Doxorubicin, doxorubicinol, cardiotoxicity, breast cancer, volumetric absorptive microsampling, LC-MS/MS.

<b>Background and Objective:</b> Doxorubicin is an anticancer therapy belonging to the anthracycline class, which has clinical activity in breast cancer. Doxorubicin can cause cardiotoxic effects due to the formation of doxorubicinol as its main metabolite. The purpose of this study was to obtain the optimum sample preparation conditions for the analysis of doxorubicin in VAMS and as a form of therapeutic drug monitoring (TDM) in patients with cancer breasts. <b>Materials and Methods:</b> Analyze doxorubicin and doxorubicinol levels with Volumetric Absorptive Microsampling (VAMS) in patients' cancer breasts receiving doxorubicin in their therapeutic regimen. The sample was analyzed using Ultra Performance Liquid Chromatography tandem Mass Spectrometry (LC-MS/MS). The method uses deep linear range concentrations of 8-200 ng/mL for doxorubicin and 3-100 ng/mL for doxorubicinol. <b>Results:</b> Multiple reaction monitoring (MRM) value set at m/z 544.22>396.9 for doxorubicin; m/z 546.22>398.9 for doxorubicinol and m/z 528.5>362.95 for daunorubicin. The LLOQ value obtained was 8 ng/mL for doxorubicin and 3 ng/mL for doxorubicinol with linearity of 0.9904 for doxorubicin and 0.9902 for doxorubicinol. Analysis results show doxorubicin levels were in the range of 9.47 ng/mL to 87.84 ng/mL and doxorubicinol range between 4.24 and 54.02 ng/mL. <b>Conclusion:</b> Dosage cumulative doxorubicin ranges between 47.93 and 346.09 mg/m²; with this, the risk of cardiomyopathy in the patients surveyed is under 4%, according to the literature.

Matrix Metalloproteinase-9 as Prognostic Factor for the Treatment of HER-2 Enriched Breast Cancer.

BACKGROUND: MMP-9 plays a significant role in invasion and migration of tumor cells and metastasis. A combination MMP-9 biomarker with HER2 and Ki-67 is expected to provide more specificity in prognosis for better breast cancer (BC) treatment options. METHODS: A retrospective cohort of 34 patients with HER2 enriched breast cancer were studied from January 2016 until December 2020. Assessment MMP-9 by IHC using Monoclonal Mouse Anti-Human MMP-9 Antigen with semiquantitative immunoreactive scores methods was done. RESULTS: The samples included patients aged 29 to 66 years. Patients' educational level was mostly high school graduation (n=17; 50%). Distant metastases occurred in 10 (29.4%) patients,  histopathological Grade II was found in 29 (85.7%) patients, positive LVI was found in 18 (52.9%) patients, and high proliferation rate (Ki67 > 20%) was found in 32 (94.1%) patients. All the patients underwent MRM with a history of chemotherapy in 29 (85.3%) patients, radiotherapy in 14 (41.2%) patients, and targeted therapy in only seven (20.6%) patients. Most of the patients had locally advanced stage III (n=21; 61.8%). The MMP-9 High expression was found in 20 (58.8%) patients and 14 (41.2%) patients had a low expression. A significant relationship was found between MMP-9 expression and DFS (p=0.023); while a significant relationship was found with OS (p=0.093). The mean DFS for High expression MMP-9 was 37.3 months and 45.3 months for low expression. The mean OS was 37.6 months for High-intensity MMP-9 and 42.7 months for Low-intensity. CONCLUSIONS: The MMP-9 expression (p=0.037), age group<40 years old (p=0.024), and the history of radiotherapy (p=0.035) were significantly related to he occurrence of distant metastases. The MMP-9 High expression had a 7.5 times greater risk of distant metastases. IMPACTS: The MMP-9 can be used as a prognostic factor for distant metastasis in HER2 Enriched BC.

The correlation between the level of 3-hydroxypropyl mercapturic acid, CYP2B6 polymorphisms, and hematuria occurrences after cyclophosphamide administration and its bioanalytical methods: A systematic review.

