Publications by authors named "Katie Thoren"

35 Publications

Minimal residual disease in multiple myeloma: defining the role of next generation sequencing and flow cytometry in routine diagnostic use.

Pathology 2021 Apr 3;53(3):385-399. Epub 2021 Mar 3.

Haematology Service, Peter MacCallum Cancer Centre, East Melbourne, Vic, Australia; Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, Vic, Australia.

For patients diagnosed with multiple myeloma (MM) there have been significant treatment advances over the past decade, reflected in an increasing proportion of patients achieving durable remissions. Clinical trials repeatedly demonstrate that achieving a deep response to therapy, with a bone marrow assessment proving negative for minimal residual disease (MRD), confers a significant survival advantage. To accurately assess for minute quantities of residual cancer requires highly sensitive methods; either multiparameter flow cytometry or next generation sequencing are currently recommended for MM response assessment. Under optimal conditions, these methods can detect one aberrant cell amongst 1,000,000 normal cells (a sensitivity of 10). Here, we will review the practical use of MRD assays in MM, including challenges in implementation for the routine diagnostic laboratory, standardisation across laboratories and clinical trials, the clinical integration of MRD status assessment into MM management and future directions for ongoing research.
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http://dx.doi.org/10.1016/j.pathol.2021.02.003DOI Listing
April 2021

Using MALDI-TOF mass spectrometry in peripheral blood for the follow up of newly diagnosed multiple myeloma patients treated with daratumumab-based combination therapy.

Clin Chim Acta 2021 May 2;516:136-141. Epub 2021 Feb 2.

Clinical Chemistry Service, Department of Laboratory Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA. Electronic address:

Background: Daratumumab-based combination therapies have shown high rates of complete response (CR) and minimal residual disease negativity in patients with multiple myeloma. However, daratumumab, an IgGκ monoclonal antibody, interferes with electrophoretic techniques making it difficult to reliably define residual disease versus CR, especially in patients with IgGκ multiple myeloma.

Methods: Enrichment with polyclonal sheep antibody-coated magnetic microparticles combined with MALDI-TOF mass spectrometry (MALDI-TOF MS) analysis was used to detect M-proteins in serial samples from newly diagnosed multiple myeloma patients treated with daratumumab-based therapy. The performance of the MALDI-TOF MS assay was compared to that of a routine test panel (serum protein electrophoresis (SPEP), immunofixation (IFE) and serum free light chain (FLC)).

Results: Comparison of MALDI-TOF MS to SPEP/IFE/FLC showed a concordance of 84.9% (p < 0.001). When MALDI-TOF MS and FLC results were combined, the M-protein detection rate was the same or better than the routine test panel. For the 9 patients who obtained CR during follow-up, MALDI-TOF MS detected an M-protein in 46% of subsequent samples. Daratumumab could be distinguished from the M-protein in 215/222 samples.

Conclusion: MALDI-TOF MS is useful in assessing CR in patients treated with monoclonal antibody-based therapies.
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http://dx.doi.org/10.1016/j.cca.2021.01.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7994191PMC
May 2021

Lifetime Pesticide Use and Monoclonal Gammopathy of Undetermined Significance in a Prospective Cohort of Male Farmers.

Environ Health Perspect 2021 Jan 6;129(1):17003. Epub 2021 Jan 6.

Myeloma Service, Memorial Sloan Kettering Cancer Center, New York, New York, USA.

Background: Farmers have a higher incidence of multiple myeloma, and there is suggestive evidence of an elevated prevalence of its precursor, monoclonal gammopathy of undetermined significance (MGUS), relative to the general population. Pesticide exposures are suspected to play a role; however, the biologic plausibility for associations with multiple myeloma remains unclear.

Objectives: Our objectives were to examine the prevalence of MGUS and evaluate associations with a wide range of pesticides in a large sample of farmers.

Methods: We obtained sera and assessed MGUS among 1,638 male farmers of age in the Agricultural Health Study (AHS), a prospective cohort in Iowa and North Carolina. Odds ratios (ORs) and 95% confidence intervals (CIs) were computed to estimate associations with MGUS for recent use (within the 12 months before phlebotomy) and cumulative intensity-weighted lifetime days of use of specific pesticides.

Results: The age-standardized MGUS prevalence was significantly elevated among AHS farmers (7.7%) compared with demographically similar men in the National Health and Nutrition Examination Survey (2.8%) or Olmsted County, Minnesota (3.8%; ). Recent use of permethrin was associated with MGUS [recent use vs. no recent use, (95% CI: 1.06, 3.13)], especially among those who had also used it in the past [recent and past use vs. never use, (95% CI: 1.32, 4.69)]. High intensity-weighted lifetime use of the organochlorine insecticides aldrin and dieldrin was associated with MGUS relative to those who never used either of these pesticides [ (95% CI: 1.29, 4.54); ]. We also observed a positive association with high lifetime use of petroleum oil/distillates as an herbicide, as well as an inverse association with fonofos use.

Discussion: This is the largest investigation of MGUS in farmers and the first to identify an association with MGUS for permethrin, a pyrethroid insecticide previously associated with multiple myeloma. Given the continued widespread use of permethrin in various residential and commercial settings, our findings may have important implications for exposed individuals in the general population. https://doi.org/10.1289/EHP6960.
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http://dx.doi.org/10.1289/EHP6960DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7787072PMC
January 2021

Dickkopf-1 Can Lead to Immune Evasion in Metastatic Castration-Resistant Prostate Cancer.

JCO Precis Oncol 2020 29;4. Epub 2020 Sep 29.

Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, NY.

Purpose: Metastatic castration-resistant prostate cancer (mCRPC) with low androgen receptor (AR) and without neuroendocrine signaling, termed double-negative prostate cancer (DNPC), is increasingly prevalent in patients treated with AR signaling inhibitors and is in need of new biomarkers and therapeutic targets.

Methods: Candidate genes enriched in DNPC were determined using differential gene expression analysis of discovery and validation cohorts of mCRPC biopsies. Laboratory studies were carried out in human mCRPC organoid cultures, prostate cancer (PCa) cell lines, and mouse xenograft models. Epigenetic studies were carried out in a rapid autopsy cohort.

