PURPOSE:High-dose methotrexate (HD-MTX) is the backbone of curative therapy for CNS lymphoma. Because of toxicity, MTX is administered in the inpatient setting along with hyperhydration and monitoring until MTX clearance is documented (3-5 days). Frequent hospitalizations result in patient time away from work, home, and exposure to potential iatrogenic/nosocomial complications. Here, we aim to demonstrate feasibility of HD-MTX administration in the outpatient setting with low-dose glucarpidase facilitating clearance. METHODS:This is a prospective nonrandomized study of outpatient HD-MTX followed by glucarpidase 2000u (ClinicalTrials.gov identifier: NCT03684980). Eligible patients had CNS lymphoma, creatinine <1.3 mg/dL, and previously tolerated HD-MTX. Patients were enrolled between May 2020 December 2021 for one HD-MTX treatment. Patients could re-enroll for subsequent doses of HD-MTX as eligibility and slots permitted. MTX 3.5 g/m2 was administered once over 2 hours, preceded by standard hydration and followed by an additional 2 hours of dextrose 5% in water with NaHCO3 75 mEq at 150 cc/h. Glucarpidase 2000u was administered once in the clinic 24 hours later. The primary end point was MTX level 48 hours after HD-MTX. RESULTS:Twenty doses of outpatient HD-MTX with glucarpidase were administered to seven patients. After 20 of 20 (100%) treatments, serum MTX levels were reduced to <100 nmol/L. Treatments were well-tolerated, and no admissions were required. One patient received additional outpatient hydration for elevated creatinine. Development of antiglucarpidase antibody was rare and did not affect treatment. CONCLUSION:Outpatient HD-MTX with glucarpidase is safe and well-tolerated and has the potential to alter standard treatment for CNS lymphoma.

Treosulfan is a structural analog of the alkylating agent busulfan which has been shown in clinical trials to exhibit comparable myeloablative activity while causing fewer serious side effects. Treosulfan is currently being considered for FDA approval in combination with fludarabine, one of the most commonly used myeloablative agents, as a conditioning regimen prior to hematopoietic stem cell transplantation (HSCT). Because plasma concentrations of both treosulfan and fludarabine exhibit significant interindividual variability, therapeutic drug monitoring (TDM) is indicated to ensure dosages are administered that maximize efficacy while minimizing toxicity. In this chapter, we describe a rapid, accurate assay to detect treosulfan and fludarabine simultaneously in human plasma using turbulent flow liquid chromatography coupled to electrospray ionization tandem mass spectrometry (TFLC-ESI-MS/MS). Treosulfan and fludarabine are extracted from only 100 μL of acidified plasma via protein precipitation with methanol containing isotope-labeled internal standards. The extract is injected into the TFLC-ESI-MS/MS system, and the analytes are quantified using multiple reaction monitoring and a six-point calibration curve.

Melphalan and busulfan are DNA-alkylating agents that are often used concurrently in hematopoietic stem cell transplant (HCT) conditioning regimens. Studies have demonstrated that this combination of alkylating agents is very effective and well-tolerated prior to HCT. This combination is widely used for acute leukemia, advanced lymphoid malignancies, and multiple myeloma. Our goal was to develop an assay for the rapid measurement of both compounds simultaneously in the hopes that rapidly measuring their concentrations could possibly shorten the length of hospital stay. It would also simplify specimen handling in the clinic and the laboratory, reduce the amount of blood drawn, and allow for rapid reporting of the drug levels, thereby facilitating rapid dose adjustments.This chapter describes a validated method that measures both compounds simultaneously. Melphalan and busulfan were extracted from plasma with methanol containing deuterated internal standards. Turbulent flow chromatography coupled with reversed-phase HPLC was used for separation, while the mass spectrometer was set in the positive ion mode. This method has proven accurate and rapid and allowed for timely dose adjustments. The assay was linear over the clinically relevant ranges; the analytical measurement range for busulfan and melphalan was 10-5000 ng/mL and 10-15,000 ng/mL, respectively. Specimens containing elevated drug levels were diluted yielding a final clinically reportable range of 10-25,000 ng/mL for busulfan and 10-75,0000 ng/mL for melphalan. This method is very suitable for simultaneous measurements of these drugs and is currently being used to support pharmacokinetic studies.

