV. CF10 retained strong activity in (5-FU+LV)-R cells. Conclusions: Overall, we have validated that cancer cells can be cultured under conditions with human-like folate levels and selected for resistance to 5-FU+LV under folate-restricted conditions. The causes of 5-FU+LV resistance are multi-factorial and include TS overexpression induced by 5-FU+LV treatment that may be mediated by O-GlcNAc conjugation. We demonstrated that CF10 is more potent than 5-FU, and importantly its potency advantage is enhanced in cells selected for 5-FU+LV resistance. Our studies support CF10 translation for treatment of metastatic CRC in patients with progressive disease following treatment with combination therapy that includes 5-FU+LV. Citation Format: Charles C. Okechukwu, Xue Ma, William H. Gmeiner. Role of O-GlcNAc conjugation and TS translational repression in 5-FU/LV resistance in CRC and CF10 response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2000.

Abstract 1189: Modulation of the MSS and MSI colorectal cancer immune microenvironment with FOLFOX and FOLFIRI -/+ anti-PD-1 immunotherapy

Abstract Metastatic colorectal cancer (mCRC) is a deadly disease with a five-year survival rate of 14%. Most patients receive 5-FU (F) and folinic acid (FOL) combined with oxaliplatin (OX), irinotecan (IRI), or both (OXIRI). Immune checkpoint inhibitors (ICIs) are effective against microsatellite instable (MSI) tumors, but 95% of mCRC tumors are microsatellite stable (MSS) and ICI-resistant. Though the primary targets of 5-FU, irinotecan, and oxaliplatin are well-understood, their effects on the tumor microenvironment (TME) remain incompletely characterized. Chemotherapy-mediated immune stimulation in MSS CRC has been observed in preclinical models and clinical trials. We hypothesize that combination chemotherapy treatment modulates the immune microenvironment of CRC -/+ ICI, with differences across subtype and treatment regimen. Chemotherapy-dependent changes in cancer cell gene expression, PD-L1 levels, cytokine secretion, and T cell activation were measured using in vitro models of MSS and MSI CRC. A preliminary evaluation of spleen and tumor T cells and dendritic cells (DCs) in murine models of MSS and MSI CRC after immunotherapy -/+ chemotherapy was conducted. CD8+ T cells, DCs, PD-L1, CD69, and GM-CSF expression were measured in clinical specimens of chemotherapy-treated MSS mCRC patients. In MSS CRC, FOX increased GM-CSF and activated CD8+ T cells in vitro and activated splenic CD8+ and CD4+ T cells in vivo. FOX and FIRI suppressed an anti-PD-1-mediated enhancement of type 1 cDCs in the tumor in vivo. FOLFOX was associated with increased tumor CD8+ T cells and decreased GM-CSF in MSS mCRC biopsies. FIRI treatment increased PD-L1 in vitro. FOLFIRI was not associated with increased tumor CD8+ T cells in MSS mCRC biopsies. In MSI CRC, FOX increased tumor CD8+ T cells in vivo. Here, we elucidate novel effects of clinically relevant chemotherapy combinations on the TME in MSS and MSI CRC. These data point to a FOX-specific mechanism by which CD8+ T cell infiltration into MSS mCRC tumors is enhanced, but their activation is halted potentially by GM-CSF suppression and/or type 1 cDC depletion in the TME. These findings contribute to our understanding of the mechanisms of chemotherapy-dependent immune modulation and bring the field closer to harnessing these effects for therapeutic gain. Citation Format: Lindsey Carlsen, Maximilian Pinho-Schwermann, Leiqing Zhang, Andrew Elliott, Kelsey E. Huntington, William J. MacDonald, Brooke Verschleiser, Laura Jinxuan Wu, Wafik S. El-Deiry. Modulation of the MSS and MSI colorectal cancer immune microenvironment with FOLFOX and FOLFIRI -/+ anti-PD-1 immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1189.