Results: Dickkopf-1 (DKK1) expression is increased in DNPC relative to prostate-specific antigen (PSA)-expressing mCRPC in the Stand Up to Cancer/Prostate Cancer Foundation discovery cohort (11.2 0.28 reads per kilobase per million mapped reads; < 0.05; n = 117) and in the University of Washington/Fred Hutchinson Cancer Research Center cohort (9.2 0.99 fragments per kilobase of transcript per million mapped reads; < .0001). DKK1 expression can be regulated by activated Wnt signaling in vitro and correlates with activating canonical Wnt signaling mutations and low PSA mRNA in mCRPC biopsies ( < .05). hypomethylation was associated with increased DKK1 mRNA expression (Pearson = -0.66; < .0001) in a rapid autopsy cohort (n = 7). DKK1-high mCRPC biopsies are infiltrated with significantly higher numbers of quiescent natural killer (NK) cells ( < .005) and lower numbers of activated NK cells ( < .0005). Growth inhibition of the human PCa model PC3 by the anti-DKK1 monoclonal antibody DKN-01 depends on the presence of NK cells in a severe combined immunodeficient xenograft mouse model.

Conclusion: These results support DKK1 as a contributor to the immunosuppressive tumor microenvironment of DNPC. These data have provided the rationale for a clinical trial targeting DKK1 in mCRPC (ClinicalTrials.gov identifier: NCT03837353).
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http://dx.doi.org/10.1200/PO.20.00097DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7529521PMC
September 2020

Defining the undetectable: The current landscape of minimal residual disease assessment in multiple myeloma and goals for future clarity.

Blood Rev 2021 Mar 10;46:100732. Epub 2020 Jul 10.

Memorial Sloan Kettering Cancer Center, USA.

Multiple Myeloma, the second most prevalent hematologic malignancy, yet lacks an established curative therapy. However, overall response rate to modern four-drug regimens approaches 100%. Major efforts have thus focused on the measurement of minute quantities of residual disease (minimal residual disease or MRD) for prognostic metrics and therapeutic response evaluation. Currently, MRD is assessed by flow cytometry or by next generation sequencing to track tumor-specific immunoglobulin V(D)J rearrangements. These bone marrow-based methods can reach sensitivity thresholds of the identification of one neoplastic cell in 1,000,000 (10). New technologies are being developed to be used alone or in conjunction with established methods, including peripheral blood-based assays, mass spectrometry, and targeted imaging. Data is also building for MRD as a surrogate endpoint for overall survival. Here, we will address the currently utilized MRD assays, challenges in validation across labs and clinical trials, techniques in development, and future directions for successful clinical application of MRD in multiple myeloma.
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http://dx.doi.org/10.1016/j.blre.2020.100732DOI Listing
March 2021

Tracking of low disease burden in multiple myeloma: Using mass spectrometry assays in peripheral blood.

Best Pract Res Clin Haematol 2020 03 11;33(1):101142. Epub 2020 Jan 11.

Department of Laboratory Medicine, Memorial Sloan Kettering Cancer Center, 327 East 64th St, New York, NY, 10065, USA. Electronic address:

Efforts over the last 5 years have demonstrated that it is technically feasible to detect low levels of monoclonal proteins in peripheral blood using mass spectrometry. These methods are based on the fact that an M-protein has a specific amino acid sequence, and therefore, a specific mass. This mass can be tracked over time and can serve as a surrogate marker of the presence of clonal plasma cells. This review describes the use of mass spectrometry to detect M-proteins in multiple myeloma to date, identifies the challenges of using this biomarker, and describes potential strategies to overcome these challenges. We discuss the work that must be done for these techniques to be incorporated into clinical practice for tracking of low disease burden in multiple myeloma.
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http://dx.doi.org/10.1016/j.beha.2020.101142DOI Listing
March 2020

Comparison of MALDI-TOF mass spectrometry analysis of peripheral blood and bone marrow-based flow cytometry for tracking measurable residual disease in patients with multiple myeloma.

Br J Haematol 2020 06 5;189(5):904-907. Epub 2020 Feb 5.

Clinical Chemistry Service, Department of Laboratory Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA.

Matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) may soon replace routine electrophoretic methods for monitoring monoclonal proteins in patients with multiple myeloma. To further evaluate the clinical utility of this assay, we compared the performance of MALDI-TOF-MS head-to-head with an established bone marrow-based measurable residual disease assay by flow cytometry (Flow-BM-MRD), using Memorial Sloan Kettering Cancer Center's 10-color, single-tube method. Our results suggest that MALDI-TOF-MS adds value to bone marrow-based MRD testing and may be most useful for early detection of relapse in peripheral blood compared to current electrophoretic methods.
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http://dx.doi.org/10.1111/bjh.16443DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7275888PMC
June 2020

A novel method for the laboratory workup of anaphylactic transfusion reactions in haptoglobin-deficient patients.

Transfusion 2020 04 23;60(4):682-687. Epub 2020 Jan 23.

Department of Laboratory Medicine, Memorial Sloan Kettering Cancer Center, New York, New York.

Background: Patients with congenital haptoglobin deficiency can develop anti-haptoglobin antibodies after exposure to blood products, and they can suffer from life-threatening anaphylactic transfusion reactions. Here, we present a case of a 57-year-old Chinese male with myelodysplastic syndrome who manifested an anaphylactic transfusion reaction during the transfusion of platelets. The only abnormality detected during his reaction laboratory workup was an undetectable haptoglobin level in the absence of evidence of hemolysis.

Study Design And Methods: Surface plasmon resonance (SPR) was explored as a method to be able to detect the presence of anti-haptoglobin antibodies in serum. First, haptoglobin was immobilized to the surface of an SPR sensor chip. The patient's serum sample was injected, and the binding response was monitored in real time. Serum samples from five healthy volunteers were used as negative controls. Binding specificity was assessed in competition experiments using soluble haptoglobin. Anti-IgG, -IgA, -IgM, -IgD and -IgE antibodies were used to identify the antibody isotype.

Results: An IgG anti-haptoglobin antibody was detected in the patient's serum with SPR.