N,N',N''-Triethylenethiophosphoramide (thioTEPA) is a polyfunctional, organophosphorus alkylating agent that has been a primary treatment of multiple solid malignancies for many years and more recently as part of conditioning regimens prior to hematopoietic stem cell transplantation for a variety of hematologic malignancies. In vivo, thioTEPA is quickly metabolized to N,N',N″-triethylenephosphoramide (TEPA). ThioTEPA and TEPA have similar alkylating activity and both exhibit outstanding central nervous system penetration. Therefore, it is possible and desirable to monitor both compounds in plasma and cerebrospinal fluid (CSF).This chapter describes a method to measure both compounds simultaneously. ThioTEPA and TEPA are extracted with solvent from plasma and CSF by the addition of deuterated internal standards prepared in methanol. Chromatographic separation is attained using a C18 column and mass spectrometry which is performed in the positive ion mode. Herein, we describe a fast, accurate, and sensitive assay to quantify both compounds in plasma and CSF by turbulent flow LC-MS/MS which allows for fast and accurate therapeutic drug monitoring and timely dose modifications.

Allogeneic hematopoietic cell transplantation (allo-HCT) is a potentially curative therapeutic treatment for many patients with high-risk hematologic malignancies and bone marrow failure syndromes. While allo-HCT can be highly effective, it is met with significant bone marrow conditioning regimen-related toxicities and complications such as infections related to poor immune reconstitution. This chapter describes the measurement of clofarabine and fludarabine concentrations to support clinical trials whose goal is to determine the optimal therapeutic ranges in order to maximize effectiveness while minimizing variability and regimen-related adverse events and toxicities. Moreover, the same holds true for patients receiving fludarabine as part of their lymphodepleting chemotherapy for chimeric antigen receptor T-cells (CAR T-cells). It is believed that one of the causes of variable outcomes after CAR T-cell therapy is lymphodepletion due to the variable fludarabine concentrations.This chapter describes a HPLC-MS/MS method to measure both compounds simultaneously. Clofarabine and fludarabine are extracted with solvent from plasma by the addition of deuterated internal standards prepared in methanol. Chromatographic separation is attained using a reversed-phase column followed by mass spectrometry which is performed in the positive ion mode. Herein, the described method to quantify both compounds in plasma is fast, accurate, and sensitive and allows for rapid drug concentration monitoring and timely dose adjustments.

Allogeneic hematopoietic cell transplantation (allo-HCT) is a potentially curative therapeutic treatment for patients with high-risk hematologic malignancies and bone marrow failure syndromes. While allo-HCT can be highly effective, it is met with significant regimen-related toxicities and complications such as graft-versus-host disease (GVHD), poor immune reconstitution, and infections. Prednisone is the preferred treatment for patients with both acute and chronic GVHD. While effective, high-dose prednisone can cause many complications, including weight gain, skin fragility, muscle weakness, bone demineralization, hyperglycemia, insomnia, and psychosis. Optimizing prednisone (and prednisolone) dosing by measuring their concentrations and calculating their pharmacokinetic parameters will allow for personalized treatments for patients, producing more effective and safer treatments for GVHD. This chapter describes a method to measure both compounds simultaneously. Prednisone and prednisolone are extracted from serum by the addition of methanol containing deuterated internal standards. Chromatographic separation is achieved using a reversed-phase HPLC column followed by tandem mass spectrometry performed in the positive ion mode. This assay is fast, accurate, sensitive and allows for rapid drug measurements and timely dose modifications.