Abstract 2522: Predictive value of tumor mutational burden (TMB) in patients with metastatic microsatellite-stable (MSS) colorectal cancer (CRC) given first-line oxaliplatin-based chemotherapy and immune checkpoint blockade (ICB)

Abstract Purpose: Most CRC patients harbor primarily non-immunogenic MSS tumor. The randomized METIMMOX trial (NCT03388190) examined if patients with previously untreated, unresectable abdominal metastases from MSS-CRC may achieve therapeutic efficacy from short-course oxaliplatin-based chemotherapy (FLOX) and sequential ICB (nivolumab) repeatedly. Half of study patients assigned to this experimental treatment had significantly extended progression-free survival (PFS) while the other half had shortened PFS, compared to the control-group patients given standard FLOX chemotherapy with median PFS 9.3 months. Here we explored if somatic mutations (mut) unveiled by next-generation sequencing might provide insights into responsiveness to the METIMMOX regimen. Procedures: Patients had MSS-CRC with infradiaphragmatic metastases deemed unresectable and were eligible for first-line oxaliplatin-based therapy. They were randomly assigned to the control group of FLOX (oxaliplatin, 5-fluorouracil, folinic acid) Q2W or the experimental group of alternating 2 cycles each of FLOX Q2W and nivolumab Q2W, with prespecified break periods. Radiologic response assessment was done every 8 weeks with PFS as the primary endpoint. Diagnostic tumor biopsies and baseline metastasis biopsies were sequenced with the TruSight Oncology 500 assay. TMB was calculated using only coding, non-synonymous single-nucleotide variants and insertions/deletions with variant allele frequency ≥5% and sequencing depth ≥50 ×, applying the effective panel size as the megabases (Mb) denominator. Results: Sequencing data were acquired from 20 experimental-group patients with median PFS 7.9 months (minimum 2.0, maximum 41.6). Median TMB was 5.1 mut/Mb (minimum 0.8, maximum 11.8), concordant in patient-paired primary tumor and metastasis specimens. The TMB was associated with treatment outcome; the higher TMB, the better PFS with hazard ratio 0.83 (95% confidence interval (CI) 0.70-0.98; p = 0.026, Cox regression) for a PFS event with increasing TMB. TMB cut-off as low as 5.0 mut/Mb distinguished between patient groups (p = 0.007, log-rank test) with median PFS 34.9 months (95% CI 10.6-59.2; TMB&amp;gt;5, n = 10) and median PFS 4.6 months (95% CI 2.7-6.5; TMB≤5, n = 10). In the TMB&amp;gt;5 group, 8/10 were KRAS- or BRAF-mutant cases. In the TMB≤5 group, 9/10 metastasis specimens were RAS/BRAF-wildtype cases. Conclusions: TMB &amp;gt;5 mut/Mb was associated with tumor KRAS or BRAF driver mutation and remarkably long PFS in first-line treatment of patients with abdominal metastases from MSS-CRC with alternating short-course oxaliplatin-based chemotherapy and ICB. Citation Format: Anne Hansen Ree, Paula A. Bousquet, Hilde L. Nilsen, Torben Lüders, Shixiong Wang, Tina Visnovska, Diana L. Bordin, Eirik Høye, Hanne M. Hamre, Christian Kersten, Eva Hofsli, Marianne G. Guren, Halfdan Sorbye, Kjersti Flatmark, Sebastian Meltzer. Predictive value of tumor mutational burden (TMB) in patients with metastatic microsatellite-stable (MSS) colorectal cancer (CRC) given first-line oxaliplatin-based chemotherapy and immune checkpoint blockade (ICB) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2522.