Conclusion: SPR provided a rapid, readily available method for the detection of an IgG anti-haptoglobin antibody in an anhaptoglobinemic individual. This confirmed the underlying etiology of the anaphylactic nonhemolytic transfusion reaction and justified the necessity of stringently washed cellular products for all future transfusions and strong caution for future use of plasma-containing products.
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http://dx.doi.org/10.1111/trf.15657DOI Listing
April 2020

Identification of gamma heavy chain disease using MALDI-TOF mass spectrometry.

Clin Biochem 2020 Mar 26;77:57-61. Epub 2019 Dec 26.

Department of Laboratory Medicine, Memorial Sloan Kettering Cancer Center, 327 E. 64th St., New York, NY 10065, USA.

We describe the use of MALDI-TOF mass spectrometry in the analysis of a suspected case of gamma heavy chain disease. The patient had an abnormal serum immunofixation result where a monoclonal gamma heavy chain band was present without a corresponding light chain. Analysis by MALDI-TOF mass spectrometry revealed large peaks in the spectrum following IgG-specific purification. The m/z values of the peaks were outside the expected range for normal heavy chains or light chains. Corresponding peaks were not present in mass spectra of the kappa- or lambda-specific purifications. MALDI-TOF MS confirmed the presence of a truncated heavy chain without associated light chains. This case report demonstrates the value of mass spectrometry in interpreting challenging cases such as the identification of heavy chain disease.
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http://dx.doi.org/10.1016/j.clinbiochem.2019.12.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7046309PMC
March 2020

Distinguishing Drug from Disease by Use of the Hydrashift 2/4 Daratumumab Assay.

J Appl Lab Med 2019 03 31;3(5):857-863. Epub 2018 May 31.

Clinical Chemistry Service, Department of Laboratory Medicine, Memorial Sloan Kettering Cancer Center, New York, NY.

Background: Daratumumab, a monoclonal antibody used to treat relapsed or refractory multiple myeloma, can interfere with protein electrophoresis and immunofixation assays. False-positive immunofixation results due to daratumumab can cause uncertainty regarding the status of a patient's disease and lead to potential misclassification of their response to therapy. The Hydrashift 2/4 Daratumumab assay (Sebia) was recently cleared by the Food and Drug Administration for resolving daratumumab interference on immunofixation. Here, we evaluate the performance of the Hydrashift assay in multiple myeloma patients receiving treatment with daratumumab-based regimens.

Methods: Waste serum samples from multiple myeloma patients (n = 40) receiving daratumumab were analyzed by standard immunofixation and the Hydrashift assay. Results from these tests were compared and were evaluated along with pretreatment serum protein electrophoresis and immunofixation results, if available.

Results: The Hydrashift assay shifted the migration of daratumumab in patient samples. In 27 cases, the patient's M protein was distinguishable from daratumumab by standard immunofixation. In these cases, the Hydrashift assay confirmed that the IgGκ band was daratumumab and helped identify the presence of treatment-related oligoclonal bands. There were 11 instances in which the patient's IgGκ M protein comigrated with daratumumab. In all 11 cases, the Hydrashift assay confirmed the presence of residual M protein. Finally, in 2 patients whose pretreatment immunofixation results were not available, the Hydrashift assay confirmed that the IgGκ band visible on immunofixation was due to daratumumab alone.

Conclusions: The Hydrashift 2/4 Daratumumab assay is a useful tool to clarify the source of an IgGκ band on immunofixation and allow a patient's M protein to be viewed without interference.
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http://dx.doi.org/10.1373/jalm.2018.026476DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7484995PMC
March 2019

Evaluation of CisBio ELISA for Chromogranin A Measurement.

J Appl Lab Med 2019 07 17;4(1):11-18. Epub 2019 Apr 17.

Department of Laboratory Medicine, Memorial Sloan Kettering Cancer Center, New York, NY.

Background: Chromogranin A (CgA) is a nonspecific marker for the presence of neuroendocrine tumors and neuroendocrine differentiation. The objective of this study was to evaluate the performance of the CisBio CgA ELISA.

Methods: Precision, linearity, limit of blank, and recovery of the CisBio CgA ELISA were evaluated. Seventy waste serum samples obtained from the clinical laboratory at Memorial Sloan Kettering Cancer Center were analyzed by the CisBio CgA ELISA. Results were compared to those obtained from a reference laboratory that used a proprietary ELISA for serum CgA measurement. Paired waste plasma samples were also collected from 24 of these patients to assess possible differences between CgA in serum and plasma. Finally, a preliminary reference range study was performed with samples from healthy volunteers in serum (n = 60) and plasma (n = 60).

Results: Within-run and between-run precision ranged from 3.0% to 5.1% and 4.8% to 12.9%, respectively. The limit of blank was 2.4 ng/mL. Recovery ranged from 88% to 102%. A statistically significant bias was observed when the CisBio CgA assay results were compared to those of a reference laboratory. Comparison of the 2 assays yielded a slope of 9.05, intercept of -18.0, and a correlation coefficient of 0.955. CgA values in serum correlated well to values measured in plasma.

Conclusions: The analytical performance of the CisBio CgA ELISA was acceptable. However, CgA results are method-specific owing to lack of standardization and use of different antibodies. This lack of standardization results in several challenges for the clinical laboratory when evaluating a CgA assay.
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http://dx.doi.org/10.1373/jalm.2018.028027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7485006PMC
July 2019

Association of Immune Marker Changes With Progression of Monoclonal Gammopathy of Undetermined Significance to Multiple Myeloma.

JAMA Oncol 2019 Jul 18. Epub 2019 Jul 18.

Myeloma Section, Department of Medicine, University Hospital of Malmo, Malmo, Sweden.

Importance: Multiple myeloma is consistently preceded by monoclonal gammopathy of undetermined significance (MGUS). Risk models that estimate the risk of progression from MGUS to multiple myeloma use data from a single time point, usually the initial workup.

Objective: To longitudinally investigate the alterations of serum immune markers with stable vs progressive MGUS.