BackgroundDysgeusia is a common but understudied complication in patients undergoing autologous hematopoietic cell transplantation (auto‐HCT). We assessed the feasibility of using chemical gustometry (CG) to measure dysgeusia and explored its associations with symptom burden, nutrition, chemotherapy pharmacokinetics (PK), and the oral microbiome.MethodsWe conducted a single‐center, prospective feasibility study (NCT03276481) of patients with multiple myeloma undergoing auto‐HCT. CG was performed longitudinally testing five flavors (sweet, sour, salty, bitter, umami) to calculate a total taste score (maximum score, 30). We measured caloric intake and patient‐reported symptoms, assessing their correlation with oral microbiota composition and salivary and blood melphalan PK exposure.ResultsAmong all 45 patients, 39 (87%) completed at least four (>60%) and 22 (49%) completed all six CG assessments. Median total CG scores remained stable over time but were lowest at day +7 (27, range 24–30) with recovery by day +100. Symptom burden was highest by day +10 (area under the curve, 2.9; range, 1.0–4.6) corresponding with the lowest median overall caloric intake (1624 kcal; range, 1345–2267). Higher serum/salivary melphalan levels correlated with higher patient‐reported dysgeusia and lower caloric intake. Oral microbiota α‐diversity was stable early and increased slightly by day +100.ConclusionsAssessment of dysgeusia by CG is feasible after auto‐HCT. Most dysgeusia, symptom burden, and lowest caloric intake occurred during the blood count nadir. Higher melphalan concentrations correlated with more dysgeusia and poorer caloric intake. Future studies will aim to modulate melphalan exposure by PK‐targeted dosing and characterize patient taste preferences to personalize diets for improved nutritional intake.Lay summary Taste changes after cancer treatments are very common. We used chemical gustometry (taste testing) to study taste changes and to better understand why patients with multiple myeloma experience this symptom after autologous hematopoietic cell transplantation. We found that taste testing was feasible, taste changes peaked when blood counts were lowest, and most patients recovered their taste by 100 days after transplantation. Taste changes correlated with lower food intake and with higher levels of chemotherapy in the body. Future work will focus on using personalized chemotherapy doses to reduce taste changes and to match patients' individual taste preferences with their diets.

Abstract Background High-dose methotrexate (HD-MTX) has broad use in the treatment of central nervous system (CNS) malignancies but confers significant toxicity without inpatient hydration and monitoring. Glucarpidase is a bacterial recombinant enzyme dosed at 50 units (u)/kg, resulting in rapid systemic MTX clearance. The aim of this study was to demonstrate feasibility of low-dose glucarpidase to facilitate MTX clearance in patients with CNS lymphoma (CNSL). Methods Eight CNSL patients received HD-MTX 3 or 6 g/m2 and glucarpidase 2000 or 1000u 24 h later. Treatments repeated every 2 weeks up to 8 cycles. Results Fifty-five treatments were administered. Glucarpidase 2000u yielded > 95% reduction in plasma MTX within 15 min following 33/34 doses (97.1%) and glucarpidase 1000u yielded > 95% reduction following 15/20 doses (75%). Anti-glucarpidase antibodies developed in 4 patients and were associated with MTX rebound. In CSF, glucarpidase was not detected and MTX levels remained cytotoxic after 1 (3299.5 nmol/L, n = 8) and 6 h (1254.7 nmol/L, n = 7). Treatment was safe and well-tolerated. Radiographic responses in 6 of 8 patients (75%) were as expected following MTX-based therapy. Conclusions This study demonstrates feasibility of planned-use low-dose glucarpidase for MTX clearance and supports the hypothesis that glucarpidase does not impact MTX efficacy in the CNS. Clinical trial registration NCT03684980 (Registration date 26/09/2018).

Adult and pediatric endocrinology and oncology often requires measuring serum estrogens and testosterone at very low concentrations. Conventional immunoassay methods often lack the required performance to meet this analytical need, and mass spectrometry techniques must be employed. Our aim was to develop a sensitive HPLC-MS/MS assay for both estradiol (E2) and testosterone (Te) in serum, utilizing commercially available calibrators and without the need for chemical derivatization. Serum samples, after the addition of an internal standard, are combined with a hexane:ethyl acetate extraction solution. The samples are vortexed, and the organic layer is decanted into a clean sample tube and evaporated to dryness under a stream of nitrogen. The samples are reconstituted in a water:methanol solution and separated chromatographically using a reversed-phase HPLC column. Subsequent mass spectrometry is performed using both positive ion mode for Te and negative ion mode for E2.