Abstract 2545: KRAS co-mutational landscape dictates treatment outcomes in pancreatic cancer (PC) patients

Abstract Background: KRAS is commonly mutated in PC patients, with a prevalence of ~80%. A holistic understanding of the landscape of KRAS mutations, co-occurring mutations (co-mutations) in other driver genes, and their impact on patient outcomes treated with current standard-of-care (SoC) therapies may inform better treatment decisions. Methods: In this study, we use a real-world clinico-genomics PC database (ConcertAI’s Genome360TM PC dataset; N=1302) to:1.Examine the distribution of KRAS mutations.2.Identify the genes most frequently co-mutated with KRAS, both positively and negatively, based on their odds ratios.3.Evaluate patient outcomes (overall survival (OS) and progression-free survival (PFS)) for the two most common first-line SoC therapies (FOLFIRINOX: folinic acid, fluorouracil, irinotecan hydrochloride, oxaliplatin, and GEMPAC: gemcitabine, paclitaxel) stratified by the mutational status of KRAS and TP53. Results: The top 5 most observed KRAS mutations and the top 3 positively (OR&amp;gt;1) and negatively (OR&amp;lt;1) KRAS co-mutated genes are shown in Table 1. 80%, 71%, and 65% of PC patients had pathogenic mutations in KRAS, TP53, and both genes, respectively. In the entire cohort, FOLFIRINOX (n=327, OS/PFS=1.26/0.58 years) was more effective (p-value&amp;lt;0.05) than GEMPAC (n=305, OS/PFS=0.79/0.44 years). However, the underlying genetic makeup (mutational status of KRAS and TP53) significantly affects these outcomes (Table 1). The KRAS+/TP53− (pathogenic mutations in KRAS, but not in TP53) cohort has the worst prognosis, and the advantage of FOLFIRINOX as the treatment of choice is not observed in this cohort. Table 1. Evaluation of KRAS mutational landscape in PC and its impact on treatment outcomes. Top 5 KRAS mutations KRAS mutations G12D G12V G12R Q61H Q61R % PC patients tested positive 42.51 32.52 14.33 4.62 1.79 Top 3 positively and negatively co-mutated genes with KRAS KRAS co-mutated genes Positive association Negative association TP53 CDKN2A SMAD4 BRAF TERT PTEN Odds Ratio 8.56 6.93 6.66 0.05 0.27 0.32 Treatment outcomes for various genetic states of KRAS and TP53 Mutational status OS (years) PFS (years) FOLFIRINOX GEMPAC FOLFIRINOX GEMPAC KRAS+ TP53+ (850) 1.13 (196) 0.68 (188) 0.58 (196) 0.39 (188) KRAS− TP53− (155) 2.08 (40) 1.11 (27) 0.66 (40) 0.51 (27) KRAS+ TP53− (190) 0.77 (33) 0.93 (37) 0.51 (33) 0.33 (37) KRAS− TP53+ (81) 2.14 (15) 0.84 (24) 0.54 (15) 0.4 (24) Conclusions: This study provides insights into the genetic makeup of patients with KRAS mutations and how co-mutation with TP53 affects outcomes of SoC treatments. It highlights the need for comprehensive genetic profiling to make better therapy decisions. Citation Format: Pritha Ghosh, Rohini George, Neeraj Singh, Smita Agrawal. KRAS co-mutational landscape dictates treatment outcomes in pancreatic cancer (PC) patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2545.

Assessment of the safety and feasibility of neoadjuvant chemotherapy (2 cycles of FLOT chemotherapy + chemoradiotherapy) followed by surgery in the treatment of locally advanced gastric cancer