Design, Setting, And Participants: This prospective cross-sectional cohort study included 77 469 adult participants aged 55 to 74 years in the screening arm of the National Cancer Institute Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial who had a diagnosis of progressing MGUS (n = 187) or stable MGUS (n = 498), including light-chain subtype, from November 1993, through December 2011. For each participant, all available serially stored prediagnostic serum samples (N = 3266) were obtained. Data analysis was performed from April 2018, to December 2018.

Main Outcomes And Measures: Serum protein and monoclonal immunoglobulin levels, serum free light chains, and serum light chains within each immunoglobulin class were measured.

Results: Of 685 individuals included in the study, 461 (67.3%) were men; the mean (SD) age was 69.1 (5.6) years. In cross-sectional modeling, risk factors associated with progressive MGUS were IgA isotype (adjusted odds ratio [OR], 1.80; 95% CI, 1.03-3.13; P = .04), 15 g/L or more monoclonal spike (adjusted OR, 23.5; 95% CI, 8.9-61.9; P < .001), skewed (<0.1 or >10) serum free light chains ratio (adjusted OR, 46.4; 95% CI, 18.4-117.0; P < .001), and severe immunoparesis (≥2 suppressed uninvolved immunoglobulins) (adjusted OR, 19.1; 95% Cl, 7.5-48.3; P < .001). Risk factors associated with progressive light-chain MGUS were skewed serum free light chains ratio (adjusted OR, 44.0; 95% CI, 14.2-136.3; P < .001) and severe immunoparesis (adjusted OR, 48.6; 95% CI, 9.5-248.2; P < .001). In longitudinal analysis of participants with serial samples prior to progression, 23 of 43 participants (53%) had high-risk MGUS before progression; 16 of these 23 (70%) experienced conversion from low-risk or intermediate-risk MGUS within 5 years. Similar results were found for light-chain MGUS.

Conclusions And Relevance: The findings of evolving risk patterns support annual blood testing and risk assessment for patients with MGUS or light-chain MGUS.
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http://dx.doi.org/10.1001/jamaoncol.2019.1568DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6646992PMC
July 2019

Pituitary as a Source of HCG: Residual Levels After Bilateral Testicular Tumor Removal.

J Investig Med High Impact Case Rep 2019 Jan-Dec;7:2324709619841414

2 Memorial Sloan Kettering Cancer Center, Cornell-Weil School of Medicine, New York, NY, USA.

Context: Challenging clinical scenario in which elevated β-human chorionic gonadotropin (HCG, subsequently termed HCG) levels suggested occult tumor metastases after removal of bilateral testicular cancers and metastases from them and as well as after chemotherapy.

Case Report: A 22-year-old male, post excision of bilateral testicular tumors, who had no imaging or clinical evidence of residual tumor but an elevated HCG raising the question of the presence and location of occult tumor metastases. Clinical Questions. Does luteinizing hormone (LH) cross-react with HCG in current assays? What levels of testosterone and estradiol are necessary to suppress LH and follicle-stimulating hormone (FSH) in a male patient with bilateral orchiectomy, and therefore lacking inhibin? Does the pituitary secrete HCG and under what circumstances?

Assessment: Current HCG assays no longer cross-react with LH as did prior assays, but the presence of heterophile antibodies and other factors such as biotin can still cause false positive HCG levels. In the chronic post-orchiectomy state, the pituitary is relatively resistant to LH and FSH suppression by testosterone. The pituitary secretes HCG in very small amounts unless interruption of negative feedback results in high LH and FSH whereupon HCG levels become elevated. Clinical Conclusion. A GnRH antagonist suppressed both LH and HCG in this patient indicating that the elevated HCG was secreted by the pituitary and not by occult tumor metastases. Further credence for this conclusion resulted from the lack of a progressive increase in HCG levels over a 4-year period of follow-up and from no evidence of metastatic tumors on serial imaging.
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http://dx.doi.org/10.1177/2324709619841414DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6480980PMC
June 2020

Standard Antithymocyte Globulin Dosing Results in Poorer Outcomes in Overexposed Patients after Ex Vivo CD34 Selected Allogeneic Hematopoietic Cell Transplantation.

Biol Blood Marrow Transplant 2019 08 1;25(8):1526-1535. Epub 2019 Mar 1.

Adult Bone Marrow Transplant Service, Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, New York; Department of Medicine, Weill Cornell Medical College, New York, New York.

Antithymocyte globulin (ATG) use mitigates the risk of graft rejection and graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (allo-HCT), but ATG overexposure in the setting of lymphopenia negatively affects immune recovery. We hypothesized that standard empiric weight-based dosing of ATG, used to prevent graft rejection in ex vivo CD34-selected allo-HCT, may lead to serious adverse consequences on outcomes in certain patients. We evaluated 304 patients undergoing myeloablative-conditioned ex vivo CD34-selected allo-HCT with HLA-matched donors for the treatment of hematologic malignancies. Patients received rabbit ATG at a dose of 2.5 mg/kg/day i.v. on days -3 and/or -2. An ATG dosing cutoff of 450 mg was used for statistical analyses to assess the relationship between ATG and overall survival (OS). Among all patients, median total ATG dose was 360 mg (range, 130 to 510 mg); 279 (92%) received a total dose of ATG ≤450 mg, and 25 (8%) received a total dose >450 mg. On the first day of ATG administration (day -3), the median absolute lymphocyte count was .0 K/µL. For patients who received a total dose of ATG >450 mg or ≤450 mg, the incidences of acute and late-acute GVHD grade II-IV were statistically similar. At 3 years post-HCT, for patients who received a total dose of ATG >450 mg or ≤450 mg, nonrelapse mortality (NRM) rates were 35% and 18%, respectively (P = .029), disease-free survival (DFS) rates were 37% and 61%, respectively (P = .003), and OS rates were 40% and 67%, respectively (P = .001). Among all patient and HCT characteristics in multivariable analyses, receipt of a total dose of ATG >450 mg was associated with an increased risk of NRM (hazard ratio [HR], 2.9; P = .01), shorter DFS (HR, 2.0; P = .03), and inferior OS (HR, 2.1; P = .01). In summary, the use of weight-based ATG at a time of relative lymphopenia before ex vivo CD34-selected allo-HCT results in overdosing in heavier patients, leading to higher NRM and lower DFS and OS. Further pharmacokinetic investigation in this setting is critical to determining the optimal dosing strategy for ATG.
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http://dx.doi.org/10.1016/j.bbmt.2019.02.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7302932PMC
August 2019

MALDI-TOF mass spectrometry distinguishes daratumumab from M-proteins.