BACKGROUND: Cyclophosphamide (CPA) is a cytotoxic prodrug that needs to be metabolized by cytochrome P450 enzymes, like CYP2B6. Unfortunately, CYP2B6 is a very polymorphic enzyme and can cause a change in 3-hydroxypropyl mercapturic acid (3-HPMA), the most found CYP metabolite in urine levels. Change in 3-HPMA levels can also indicate the level change in its precursor, acrolein, which is responsible for the hematuria incidence after CPA administration.This review's purpose is to obtain a conclusion about the optimal 3-HPMA analysis method in urine after the administration of cyclophosphamide using liquid chromatography-tandem mass spectrometry (LC-MS/MS) through literature review from previous studies. Also, this review was written to examine the relationship between levels of 3-HPMA in urine, polymorphisms of CYP2B6 enzymes, and the incidence of hematuria after cyclophosphamide administration in cancer patients. METHODS: Major databases, such as Universitas Indonesia's library database ScienceDirect, PubMed/Medline, Frontiers Media, and Google Scholar database, were used to find both published and unpublished studies without a time limit until 2020. Studies on pharmacokinetics, pharmacodynamics, drug therapy monitoring of cyclophosphamide, bioanalysis, and polymerase chain reaction (PCR) published in Indonesian and English were included. Meanwhile, non-related studies or studies written in other languages besides Indonesian and English were excluded. Two independent reviewers screened the titles, abstracts, and full-text manuscripts. Data obtained from eligible sources were used to answer the purpose of this review in a narrative form. RESULTS: The authors found 436 related studies from various databases and websites. Then, the authors narrowed it down into 62 pieces of literature by removing the duplicates and reviewing the abstracts and full-text manuscripts. Out of 62 sources, the authors found 30 studies that explained 3-HPMA analysis using LC/MS-MS, CYP2B6 polymorphisms, and hematuria occurrences. The authors used those 30 studies to build a conclusion regarding the purpose of this study. We strengthened the results with some additional information from the other 32 eligible sources. CONCLUSIONS: The authors conclude that according to literature searches from previous studies, the optimal 3-HPMA analysis method in urine after cyclophosphamide administration using LC-MS/MS is using triple quadrupole LC-MS/MS; source of positive ion electrospray ionization (ESI); mobile phase combination of 0.1% formic acid in water (A) - 0.1% formic acid in acetonitrile (90:10 v/v) (B); the Acquity® BEH C18 column (2.1 × 100 mm; 1.7 µm); injection volume of 10 µl; flow rate of 0.2 ml/minute; gradient elution method. Detection was carried out using mass spectrometry with m/z ratio of 222.10 > 90 for 3-HPMA and m/z 164.10 > 122 for n-acetylcysteine (NAC). The optimum sample preparation method is acidification and dilution ratio of 1:5 v/v. Also, there is a relationship between 3-HPMA levels, CYP2B6 polymorphisms, and the occurrences of hematuria after the administration of cyclophosphamide, which is a type of CYP2B6 polymorph, namely CYP2B6∗6, can increase cyclophosphamide hydroxylation so that it can increase the levels of acrolein and 3-HPMA, as its metabolites, and risk of hematuria. ETHICS AND DISSEMINATION: This research does not use human participants, human data, or human tissue for being directly studied for the review. Therefore, ethics approval and consent to participate are not applicable. REGISTRATION: This research has not been registered yet.

Determination of O6-Methylguanine in dried blood spot of breast cancer patients after cyclophosphamide administration.

Cyclophosphamide is a nitrogen mustard class of drugs that are often used in cancer chemotherapy. However, the use of Cyclophosphamide in high doses over a long period has been shown to increase the risk of developing secondary cancer. This can be indicated by the formation of mutagenic DNA adducts, such as O6-Methylguanine. Therefore, this adduct can be used as a biomarker for secondary cancer in patients receiving Cyclophosphamide. Bio sampling was carried out by using the Dried Blood Spot (DBS) method, followed by DNA extraction by using QIAamp DNA mini kit, and acid hydrolysis to obtain O6-Methylguanine. Analysis of O6-Methylguanine was performed by using the UPLC-MS/MS instrument with the conditions developed by Vianney, Harahap, & Suryadi (2021). Partial validation was carried out before the analysis. The results obtained from the calibration curve, accuracy, and precision validation test met the FDA requirements. The analysis method was then implemented in 16 breast cancer patients who received the Cyclophosphamide regimen. The O6-Methylguanine was successfully detected and quantified in all of the samples in the range of 0.55-6.66 ng/mL. It shows that the O6-Methylguanine accumulation in cancer patients receiving Cyclophosphamide is very likely to occur and the analysis method proposed by Vianney, Harahap, & Suryadi (2021) is potential to be used for Therapeutic Drug Monitoring in this group of patients.

Phenotyping Study of Cyclophosphamide 4-Hydroxylation in Malay Cancer Patients.