The aim of the study was to analyze of the safety and feasibility of neoadjuvant chemotherapy (2 cycles of FLOT chemotherapy + chemoradiotherapy) followed by surgery in the treatment of locally advanced gastric cancer. Material and Methods. The phase II clinical trial included 47 patients with histologically verified locally advanced gastric cancer (cT3/T4a-b: 26/21 and cN0/N1–3: 20/27) treated at the A.F. Tsyba MRRC from 2018 to 2021. Lesion location was: upper third in 19 patients (esophageal invasion in 9 patients), middle third in 13 patients, lower third in 14 patients, and entire stomach in 1 patient. The tumor grade was G3 in 23 patients, G2 in 13 patients and G1 in 7 patients. Signet ring cell carcinoma was revealed in 4 patients. The patients received 2 cycles of induction chemotherapy with FLOT regimen (85 mg/m2 oxaliplatin + 200 mg/m2 calcium folinate + 50 mg/m2 docetaxel on day 1 + 2600 mg/m2 fluorouracil as 24-hour infusion from day 1; every 2 weeks) followed by 3d-conformal external beam radiotherapy (46 Gy in daily fractions of 2 Gy) combined with chemotherapy with capecitabine and oxaliplatin). The patients then underwent follow-up examination to exclude disease progression and to plan surgery. Postoperative complications were analyzed using the Clavien-dindo classification. Results. Induction chemotherapy followed by concurrent chemoradiotherapy was well tolerated. Forty-five (95.7 %) patients underwent surgery, 97.7 % of them underwent radical surgery. Postoperative complications were observed in 11 (23.4 %) patients. Grade III and more severe complications were observed in 3 (6.4 %) patients. It should be noted that postoperative mortality rate was low, amounting to 2.2 % (1 patient). Conclusion. Induction FLOT polychemotherapy followed by concurrent chemoradiotherapy was shown to be safe, feasible, and tolerable. Moreover, this treatment regimen did not reduce the frequency of R0 surgeries and did not increase incidence and severity of postoperative complications.

Abstract 4730: Pharmacodynamic determinants of anti tumor activity of SHMT2 inhibitors

Abstract These studies investigate factors that impact the antitumor activity of SHMT2 targeting including drug accumulation, metabolism, expression of one carbon (C1) related enzymes, and tumor hypoxia. Mitochondrial C1 metabolism is upregulated in a variety of cancer types including leukemias and solid tumors. The mitochondrial C1 enzymes serine hydroxymethyltransferase (SHMT) 2 and 5,10-methylene tetrahydrofolate (THF) dehydrogenase 2 (MTHFD2) are among the top 5 overexpressed metabolic enzymes in tumors vs normal tissues in a wide range of tumor types. Thus, SHMT2 and MTHFD2 are potential therapeutic targets for cancer, sparing normal tissues that are less reliant on mitochondrial C1 metabolism. SHMT2 metabolizes serine and THF to produce glycine and 5,10-methylene THF; MTHFD2 metabolizes 5,10-methylene THF to NADH and 10-formyl THF which is converted to formate by MTHFD1L. Thus, mitochondrial C1 metabolism is the principal source of C1 units for cellular biosynthesis and provides the majority of glycine for protein, purine and glutathione synthesis. We previously discovered novel 5-substituted pyrrolo[3,2-d]pyrimidine antifolate compounds which exhibit primary inhibition at mitochondrial SHMT2 along with cytosolic SHMT1 and de novo purine biosynthesis at glycinamide ribonucleotide and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferases. The lead compound AGF347 is taken up into cells by the facilitative transporters reduced folate carrier (SLC19A1) and the proton coupled folate transporter (SLC46A1) and like folate cofactors is metabolized to polyglutamate (PG) forms by folylpolyglutamate synthase (FPGS) which promotes retention in cytosolic and mitochondrial compartments and target enzyme engagement. We investigated the impact of folate transporter expression on metabolism to PGs for SHMT2 targeted antifolates. Folate transporter expression inversely correlated with anti-tumor efficacy of SHMT2 targeted antifolates and overexpression of FPGS resulted in dramatically increased sensitivities. However, for pyrazolopyran inhibitors of SHMT2 (e.g. SHIN1) which enter cells by diffusion and are not metabolized to polyglutamates, increased transporter and FPGS levels dramatically decreased anti-tumor activity. As SHMT2 is a target of HIF1α, we studied the impact of multitargeting SHMT2 and purine biosynthesis by pyrrolo[3,2-d]pyrimidine antifolates under hypoxic conditions. Our results establish that multi-targeting SHMT2 and purine biosynthesis preserves anti-tumor activity under hypoxia. Collectively, our results demonstrate that SHMT2 targeted pyrrolo[3,2-d]pyrimidine antifolates exhibit increased antitumor efficacies in cells expressing low folate transporter expression and is further enhanced by increased FPGS activity. Additionally, we find multitargeting of SHMT2 and de novo purine biosynthesis manifests as antitumor activity even under hypoxic conditions. Citation Format: Mathew Joseph Schneider, Carrie O'Connor, Xun Bao, Md. Junayed Nayeen, Zhanjun Hou, Jing Li, Aleem Gangjee, Larry Matherly. Pharmacodynamic determinants of anti tumor activity of SHMT2 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4730.