Clin Chim Acta 2019 May 18;492:91-94. Epub 2019 Feb 18.

Clinical Chemistry Service, Department of Laboratory Medicine, Memorial Sloan Kettering Cancer Center, 327 E. 64th St., New York, NY 10065, United States of America. Electronic address:

Background: Daratumumab, a therapeutic IgG kappa monoclonal antibody, can cause a false positive interference on electrophoretic assays that are routinely used to monitor patients with monoclonal gammopathies. In this study, we evaluate the ability of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to distinguish daratumumab from disease-related IgG kappa monoclonal proteins (M-protein).

Methods: Waste clinical samples from 31 patients who were receiving daratumumab and had a history of IgG kappa monoclonal gammopathy were collected. Immunoglobulins were purified from serum and analyzed by MALDI-TOF MS. Mass spectra were assessed for the presence of distinct monoclonal proteins. For samples in which only one monoclonal peak was identified near the expected m/z of daratumumab, the Hydrashift 2/4 Daratumumab Assay was used to confirm the presence of an M-protein.

Results: Using MALDI-TOF MS, daratumumab could be distinguished from M-proteins in 26 out of 31 samples (84%). Results from 2 samples were inconclusive since the M-protein was not detected by the Hydrashift assay and may also be undetectable by MALDI-TOF MS. Comparatively, daratumumab was distinguishable from M-proteins in 14 out of 31 samples (45%) by immunofixation.

Conclusions: MALDI-TOF MS offers greater specificity compared to immunofixation for distinguishing daratumumab from M-proteins.
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http://dx.doi.org/10.1016/j.cca.2019.02.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6524149PMC
May 2019

Mass spectrometry methods for detecting monoclonal immunoglobulins in multiple myeloma minimal residual disease.

Authors:
Katie L Thoren

Semin Hematol 2018 01 26;55(1):41-43. Epub 2018 Feb 26.

Department of Laboratory Medicine, Memorial Sloan Kettering Cancer Center, New York, NY. Electronic address:

Mass spectrometry methods that can detect low levels of monoclonal immunoglobulin in serum have recently been developed. These assays are based on the principle that each immunoglobulin has a unique amino acid sequence and therefore, has a unique mass. This mass can be used as a surrogate marker in order to monitor a patient's disease over time and at low levels. Here, we explain these methods, discuss their advantages and disadvantages and how they may be used to monitor monoclonal immunoglobulins for minimal residual disease detection in multiple myeloma.
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http://dx.doi.org/10.1053/j.seminhematol.2018.02.008DOI Listing
January 2018

Multiple Myeloma and Its Precursor Disease Among Firefighters Exposed to the World Trade Center Disaster.

JAMA Oncol 2018 06;4(6):821-827

Bureau of Health Services, Fire Department of the City of New York, Brooklyn, New York.

Importance: The World Trade Center (WTC) attacks on September 11, 2001, created an unprecedented environmental exposure to known and suspected carcinogens suggested to increase the risk of multiple myeloma. Multiple myeloma is consistently preceded by the precursor states of monoclonal gammopathy of undetermined significance (MGUS) and light-chain MGUS, detectable in peripheral blood.

Objective: To characterize WTC-exposed firefighters with a diagnosis of multiple myeloma and to conduct a screening study for MGUS and light-chain MGUS.

Design, Setting, And Participants: Case series of multiple myeloma in firefighters diagnosed between September 11, 2001, and July 1, 2017, together with a seroprevalence study of MGUS in serum samples collected from Fire Department of the City of New York (FDNY) firefighters between December 2013 and October 2015. Participants included all WTC-exposed FDNY white, male firefighters with a confirmed physician diagnosis of multiple myeloma (n = 16) and WTC-exposed FDNY white male firefighters older than 50 years with available serum samples (n = 781).

Exposures: WTC exposure defined as rescue and/or recovery work at the WTC site between September 11, 2001, and July 25, 2002.

Main Outcomes And Measures: Multiple myeloma case information, and age-adjusted and age-specific prevalence rates for overall MGUS (ie, MGUS and light-chain MGUS), MGUS, and light-chain MGUS.

Results: Sixteen WTC-exposed white male firefighters received a diagnosis of multiple myeloma after September 11, 2001; median age at diagnosis was 57 years (interquartile range, 50-68 years). Serum/urine monoclonal protein isotype/free light-chain data were available for 14 cases; 7 (50%) had light-chain multiple myeloma. In a subset of 7 patients, myeloma cells were assessed for CD20 expression; 5 (71%) were CD20 positive. In the screening study, we assayed peripheral blood from 781 WTC-exposed firefighters. The age-standardized prevalence rate of MGUS and light-chain MGUS combined was 7.63 per 100 persons (95% CI, 5.45-9.81), 1.8-fold higher than rates from the Olmsted County, Minnesota, white male reference population (relative rate, 1.76; 95% CI, 1.34-2.29). The age-standardized prevalence rate of light-chain MGUS was more than 3-fold higher than in the same reference population (relative rate, 3.13; 95% CI, 1.99-4.93).

Conclusions And Relevance: Environmental exposure to the WTC disaster site is associated with myeloma precursor disease (MGUS and light-chain MGUS) and may be a risk factor for the development of multiple myeloma at an earlier age, particularly the light-chain subtype.
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http://dx.doi.org/10.1001/jamaoncol.2018.0509DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6145680PMC
June 2018

Voxelotor (GBT440) produces interference in measurements of hemoglobin S.

Clin Chim Acta 2018 Jul 27;482:57-59. Epub 2018 Mar 27.

Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN, United States. Electronic address:

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http://dx.doi.org/10.1016/j.cca.2018.03.032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7485007PMC
July 2018

Quantitation of Infliximab and Detection of Antidrug Antibodies in Serum by Use of Surface Plasmon Resonance.