BACKGROUND AND OBJECTIVES:High-dose melphalan is an integral part of conditioning chemotherapy prior to both autologous and allogeneic hematopoietic cell transplantation. While underexposure may lead to relapse, overexposure may lead to toxicities include mucositis, diarrhea, bone marrow suppression, and rarely sinusoidal obstruction syndrome. In this study, we describe the population pharmacokinetics of high-dose melphalan as a first step towards individualized dosing.METHODS:Melphalan samples were collected in patients receiving an allogeneic or autologous hematopoietic cell transplantation between August 2016 and August 2020 at the Memorial Sloan Kettering Cancer Center. A population-pharmacokinetic model was developed using NONMEM.RESULTS:Based on a total of 3418 samples from 452 patients receiving a median cumulative dose of 140 mg/m2, a two-compartment population-pharmacokinetic model was developed. Fat-free mass was a covariate for clearance, central volume of distribution, and inter-compartmental clearance, while glomerular filtration rate predicted clearance. Simulation studies showed that based on fixed body surface area-based dosing, renal impairment has a higher impact in increasing melphalan exposure compared with obesity.CONCLUSIONS:The proposed model adequately describes the population pharmacokinetics of melphalan in adult patients receiving a hematopoietic cell transplantation. This model can be used to define the therapeutic window of melphalan, and subsequently to develop individualized dosing regimens aiming for that therapeutic window in all patients.

High-dose melphalan prior to AHCT for MM and AL patients is currently given as a fixed dose based on body surface area leading to inter-patient differences in area under the curve (AUC). Increased exposure (higher AUC) leads to increased toxicity, but longer survival (Shaw et al., BBMT 2012). To optimize therapy, we evaluated the feasibility of PK-directed dosing of PGF-MEL (Acrotech Biopharma), a more stable and possibly less toxic intravenous formulation of melphalan.

Background Carmustine, etoposide, cytarabine and melphalan (BEAM) is the most common conditioning regimen for autologous hematopoietic cell transplantation (AHCT) recipients with lymphoma. Data in multiple myeloma have shown that dosing melphalan as a fixed dose based on body surface area leads to inter-patient differences with a 5-fold variability in area under the curve (AUC), with higher AUCs associated with improved outcomes but increased toxicity (Shaw BBMT 2012). We evaluated the pharmacokinetics (PK) of Evomela® (propylene glycol free melphalan, PGF-MEL) in lymphoma patients to investigate the effect of melphalan exposure on outcomes and toxicities. Methods On D-1 prior to AHCT, 140 mg/m2 of PGF-MEL was given over 30 minutes and serum sample were drawn serially at 5, 15, 30, 40, 75, and 150 min after the dose. AUC PK modeling was done with Cetara Phoenix WinNonlin, Princeton, NJ. Toxicities will be collected using CTCAE v4 through D+100 after AHCT. Results Eighty-seven patients received BEAM and had melphalan PK analysis. Among all patients, the median age was 51 (range, 19-74), 62% were male, and 53% had aggressive B-cell lymphoma. The median PK parameters were as follows: AUC 8.1 mg*h/L (range, 3.8-18.8); clearance 31.8 L/h (range 16.4-79.1); volume of distribution 33.2 L (range, 19.7-70.1); and half-life of elimination 0.7 hours (range, 0.5-1.7). The median dose of melphalan given was similar between patients ≤ median AUC and those above (260 mg [range, 200-340 mg) and 270 mg [range, 210-340 mg], respectively.) Median follow-up of survivors was 8.94 months (range 0.72-32.91). Univariate analysis showed no difference in 1-year overall survival (OS) (95.5% [84-100%] vs. 87.4% [76.5-100%], p=0.2) or progression-free survival (PFS) (78.4% [63.6-96.6%] vs. 78% [63.7-95.5%], p>0.99) when evaluating patients ≤ or above median melphalan AUC, respectively. Patients with a melphalan AUC ≤ the median had an average length of stay of 19 days (range, 5-27) vs. 21 days (range, 7-38) for those above the median exposure (OR 1.19 [1.05-1.38], p = 0.004). Toxicity data collection is ongoing. Conclusion PGF-MEL demonstrated a 5-fold variability in AUC when used as part of BEAM conditioning in AHCT for lymphoma. Early results do not yet indicate an impact on OS or PFS, but the data requires time to mature. However, melphalan exposure ≤ 8.1 mg*h/L resulted in a shorter length of hospitalization likely driven by toxicities that will be reported and presented.

