Isatuximab-Specific Immunofixation Electrophoresis Assay to Remove Interference in Serum M-Protein Measurement in Patients with Multiple Myeloma.

BACKGROUND: Isatuximab, an IgG-kappa (IgGκ) anti-cluster of differentiation 38 (CD38) monoclonal antibody approved for use in patients with relapsed or refractory multiple myeloma (MM), can potentially interfere with the visualization of endogenous monoclonal protein (M-protein) on standard immunofixation electrophoresis (IFE) and lead to inaccurate classification of a patient's response to therapy. The Hydrashift 2/4 isatuximab IFE assay (Hydrashift isatuximab assay) removes isatuximab interference from IFE. Using samples from patients enrolled in clinical trials of isatuximab-based therapy for MM, we demonstrate how the Hydrashift isatuximab assay improves the ability to detect residual M-protein and offer recommendations for when the assay is most useful. METHODS: Samples from 141 patients with a variety of known M-protein isotypes were selected and analyzed by standard IFE and the Hydrashift isatuximab assay. A positive control containing isatuximab was run on every standard IFE and Hydrashift gel. RESULTS: The Hydrashift isatuximab assay reliably shifted the migration of isatuximab in patient samples. Standard IFE was adequate for determining 104 patients' M-protein status, and the Hydrashift isatuximab assay confirmed these results. In samples from 37 patients with a history of IgGκ MM and a single IgGκ band visible on standard IFE near the isatuximab migration site, the Hydrashift isatuximab assay was able to separate isatuximab from endogenous M-protein, identifying residual M-protein in 17 samples and preventing false-positive interpretations of standard IFE in 20 samples. CONCLUSIONS: The Hydrashift isatuximab assay is most useful in patients with known IgGκ MM when a single IgGκ band appears near the isatuximab migration site on standard IFE during isatuximab-based therapy. ClinicalTrials.gov Registration Numbers: NCT03275285 and NCT03319667.

Myeloma precursor disease (MGUS) among rescue and recovery workers exposed to the World Trade Center disaster.

An elevated risk of myeloma precursor disease, monoclonal gammopathy of undetermined significance (MGUS), was identified among Fire Department of the City of New York (FDNY) World Trade Center (WTC)-exposed firefighters. Further investigation was needed to determine if these findings were reproducible in a more heterogeneous WTC-exposed rescue/recovery workers cohort, the Stony Brook University-General Responder Cohort GRC (SBU-GRC). MGUS risk was compared between the cohorts and to published general population estimates from Olmsted County, MN, USA. In this observational seroprevalence study, odds ratios (OR) and age-standardized risk ratios (RR) of MGUS (M-spike and light-chain-MGUS combined), M-spike, and light-chain-MGUS were estimated using logistic regression. Age-standardized prevalences were calculated for white males aged 50-79; RRs were estimated by comparing risk in the WTC-exposed cohort with the Olmsted County screened cohort. SBU-GRC had elevated odds of MGUS compared with FDNY (OR = 1.38; 95%CI = 1.00-1.89). The age-standardized prevalence of MGUS was 9.0/100 persons (95%CI = 7.5-10.6), over two-fold higher than the general population (RR = 2.08; 95%CI = 1.72-2.51); the age-standardized prevalence of light-chain-MGUS was 3.5-fold higher (RR = 3.54; 95%CI = 2.52-4.97). This study adds to mounting evidence supporting an association between WTC/environmental exposures and MGUS among rescue/recovery workers. Access to MGUS screenings for the entire WTC-exposed cohort could allow for treatment interventions that improve survival.

Use of a Daratumumab-Specific Immunofixation Assay to Assess Possible Immunotherapy Interference at a Major Cancer Center: Our Experience and Recommendations.

BACKGROUND: The incorporation of monoclonal antibodies, such as daratumumab, into multiple myeloma treatment regimens has led to the issue of false-positive interference in both serum protein electrophoresis (SPEP) and immunofixation (IF). The Hydrashift assay removes daratumumab interference from IF, allowing for correct interpretation. Here, we retrospectively examined the use of the Hydrashift assay at a large cancer center and provide guidelines on its most appropriate use. METHODS: 38 patients with distinct daratumumab peaks on their SPEP were selected and were used to quantify the daratumumab peak on SPEP using the Sebia Phoresis software. A retrospective review of all Hydrashift assays ordered at our institution from July 2018 to March 2020 was performed. Data collected included patient clone type, IF migration patterns, and Hydrashift result. Serial quantification of SPEP results was performed as the corresponding IF transitioned from a true positive to a false positive. RESULTS: Daratumumab adds a maximum magnitude of 0.20 g/dL on SPEP. Serial SPEP quantification showed IF transitioned from true positive to false positive when M-spikes ranged from 0.09 g/dL to 0.11 g/dL. Over 20 months, our laboratory performed 280 Hydrashift assays on 96 patients, 43/96 of whom had comigrating daratumumab/IgG-K IF bands. CONCLUSIONS: The Hydrashift assay is typically unnecessary in patients with large M-spikes, >0.25 g/dL, regardless of clone type. When patient history is available, we recommend the Hydrashift assay be used in patients with comigrating daratumumab/IgG-K bands with M-spikes of <0.25 g/dL.