J Appl Lab Med 2018 Mar;2(5):725-736

Department of Laboratory Medicine, University of California San Francisco, San Francisco, CA.

Background: Monitoring infliximab (IFX) concentrations and the presence of antidrug antibodies (ADA) is important for patient management. We developed a method to measure IFX and ADA in serum in a single injection using surface plasmon resonance (SPR).

Methods: Using the Bio-Rad ProteOn XPR36, tumor necrosis factor-α and IFX were covalently immobilized onto separate lanes of a chip surface. Diluted serum was injected over both lanes, followed by an injection of goat antihuman antibody. The binding response was used to quantify IFX or detect ADA. The analytical performance of the assay was determined. Using 50 patient samples, SPR results were compared with results from a reporter gene assay (RGA).

Results: For the quantification of IFX, the functional sensitivity was 0.5 μg/mL. The total precision was <10% for all concentrations tested. IFX concentrations measured by SPR correlated well with RGA (R = 0.862), but a bias was observed (slope = 0.61). SPR detected 14 ADA-positive samples. Compared with RGA for ADA detection, there were 6 true-positive, 8 false-positive, 5 false-negative, and 31 true-negative findings.

Conclusion: SPR can be used to measure biological drug concentrations and detect ADA in serum. This technique may provide complementary information to current methods used to detect ADA.
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http://dx.doi.org/10.1373/jalm.2017.024380DOI Listing
March 2018

Suspect Screening Using LC-QqTOF Is a Useful Tool for Detecting Drugs in Biological Samples.

J Anal Toxicol 2018 May;42(4):207-213

Department of Laboratory Medicine, University of California San Francisco, 1001 Potrero Avenue NH 2M16, San Francisco, CA 94110, USA.

High-resolution mass spectrometers (HRMS), including quadrupole time of flight mass analyzers (QqTOF), are becoming more prevalent as screening tools in clinical and forensic toxicology laboratories. Among other advantages, HRMS instruments can collect untargeted, full-scan mass spectra. These datasets can be analyzed retrospectively using a combination of techniques, which can extend the drug detection capabilities. Most laboratories using HRMS in production settings perform untargeted data collection, but analyze data in a targeted manner. To perform targeted analysis, a laboratory must first analyze a reference standard to determine the expected characteristics of a given compound. In an alternate technique known as suspect screening, compounds can be tentatively identified without the use of reference standards. Instead, predicted and/or intrinsic characteristics of a compound, such as the accurate mass, isotope pattern, and product ion spectrum are used to determine its presence in a sample. The fact that reference standards are not required a priori makes this data analysis approach very attractive, especially for the ever-changing landscape of novel psychoactive substances. In this work, we compared the performance of four data analysis workflows (targeted and three suspect screens) for a panel of 170 drugs and metabolites, detected by LC-QqTOF. We found that retention time was not required for drug identification; the suspect screen using accurate mass, isotope pattern, and product ion library matching was able to identify more than 80% of the drugs that were present in human urine samples. We showed that the inclusion of product ion spectral matching produced the largest decrease in false discovery and false negative rates, as compared to suspect screening using mass alone or using just mass and isotope pattern. Our results demonstrate the promise that suspect screening holds for building large, economical drug screens, which may be a key tool to monitor the use of emerging drugs of abuse, including novel psychoactive substances.
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http://dx.doi.org/10.1093/jat/bkx107DOI Listing
May 2018

Prognostic significance of baseline metabolic tumor volume in relapsed and refractory Hodgkin lymphoma.

Blood 2017 11 5;130(20):2196-2203. Epub 2017 Sep 5.

Lymphoma Service and.

Identification of prognostic factors for patients with relapsed/refractory Hodgkin lymphoma (HL) is essential for optimizing therapy with risk-adapted approaches. In our phase 2 study of positron emission tomography (PET)-adapted salvage therapy with brentuximab vedotin (BV) and augmented ifosfamide, carboplatin, and etoposide (augICE), we assessed clinical factors, quantitative PET assessments, and cytokine and chemokine values. Transplant-eligible patients with relapsed/refractory HL received 2 (cohort 1) or 3 (cohort 2) cycles of weekly BV; PET-negative patients (Deauville score ≤2) proceeded to autologous stem cell transplantation (ASCT) whereas PET-positive patients received augICE before ASCT. Serum cytokine and chemokine levels were measured at baseline and after BV. Metabolic tumor volume (MTV) and total lesion glycolysis were measured at baseline, after BV, and after augICE. Sixty-five patients enrolled (45, cohort 1; 20, cohort 2); 49 (75%) achieved complete response and 64 proceeded to ASCT. Three-year overall survival and event-free survival (EFS) were 95% and 82%, respectively. Factors predictive for EFS by multivariable analysis were baseline MTV (bMTV) ( < .001) and refractory disease ( = .003). Low bMTV (<109.5 cm) and relapsed disease identified a favorable group (3-year EFS, 100%). For patients who received a transplant, bMTV and pre-ASCT PET were independently prognostic; 3-year EFS for pre-ASCT PET-positive patients with low bMTV was 86%. In this phase 2 study of PET-adapted therapy with BV and augICE for relapsed/refractory HL, bMTV and refractory disease were independent prognostic factors for EFS. Furthermore, bMTV improved the predictive power of pre-ASCT PET. Future studies should optimize efficacy and tolerability of salvage therapy by stratifying patients according to risk factors such as bMTV.
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http://dx.doi.org/10.1182/blood-2017-06-788877DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5691245PMC
November 2017

Optimization and Validation of High-Resolution Mass Spectrometry Data Analysis Parameters.

J Anal Toxicol 2017 Jan 6;41(1):1-5. Epub 2016 Oct 6.

Department of Laboratory Medicine, University of California San Francisco, 1001 Potrero Avenue NH 2M16, San Francisco, CA 94110, USA.