BACKGROUND: Cyclophosphamide (CP) is an anticancer alkylating group (nitrogen mustard) and a prodrug that will be metabolized to form its active metabolite, 4-hydroxycyclophosphamide (4-OHCP). The various enzymes involved in its bioactivation can cause a wide range of CP expression and activity among patients and ultimately affect the metabolism, efficacy and toxicity of this drug. The effectiveness of CP therapy can be determined by 4-OHCP level in dried blood spot (DBS). AIM: The purpose of this study was to conduct the phenotyping of CP 4-hydroxylation rate in Malay cancer patients. METHODOLOGY: Phenotyping study of CP 4-hydroxylation rate to 40 subjects of Malay cancer patients was done based on the value of its bioactivity ratio (4-OHCP to CP levels). RESULTS: The result shown the cyclophosphamide 4-hydroxylation rate of 80% (n=32) subjects as ultrarapid metabolizer (UM) and 20% (n=8) as poor metabolizer (PM). CONCLUSION: Phenotyping study of CP 4-hydroxylation in Malay cancer patients can be conducted by quantifying CP bioactivity ratio (4-OHCP to CP level) in dried blood spot. In majority of Malay cancer patients, cyclophosphamide would be bioactivated through 4-hydroxylation in hepar rapidly as indicated by the high value of the bioactivity ratio or the increased CP clearance and 4-OHCP level.

Analysis of the Doxorubicin and Doxorubicinol in the Plasma of Breast Cancer Patients for Monitoring the Toxicity of Doxorubicin.

INTRODUCTION: Doxorubicin is an anthracycline antibiotic used as an anticancer agent. Long-term use of this anticancer agent could accumulate its metabolite, doxorubicinol, and cause cardiomyopathy, due to its cardiotoxicity. This cardiotoxic effect depends on the amount of doxorubicin and doxorubicinol accumulated in the body. This study aimed to analyze doxorubicin and doxorubicinol levels in the blood plasma of breast cancer patients. METHODS: Participants of this study were 30 breast cancer patients who had received doxorubicin in their therapy regimen. The samples were analyzed using ultra-high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS), with the Acquity UPLC BEH C18 Waters chromatography column (2.1 x 100 mm : 1.7 µm). Plasma (250 µL) samples were prepared by protein precipitation, using methanol. The mobile phase consisted of 0.1% acetic acid (eluent A) and acetonitrile (eluent B), with gradient elution; the flow rate was 0.15 mL/min and runtime, 7 min. RESULTS AND DISCUSSION: This method was linear in the range of 1-1000 ng/mL for doxorubicin and 0.5-500 ng/mL for doxorubicinol. This method was successfully used to analyze doxorubicin and doxorubicinol, simultaneously, using hexamethylphosphoramide as the internal standard, in the plasma of breast cancer patients. Results showed that the measured concentrations of doxorubicin and doxorubicinol ranged between 12.54-620.01 ng/mL and 1.10-27.00 ng/mL, respectively. The measured cumulative doses of doxorubicin ranged between 48.76 and 319.01 mg/m2; thus, the risk of cardiomyopathy in the surveyed patients was under 4%, according to literature.

Effect of melatonin supplementation in combination with neoadjuvant chemotherapy to miR-210 and CD44 expression and clinical response improvement in locally advanced oral squamous cell carcinoma: a randomized controlled trial.

BACKGROUND: Squamous cell carcinoma of the oral cavity (OSCC) is the sixth most common malignancy. Surgery is mainstay treatment for oral cancers. Surgery in locally advanced OSCC presents many challenges primarily because the head and neck have critical structures that can be damaged by tumor or treatment. It is thought that neoadjuvant chemotherapy (NC) in locally advanced OSCC is able to shrink tumor size. Chemoresistancy is a problem due to hypoxic microenvironment characterized by increased expression of HIF-1α. It is also regulated by miR-210 as well as increased expression of CD44 and CD133. Melatonin has a powerful antioxidant and oncostatic effects that are expected to improve tumor hypoxia and clinical response. Fifty patients with OSCC were included and randomized. miR-210 and CD44 expression were measured before and after intervention using qRT-PCR absolute quantification, and clinical response was evaluated according to RECIST 1.1 criteria. This study aims to determine the effect of melatonin in improving the clinical response of patients with locally advanced oral squamous cell carcinoma (OSCC) after neoadjuvant chemotherapy to miR-210 and CD44 expression. RESULTS: Melatonin administration reduced miR-210 levels but not significant (p = 0.767). CD44 expression also decreased in the melatonin group compared with placebo yet was not significant (p = 0.103). There was a decrease in the expression of miR-210 and CD44 followed by a decrease in the percentage of residual tumor but not significant (p = 0.114). CONCLUSION: In OSCC, the addition of 20-mg melatonin to neoadjuvant chemotherapy (NC) reduced the expression of miR-210 and CD44 and decreased the percentage of tumor residue; however, no statistically significant result was observed. TRIAL REGISTRATION: This study is registered to ClinicalTrials.gov under trial registration number: NCT04137627 with date of registration on October 22, 2019-retrospectively registered, accessible from: https://clinicaltrials.gov/ct2/show/NCT04137627.

Quantification of 3-Hydroxypropyl Mercapturic Acid in the Urine of Patients with Breast Cancer to Monitor Cyclophosphamide Toxicity.