Minimal Residual Disease in Myeloma: Application for Clinical Care and New Drug Registration.

The development of novel agents has transformed the treatment paradigm for multiple myeloma, with minimal residual disease (MRD) negativity now achievable across the entire disease spectrum. Bone marrow-based technologies to assess MRD, including approaches using next-generation flow and next-generation sequencing, have provided real-time clinical tools for the sensitive detection and monitoring of MRD in patients with multiple myeloma. Complementary liquid biopsy-based assays are now quickly progressing with some, such as mass spectrometry methods, being very close to clinical use, while others utilizing nucleic acid-based technologies are still developing and will prove important to further our understanding of the biology of MRD. On the regulatory front, multiple retrospective individual patient and clinical trial level meta-analyses have already shown and will continue to assess the potential of MRD as a surrogate for patient outcome. Given all this progress, it is not surprising that a number of clinicians are now considering using MRD to inform real-world clinical care of patients across the spectrum from smoldering myeloma to relapsed refractory multiple myeloma, with each disease setting presenting key challenges and questions that will need to be addressed through clinical trials. The pace of advances in targeted and immune therapies in multiple myeloma is unprecedented, and novel MRD-driven biomarker strategies are essential to accelerate innovative clinical trials leading to regulatory approval of novel treatments and continued improvement in patient outcomes.

Immunotyping Provides Equivalent Results to Immunofixation in a Population with a High Prevalence of Monoclonal Gammopathies.

BACKGROUND: Serum immunofixation (IF) is a common laboratory test used to diagnose and monitor patients with monoclonal gammopathies. Similarly, immunotyping (IT) by capillary electrophoresis can confirm the presence of a monoclonal protein (M-protein) and determine its isotype. The goal of this study was to compare the ability of IT and IF to detect M-proteins. METHODS: IT and IF results for 1000 waste clinical serum samples were obtained. All results were interpreted blindly by reviewers who were experienced in each technique. Results were compared by band. Results were also compared to patient history to determine if the original clone was present. We determined the sensitivity of IT and IF alone and in combination with additional tests. Finally, we evaluated the impact of reviewer training on the sensitivity of IT. RESULTS: IT and IF were concordant in 721/773 (93%) samples with a history of an intact M-protein and in 143/172 (83%) samples with a history of a free light chain (FLC) M-protein. IF was significantly more sensitive than IT for the detection of FLC M-proteins (P < 0.0001). However, IF was not more sensitive than IT for detection of intact M-proteins (P = 0.1272) or when each test was combined with the FLC ratio or urine immunofixation (P = 0.2812 and P = 0.6171, respectively). Finally, after training, inexperienced reviewers improved their IT sensitivity by 19%. CONCLUSION: IT provides equivalent results to IF for the detection of monoclonal proteins. Training and experience are critical to the accurate interpretation of IT.

Dynamics of minimal residual disease in patients with multiple myeloma on continuous lenalidomide maintenance: a single-arm, single-centre, phase 2 trial.

Background Lenalidomide maintenance improves progression-free survival for patients with multiple myeloma, although its optimal duration is unknown. Clearance of minimal residual disease (MRD) in the bone marrow results in superior outcomes, although its attainment or sustainment does not alter clinical decision-making. Studies that have evaluated MRD serially are limited in length. We therefore aimed to evaluate longitudinal changes in MRD-status (dynamics) and their association with progression-free survival in patients with multiple myeloma. METHODS: In this single-centre, single-arm, phase 2 study, we enrolled patients aged 18 years and older from the Memorial Sloan Kettering Cancer Center (New York, NY, USA) who had newly diagnosed multiple myeloma following unrestricted frontline therapy and an Eastern Cooperative Oncology Group Performance Status of 2 or lower, including patients who started maintenance before study enrolment. All participants received lenalidomide maintenance at 10 mg for 21 days of 28-day cycles until progression or unacceptable toxic effects for up to 5 years on protocol. The primary endpoint was progression-free survival at 60 months per protocol and key secondary endpoints were MRD rates after completion of the 12th, 24th, and 36th cycle of maintenance and the association between progression-free survival and annual measurement of MRD status. MRD was assessed from first-pull bone marrow aspirates at baseline and annually by flow cytometry per International Myeloma Working Group criteria, (limit of detection of at least 1 × 10-5) up to a maximum of 5 years. Patients who completed at least four cycles of treatment were included in the analysis of the primary endpoint, and patients who had completed at least one dose of treatment on protocol were assessable for secondary endpoints. The study was registered at ClinicalTrials.gov, NCT02538198, and is now closed to accrual. FINDINGS: Between Sept 8, 2015, and Jan 25, 2019, 108 patients (100 evaluable for the primary endpoint) were enrolled. Median follow-up was 40·7 months (95% CI 38·7-45·0). At 60 months, progression-free survival was 64% (95% CI 52-79). Median progression-free survival was unreached (95% CI unreached-unreached). MRD dynamics were assessed using 340 MRD assessments done over 5 years for 103 evaluable patients. Patients who sustained MRD negativity for 2 years (n=34) had no recorded disease progression at median 19·8 months (95% CI 15·8-22·3) past the 2-year maintenance landmark. By contrast, patients who lost their MRD-negative responses (n=10) were more likely to progress than those with sustained MRD negativity (HR infinite; p<0·0001) and those with persistent MRD positivity (HR 5·88, 95% CI 1·18-33·33; p=0·015) at the 2-year landmark. Haematological and non-haematological serious adverse events occurred in 19 patients (18%). The most common adverse events of grade 3 or worse were decreased lymphocyte count in 48 (44%) patients and decreased neutrophil count in 47 (44%) patients. One death occurred on study due to sepsis and heart failure and was considered unrelated to the study drug. INTERPRETATION: Serial measurements of MRD allow for dynamic assessment of risk for disease progression. Early intervention should be investigated for patients with loss of MRD negativity. Sustained MRD positivity is not categorically an unfavourable outcome and might portend prolonged stability of low-level disease. FUNDING: Memorial Sloan Kettering and Celgene.