High-resolution mass spectrometry (HRMS) has gained recognition as a valuable tool for comprehensive drug screening in a variety of biological matrices. HRMS instruments collect untargeted, accurate mass data, which permit identification of known and unknown compounds in a single analytical run. One of the most challenging aspects of implementing an HRMS drug screen is establishing appropriate data analysis parameters for identifying compounds. Unlike other types of mass spectrometry data, guidelines for HRMS data analysis and acceptability criteria have not been established. Although many laboratories have published on the utility of HRMS for drug screening, few have included details on how they determined allowable errors and set positivity criteria. Previously, we developed and validated a comprehensive 169-compound drug screen on a high-resolution quadrupole time of flight mass spectrometer. Here we report the detailed procedure that we used to determine appropriate positivity criteria for our screening procedure. Our approach was empirical; we collected data and analyzed it with commonly available software. We found that a combined scoring approach using a threshold of 70, with 70% weight given to library match and 10% weight given to each of mass error, retention time error and isotope pattern difference provided optimum drug identification efficiency of 99.2%. Our results demonstrate the importance of library matching in accurately identifying compounds, and underscore the utility of robust product ion spectra that contain information on the lineage, mass and relative abundance of fragments. The method we describe is easily adaptable to include alternative parameters that may be available in software associated with a variety of HRMS platforms. With careful selection of error limits and positivity criteria, HRMS instruments are capable of producing high-quality, high-confidence results that may reduce the need for confirmatory testing.
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http://dx.doi.org/10.1093/jat/bkw112DOI Listing
January 2017

Comparison of Information-Dependent Acquisition on a Tandem Quadrupole TOF vs a Triple Quadrupole Linear Ion Trap Mass Spectrometer for Broad-Spectrum Drug Screening.

Clin Chem 2016 Jan 9;62(1):170-8. Epub 2015 Oct 9.

Department of Laboratory Medicine, University of California-San Francisco, San Francisco, CA;

Background: Liquid chromatography high-resolution mass spectrometry (LC-HRMS) with untargeted data collection is especially attractive for general unknown drug screening owing to its ability to identify unexpected compounds. LC-HRMS offers several advantages over traditional selected reaction monitoring (SRM) techniques and could be an ideal screening platform as long as its analytical performance is comparable to that of SRM-based methods.

Methods: We developed a broad-spectrum drug screen on a high-resolution mass spectrometer [tandem quadrupole time-of-flight (QqTOF)] that collected data in an untargeted manner and compared its performance to a nominal mass instrument [triple quadrupole linear ion trap (QqLIT)] that collected data in a targeted manner. Both methods used information-dependent acquisition of product ion spectra. We evaluated the lower limits of detection and matrix effects for each method and compared their ability to identify drugs in 100 routine clinical urine samples. Additional information (patient prescription history, drug screening results, etc.) was used to confirm discordant results.

Results: QqLIT was slightly more analytically sensitive than QqTOF; however, this difference did not significantly affect compound identification in patient samples. QqLIT identified 596 drugs in the urine samples, of which 531 (89%) were confirmed. QqTOF identified 515 drugs, of which 500 (97%) were confirmed. There were 562 instances of a confirmed drug (68 unique drugs) in the 100 urine samples; the methods were concordant in 469 of these instances.

Conclusions: Overall, QqTOF performed similarly to QqLIT and could serve as an alternative method for general unknown screening.
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http://dx.doi.org/10.1373/clinchem.2015.241315DOI Listing
January 2016

Role of the α Clamp in the Protein Translocation Mechanism of Anthrax Toxin.

J Mol Biol 2015 Oct 5;427(20):3340-3349. Epub 2015 Sep 5.

Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA; Department of Chemistry, University of California, Berkeley, CA 94720, USA; Department of Microbial Pathogenesis, School of Dentistry, University of Maryland, Baltimore, 650 West Baltimore Street, Baltimore, MD 21201, USA. Electronic address:

Membrane-embedded molecular machines are utilized to move water-soluble proteins across these barriers. Anthrax toxin forms one such machine through the self-assembly of its three component proteins--protective antigen (PA), lethal factor, and edema factor. Upon endocytosis into host cells, acidification of the endosome induces PA to form a membrane-inserted channel, which unfolds lethal factor and edema factor and translocates them into the host cytosol. Translocation is driven by the proton motive force, composed of the chemical potential, the proton gradient (ΔpH), and the membrane potential (Δψ). A crystal structure of the lethal toxin core complex revealed an "α clamp" structure that binds to substrate helices nonspecifically. Here, we test the hypothesis that, through the recognition of unfolding helical structure, the α clamp can accelerate the rate of translocation. We produced a synthetic PA mutant in which an α helix was crosslinked into the α clamp to block its function. This synthetic construct impairs translocation by raising a yet uncharacterized translocation barrier shown to be much less force dependent than the known unfolding barrier. We also report that the α clamp more stably binds substrates that can form helices than those, such as polyproline, that cannot. Hence, the α clamp recognizes substrates by a general shape-complementarity mechanism. Substrates that are incapable of forming compact secondary structure (due to the introduction of a polyproline track) are severely deficient for translocation. Therefore, the α clamp and its recognition of helical structure in the translocating substrate play key roles in the molecular mechanism of protein translocation.
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http://dx.doi.org/10.1016/j.jmb.2015.08.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5125381PMC
October 2015

Hallucinogens causing seizures? A case report of the synthetic amphetamine 2,5-dimethoxy-4-chloroamphetamine.

Neurohospitalist 2015 Jan;5(1):32-4

Department of Neurology, University of California-San Francisco, San Francisco, CA, USA.

Although traditional hallucinogenic drugs such as marijuana and lysergic acid diethylamide (LSD) are not typically associated with seizures, newer synthetic hallucinogenic drugs can provoke seizures. Here, we report the unexpected consequences of taking a street-bought hallucinogenic drug thought to be LSD. Our patient presented with hallucinations and agitation progressing to status epilepticus with a urine toxicology screen positive only for cannabinoids and opioids. Using liquid chromatography high-resolution mass spectrometry, an additional drug was found: an amphetamine-derived phenylethylamine called 2,5-dimethoxy-4-chloroamphetamine. We bring this to the attention of the neurologic community as there are a growing number of hallucinogenic street drugs that are negative on standard urine toxicology and cause effects that are unexpected for both the patient and the neurologist, including seizures.
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http://dx.doi.org/10.1177/1941874414528939DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4272348PMC
January 2015

Biological variation of the osmolality and the osmolal gap.