Long-Term Low-Dose Pyrimethamine Use for the Prevention of Ocular Toxoplasmosis Recurrences: A Cohort Study

ABSTRACT Purpose To describe the effect of long-term, low-dose pyrimethamine for the prevention of ocular toxoplasmosis (OT) recurrences. Methods Sixty-three consecutive patients with inactive ocular toxoplasmosis and positive toxoplasma IgG serology were included. Pyrimethamine (25 mg) + folinic acid (15 mg) were administered every other day (three times weekly) for 12 months. Eighteen patients received the treatment for an additional six months as part of an extension study. Results Thirty-eight patients (60.3%, n = 63) were female; 38 (60.3%) had a previous history of recurrence and 37 (58.7%) had active OT within the preceding 12 months. Three (4.8%) patients had unilateral recurrences at 8, 12 and 18 months after starting intermittent pyrimethamine treatment. Five patients (7.9%) were discontinued due to hematological, renal and hepatic changes. Treatment was considered successful in 42 patients (84%). Conclusion Long-term, low-dose pyrimethamine can be considered as a treatment option for the prevention of ocular toxoplasmosis recurrence in selected patients, with only a few, mild and reversible systemic adverse events.

Simultaneous Determination of One-Carbon Folate Metabolites and One-Carbon-Related Amino Acids in Biological Samples Using a UHPLC-MS/MS Method.

One-carbon folate metabolites and one-carbon-related amino acids play an important role in human physiology, and their detection in biological samples is essential. However, poor stability as well as low concentrations and occurrence in different species in various biological samples make their quantification very challenging. The aim of this study was to develop a simple, fast, and sensitive ultra-high-performance liquid chromatography MS/MS (UHPLC-MS/MS) method for the simultaneous quantification of various one-carbon folate metabolites (folic acid (FA), tetrahydrofolic acid (THF), p-aminobenzoyl-L-glutamic acid (pABG), 5-formyltetrahydrofolic acid (5-CHOTHF), 5-methyltetrahydrofolic acid (5-CH3THF), 10-formylfolic acid (10-CHOFA), 5,10-methenyl-5,6,7,8-tetrahydrofolic acid (5,10-CH+-THF), and 4-α-hydroxy-5-methyltetrahydrofolate (hmTHF)) and one-carbon-related amino acids (homocysteine (Hcy), methionine (Met), S-ade-L-homocysteine (SAH), and S-ade-L-methionine (SAM)). The method was standardized and validated by determining the selectivity, carryover, limits of detection, limits of quantitation, linearity, precision, accuracy, recovery, and matrix effects. The extraction methods were optimized with respect to several factors: protease-amylase treatment on embryos, deconjugation time, methanol precipitation, and proteins' isoelectric point precipitation on the folate recovery. Ten one-carbon folate metabolites and four one-carbon-related amino acids were detected using the UHPLC-MS/MS technique in various biological samples. The measured values of folate in human plasma, serum, and whole blood (WB) lay within the concentration range for normal donors. The contents of each analyte in mouse plasma were as follows: pABG (864.0 nmol/L), 5-CH3THF (202.2 nmol/L), hmTHF (122.2 nmol/L), Met (8.63 μmol/L), and SAH (0.06 μmol/L). The concentration of each analyte in mouse embryos were as follows: SAM (1.09 μg/g), SAH (0.13 μg/g), Met (16.5 μg/g), 5,10-CH+THF (74.3 ng/g), pABG (20.6 ng/g), and 5-CH3THF (185.4 ng/g). A simple and rapid sample preparation and UHPLC-MS/MS method was developed and validated for the simultaneous determination of the one-carbon-related folate metabolites and one-carbon-related amino acids in different biological samples.