IntroductionDysgeusia commonly influences nutritional intake and quality of life after HCT, yet its pathobiology is poorly understood and methods to study it are not established. We report interim results of a prospective feasibility study of chemical gustometry (CG) to measure dysgeusia in pts with multiple myeloma (MM) undergoing high-dose melphalan and autologous HCT (mel-AHCT). We evaluated associations between dysgeusia and symptoms, nutrition, mel pharmacokinetics (PK), and oral microbiota composition. We hypothesized that mel penetration into saliva contributes to dysgeusia.MethodsCG was performed by the Henkin method pre-AHCT and on days (d) -1, +7, +14, +30 testing 5 flavors (sweet, sour, salty, bitter, umami) to calculate a total taste score (max value 30). CG was considered feasible if > 60% of pts could complete > 60% of all evaluations. Caloric intake (kcal) was calculated with 24-hr recall using the USDA 5-Step Multiple-Pass Method, and pt-reported symptoms were assessed using the MD Anderson Symptom Inventory (MDASI) at the above times and on d +3 and +10. Salivary microbiome diversity was assessed longitudinally by 16S sequencing and the inverse Simpson index. 6-point serum mel concentrations were used to calculate an area under the curve (AUC) and peak salivary mel was obtained 75 min post-infusion. Spearman rank-sum correlation was used to assess associations.Results22 mel-AHCT MM pts have follow-up through d +30. 18 pts (82%) completed > 60% of all assessments. Median age was 62 (range 41-75). 11 pts (50%) were male. Mel 200 mg/m2 and 140 mg/m2 was given in 19 (86%) and 3 (14%) pts, respectively. Median serum mel AUC was 12.6 mg*h/L (range 5.8-17.5); median salivary mel was 119 ng/ml (range 25-662). Higher serum mel AUC correlated with higher salivary mel levels, p=0.004, and associated with poorer caloric intake from pre-ASCT to d +30, p=0.04. Higher serum mel AUC associated with higher MDASI scores (worse symptom burden) from pre-AHCT to d +30, p=0.005, but not with taste scores. Higher salivary mel associated with poorer caloric intake from pre-AHCT to d +30, p=0.02. MDASI scores peaked d +10, coincident with nadirs in taste scores and caloric intake on d +7 and +10, respectively. Taste scores partially recovered by d +30, but often did not return to baseline (Fig 1A-C). Oral microbiota diversity was stable early after AHCT but increased by d+100. D+7 diversity did not correlate with serum mel AUC, p=0.4, or salivary mel, p=0.84 (Fig 2A-C).ConclusionCG is a feasible method to evaluate dysgeusia after AHCT. Dysgeusia peaked by d +7 and often persisted through d +30. Oral microbiota diversity was stable early after AHCT. Higher serum and salivary mel levels did not correlate with microbiota diversity but were associated with poorer caloric intake and higher MDASI scores suggesting that using targeted mel PK dosing may improve caloric intake and limit symptom burden after AHCT.

Busulfan and melphalan are cytotoxic DNA alkylating agents that are used in many hematopoietic stem cell transplantation (HCT) conditioning regimens. We report the development of an assay using turbulent flow liquid chromatography (TFLC) and tandem mass spectrometry to simultaneously measure the concentration of busulfan (Bu) and melphalan (Mel) in human plasma. The method involves precipitating proteins in the plasma specimen with an organic solvent containing deuterated internal standards of both compounds. Following centrifugation, an aliquot of the supernatant was injected into the TFLC mass spectrometry system operated in the positive ion mode. The analytical measurement range for both compounds was 10-5000 ng/mL, and with validated dilutions the reportable range was extended to 25,000 ng/mL. Intra-day and inter-day (n = 20 day) precision studies showed a coefficient of variation (CV) of <7% at several concentrations across the measurement range. To determine accuracy recovery studies were performed at several concentrations spanning the measurement range. Recoveries for both compounds were between 98 and 103%. Additionally, busulfan was compared with an existing assay and showed excellent correlation. Experiments were conducted to rule out matrix effects, carryover and interference from endogenous substances. The validated clinically reportable range (CRR) and assay precision will allow this assay to be used clinically to monitor and adjust Mel and Bu levels to ensure better therapeutic outcomes and also to support clinical trials aimed at better defining therapeutic ranges.