Clin Biochem 2014 Oct 15;47(15):130-1. Epub 2014 Aug 15.

Department of Laboratory Medicine, University of California, San Francisco, CA, USA.

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http://dx.doi.org/10.1016/j.clinbiochem.2014.08.007DOI Listing
October 2014

Domain flexibility modulates the heterogeneous assembly mechanism of anthrax toxin protective antigen.

J Mol Biol 2012 Jan 31;415(1):159-74. Epub 2011 Oct 31.

Department of Chemistry, University of California, Berkeley, CA 94720, USA.

The three protein components of anthrax toxin are nontoxic individually, but they form active holotoxin complexes upon assembly. The role of the protective antigen (PA) component of the toxin is to deliver two other enzyme components, lethal factor and edema factor, across the plasma membrane and into the cytoplasm of target cells. PA is produced as a proprotein, which must be proteolytically activated; generally, cell surface activation is mediated by a furin family protease. Activated PA can then assemble into one of two noninterconverting oligomers, a homoheptamer and a homooctamer, which have unique properties. Herein we describe molecular determinants that influence the stoichiometry of PA in toxin complexes. By tethering PA domain 4 (D4) to domain 2 with two different-length cross-links, we can control the relative proportions of PA heptamers and octamers. The longer cross-link favors octamer formation, whereas the shorter one favors formation of the heptamer. X-ray crystal structures of PA (up to 1.45 Å resolution), including these cross-linked PA constructs, reveal that a hinge-like movement of D4 correlates with the relative preference for each oligomeric architecture. Furthermore, we report the conformation of the flexible loop containing the furin cleavage site and show that, for efficient processing, the furin site cannot be moved ~5 or 6 residues within the loop. We propose that there are different orientations of D4 relative to the main body of PA that favor the formation of either the heptamer or the octamer.
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http://dx.doi.org/10.1016/j.jmb.2011.10.035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3249527PMC
January 2012

Charge requirements for proton gradient-driven translocation of anthrax toxin.

J Biol Chem 2011 Jul 20;286(26):23189-99. Epub 2011 Apr 20.

Department of Molecular & Cell Biology, University of California, Berkeley, California 94720, USA.

Anthrax lethal toxin is used as a model system to study protein translocation. The toxin is composed of a translocase channel, called protective antigen (PA), and an enzyme, called lethal factor (LF). A proton gradient (ΔpH) can drive LF unfolding and translocation through PA channels; however, the mechanism of ΔpH-mediated force generation, substrate unfolding, and establishment of directionality are poorly understood. One recent hypothesis suggests that the ΔpH may act through changes in the protonation state of residues in the substrate. Here we report the charge requirements of LF's amino-terminal binding domain (LF(N)) using planar lipid bilayer electrophysiology. We found that acidic residues are required in LF(N) to utilize a proton gradient for translocation. Constructs lacking negative charges in the unstructured presequence of LF(N) translocate independently of the ΔpH driving force. Acidic residues markedly increase the rate of ΔpH-driven translocation, and the presequence is optimized in its natural acidic residue content for efficient ΔpH-driven unfolding and translocation. We discuss a ΔpH-driven charge state Brownian ratchet mechanism for translocation, where glutamic and aspartic acid residues in the substrate are the "molecular teeth" of the ratchet. Our Brownian ratchet model includes a mechanism for unfolding and a novel role for positive charges, which we propose chaperone negative charges through the PA channel during ΔpH translocation.
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http://dx.doi.org/10.1074/jbc.M111.231167DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3123086PMC
July 2011

The unfolding story of anthrax toxin translocation.

Mol Microbiol 2011 May 28;80(3):588-95. Epub 2011 Mar 28.

Departments of Chemistry, University of California, Berkeley, CA 94720, USA.

The essential cellular functions of secretion and protein degradation require a molecular machine to unfold and translocate proteins either across a membrane or into a proteolytic complex. Protein translocation is also critical for microbial pathogenesis, namely bacteria can use translocase channels to deliver toxic proteins into a target cell. Anthrax toxin (Atx), a key virulence factor secreted by Bacillus anthracis, provides a robust biophysical model to characterize transmembrane protein translocation. Atx is comprised of three proteins: the translocase component, protective antigen (PA) and two enzyme components, lethal factor (LF) and oedema factor (OF). Atx forms an active holotoxin complex containing a ring-shaped PA oligomer bound to multiple copies of LF and OF. These complexes are endocytosed into mammalian host cells, where PA forms a protein-conducting translocase channel. The proton motive force unfolds and translocates LF and OF through the channel. Recent structure and function studies have shown that LF unfolds during translocation in a force-dependent manner via a series of metastable intermediates. Polypeptide-binding clamps located throughout the PA channel catalyse substrate unfolding and translocation by stabilizing unfolding intermediates through the formation of a series of interactions with various chemical groups and α-helical structure presented by the unfolding polypeptide during translocation.
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http://dx.doi.org/10.1111/j.1365-2958.2011.07614.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3094749PMC
May 2011

Structural basis for the unfolding of anthrax lethal factor by protective antigen oligomers.

Nat Struct Mol Biol 2010 Nov 31;17(11):1383-90. Epub 2010 Oct 31.

Department of Chemistry, University of California, Berkeley, California, USA.

The protein transporter anthrax lethal toxin is composed of protective antigen (PA), a transmembrane translocase, and lethal factor (LF), a cytotoxic enzyme. After its assembly into holotoxin complexes, PA forms an oligomeric channel that unfolds LF and translocates it into the host cell. We report the crystal structure of the core of a lethal toxin complex to 3.1-Å resolution; the structure contains a PA octamer bound to four LF PA-binding domains (LF(N)). The first α-helix and β-strand of each LF(N) unfold and dock into a deep amphipathic cleft on the surface of the PA octamer, which we call the α clamp. The α clamp possesses nonspecific polypeptide binding activity and is functionally relevant to efficient holotoxin assembly, PA octamer formation, and LF unfolding and translocation. This structure provides insight into the mechanism of translocation-coupled protein unfolding.
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http://dx.doi.org/10.1038/nsmb.1923DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3133606PMC
November 2010