BACKGROUND: The alkylating agent cyclophosphamide is used in chemotherapy regimens for various type of cancer. However, cyclophosphamide may lead to toxic side effects on the bladder, namely hemorrhagic cystitis, which can cause hematuria, and, potentially, bladder cancer. These effects are caused by acrolein, a byproduct of cyclophosphamide metabolism. In this study, a method to quantify 3-hydroxypropyl mercapturic acid (3-HPMA) in urine was developed. 3-HPMA is a stable metabolite of acrolein that serves as biomarker of acrolein. METHODS: Urine samples were collected 4 hours after cyclophosphamide administration and analyzed to determine the risk of hematuria. 3-HPMA was analyzed by reverse-phase LC-MS/MS using a triple quadrupole electrospray ionization mass spectrometer in the positive-ion mode. The mobile phase was a 90:10 (vol/vol) mixture of 0.1% formic acid in water and 0.1% formic acid in acetonitrile. Multiple reaction monitoring mode was used, with m/z 222.10 → 90.97 for 3-HPMA and 164.10 → 122.02 for the internal standard N-acetyl cysteine (NAC). Samples were prepared by acidification and dilution. RESULTS: The analytical method produced a linear response within the concentration range of 40-10,000 ng/mL. The method was validated in accordance with 2018 FDA guidelines and applied to quantify 3-HPMA in the urine of 40 patients with breast cancer. The measured concentrations ranged from 820.3 to 5596.1 ng/mg creatinine. Seven patients identified with hematuria had low 3-HPMA concentrations of 4445.824 ± 411.17 ng/mg creatinine, and 33 patients without hematuria had low 3-HPMA concentrations of 2419.4 ± 1171.8 ng/mg creatinine. CONCLUSIONS: The method was applicable for the quantification of 3-HPMA in human urine. Large variations in 3-HPMA concentrations were found in 40 patients with breast cancer treated with cyclophosphamide, with a significant difference (P < 0.05) observed between patients with hematuria and those without hematuria.

Volumetric Absorptive Microsampling as a Sampling Alternative in Clinical Trials and Therapeutic Drug Monitoring During the COVID-19 Pandemic: A Review.

An infectious disease, COVID-19, caused by a new type of coronavirus, has been discovered recently. This disease can cause respiratory distress, fever, and fatigue. It still has no drug and vaccine for treatment and prevention. Therefore, WHO recommends that people should stay at home to reduce disease transmission. Due to the quarantine, FDA stated that this could hamper drug development clinical trial protocols. Hence, an alternative sampling method that can be applied at home is needed. Currently, volumetric absorptive microsampling (VAMS) has become attention in its use in clinical and bioanalytical fields. This paper discusses the advantages and challenges that might be found in the use of VAMS as an alternative sampling tool in clinical trials and therapeutic drug monitoring (TDM) during the COVID-19 pandemic. VAMS allows easy sampling, can be done at home, storage and delivery at room temperature, and the volume taken is small and minimally invasive. VAMS is also able to absorb a fixed volume that can increase the accuracy and precision of analytical methods, and reduce the hematocrit effects (HCT). The use of VAMS is expected to be implemented immediately in clinical trials and TDM during this pandemic considering the benefits it has.

Dietary patterns and risk of oral cancer: a factor analysis study of a population in Jakarta, Indonesia.

A matched case-control, hospital-based study of oral cancer was conducted in Jakarta population. The sample included 81 cases and 162 controls. The purpose of this study was to determine the association between dietary pattern and oral cancer in a Jakarta population using factor analysis. Dietary data were collected using food frequency questionnaire and factor analysis was performed on 15 food groups resulting in four principle factors/components being retained. The first factor "preferred" was characterized by fast food, fermented food, canned food, snacks high in fat and sugar, cooked and raw vegetables, and seafood. The second factor labeled "combination" was loaded by the intake of dairy product, red meat, white meat and fruits. The third factor labeled "chemical related was loaded by processed food and monosodium glutamate and the fourth principle component consisted of drinks and grain was labeled as "traditional". The conditional logistic regression was done using STATA 8 to obtain the odds ratio (OR) of highest tertile of each component retained from factor analysis and the ORs were then adjusted with risk habits. The consumption the highest tertile of the "preferred" pattern increased the risk of oral cancer by two-times compared to the lowest tertile of consumption [adjusted odds ratio (aOR)=2.17; 95% confidence interval (CI)=1.05-4.50]. The chemical related" pattern showed higher risk of about threefold (aOR=2.56; 95% CI=1.18-5.54), while the "traditional" pattern showed an increased of risk by twofold (aOR=2.04; 95% CI=1.01-4.41). In contrast, the "combination" pattern displayed protective effects in relation to oral cancer (aOR=0.50; 95% CI=0.24-1.00). This finding suggests that factor analysis may be useful to determine the diet pattern of a big set of food type and establish the correlation with oral cancer.