Background: Taste disturbance, or dysgeusia, negatively affects quality of life and may lead to poorer nutritional status after high-dose melphalan and autologous stem cell transplantation (mel-AHCT) for patients with multiple myeloma (MM). Despite its frequency, dysgeusia has received limited prospective study. Here, we report the interim results of an on-going prospective clinical trial aimed at evaluating the feasibility of performing chemical gustometry, and quantifying the effects of dysgeusia on patient-reported symptom burden and nutritional status in MM patients undergoing mel-ASCT. We also sought to identify novel associations between mel serum and salivary pharmacokinetic (PK) levels and dysgeusia.

A very sensitive LC-MS/MS assay was developed implementing a liquid-liquid extraction step followed by mass spectrometry which was operated in both positive and negative ion modes. The assay was calibrated with readily available commercial calibrators and compared with international reference standards. This data is also presented in “Sensitive Simultaneous Quantitation of Testosterone and Estradiol in Serum by LC-MS/MS without Derivatization and Comparison with the CDC HoSt Program” (Schofield et al., 2017). This article includes the comparison of the LC-MS/MS assay with a commonly available chemiluminescencent immunoassay for the quantitation of both estradiol and testosterone. In addition we show baseline separation of estradiol and testosterone from other structurally related and/or isobaric compounds that could potentially interfere with the assay. In addition, various calibrator materials were tested and compared with internationally-recognized reference materials.

Background: Melphalan exposure as measured by area under the curve (AUC) has a 5-fold variability between patients, with higher AUCs associated with improved outcomes but increased toxicity (Shaw BBMT 2012). Propylene glycol free melphalan (Evomela®, PGF-MEL) is an intravenous formulation with improved stability and possibly less toxicity related to the solubilizing agent. We have switched to PGF-MEL as conditioning prior to AHCT for multiple myeloma (MM) and AL amyloidosis (AL) and aimed to determine the pharmacokinetics (PK) and relationship between exposure and toxicities with this formulation in order to optimize dosing and personalize care in the future.

Secondary lymphedema, a life-long complication of cancer treatment, currently has no cure. Lymphedema patients have decreased quality of life and recurrent infections with treatments limited to palliative measures. Accumulating evidence indicates that T cells play a key role in the pathology of lymphedema by promoting tissue fibrosis and inhibiting lymphangiogenesis. Here using mouse models, we show that topical therapy with tacrolimus, an anti-T-cell immunosuppressive drug, is highly effective in preventing lymphedema development and treating established lymphedema. This intervention markedly decreases swelling, T-cell infiltration and tissue fibrosis while significantly increasing formation of lymphatic collaterals with minimal systemic absorption. Animals treated with tacrolimus have markedly improved lymphatic function with increased collecting vessel contraction frequency and decreased dermal backflow. These results have profound implications for lymphedema treatment as topical tacrolimus is FDA-approved for other chronic skin conditions and has an established record of safety and tolerability.

Background: For multiple myeloma (MM) and AL amyloidosis (AL) patients undergoing autologous hematopoietic stem cell transplant (AHCT), high-dose melphalan is given at a fixed dose based on body surface area with five-fold inter-patient variability in exposure, as measured by area under the curve (AUC). Patients with higher than the median AUC (12.85mg/L*h) have prolonged survival, but increased toxicity (Shaw et al., BBMT 2012). Pharmacokinetically (PK)-directed dosing has the potential to achieve an optimal concentration in each patient. We prospectively evaluated the feasibility of PK-directed dosing of propylene glycol free melphalan (Evomela®, PGF-MEL), a more stable and possibly less toxic intravenous formulation of melphalan, to personalize therapy for maximum efficacy and tolerability.