Safety and Effectiveness of Weekly Carfilzomib, Lenalidomide, Dexamethasone, and Daratumumab Combination Therapy for Patients With Newly Diagnosed Multiple Myeloma: The MANHATTAN Nonrandomized Clinical Trial.

IMPORTANCE: Recently, the benefit of adding daratumumab to the proteasome inhibitor-based, 3-drug combination of bortezomib, lenalidomide, and dexamethasone for patients with newly diagnosed multiple myeloma who underwent high-dose melphalan chemotherapy and autologous hemopoietic cell transplant was assessed. Here, we examine the addition of daratumumab to the second-generation proteasome inhibitor-based, 3-drug combination of carfilzomib, lenalidomide, and dexamethasone. OBJECTIVE: To assess the safety and effectiveness of carfilzomib-lenalidomide-dexamethasone-daratumumab combination therapy for patients with newly diagnosed multiple myeloma, in the absence of high-dose melphalan chemotherapy and autologous hemopoietic cell transplant. DESIGN, SETTING, AND PARTICIPANTS: Clinical and correlative pilot study at the Memorial Sloan Kettering Cancer Center in New York, New York. Patients with newly diagnosed multiple myeloma were enrolled between October 1, 2018, and November 15, 2019. The median follow-up from start of treatment was 20.3 months (95% CI, 19.2-21.9 months). INTERVENTIONS: Eight 28-day cycles with intravenous carfilzomib, 20/56 mg/m2 (days 1, 8, and 15); oral lenalidomide, 25 mg, (days 1-21); dexamethasone, 40 mg weekly, orally or intravenously (cycles 1-4), and 20 mg after cycle 4; and intravenous daratumumab, 16 mg/kg (days 1, 8, 15, and 22 [cycles 1-2]; days 1 and 15 [cycles 3-6]; and day 1 [cycles 7 and 8]). MAIN OUTCOMES AND MEASURES: The primary end point was the minimal residual disease (MRD) rate, in the absence of high-dose melphalan chemotherapy and autologous hemopoietic cell transplant. Secondary end points included determining safety and tolerability, evaluating rates of clinical response per the International Myeloma Working Group, and estimating progression-free survival (PFS) and overall survival (OS) rates. RESULTS: Forty-one evaluable patients were enrolled (median age, 59 years; range, 30-70 years); 25 (61%) were female, and 20 (49%) had high-risk multiple myeloma. The primary end point (MRD negativity in the bone marrow; 10-5 sensitivity) was achieved in 29 of 41 patients (71%; 95% CI, 54%-83%), and therefore the trial was deemed successful. Median time to MRD negativity was 6 cycles (range, 1-8 cycles). Secondary end points of the overall response rate and the very good partial response or complete response rate were 100% (41 of 41 patients) and 95% (39 of 41 patients), respectively. At 11 months of the median follow-up, the 1-year PFS rate and the OS rate were 98% (95% CI, 93%-100%) and 100%, respectively. Most common (≥2 patients) grade 3 or 4 adverse events were neutropenia (12 patients [27%]), rash (4 patients [9%]), lung infection (3 patients [7%]), and increased alanine aminotransferase level (2 patients [4%]). There were no deaths. CONCLUSIONS AND RELEVANCE: In this nonrandomized clinical trial, carfilzomib-lenalidomide-dexamethasone-daratumumab combination therapy was associated with high rates of MRD negativity in patients with newly diagnosed multiple myeloma and high rates of PFS.

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

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 10-6). 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.

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

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.

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

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 ≥50 years 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%; p<0.001). Recent use of permethrin was associated with MGUS [recent use vs. no recent use, OR=1.82 (95% CI: 1.06, 3.13)], especially among those who had also used it in the past [recent and past use vs. never use, OR=2.49 (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 [OR=2.42 (95% CI: 1.29, 4.54); ptrend=0.006]. 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.

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

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 (10-6). 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.

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

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 v 0.28 reads per kilobase per million mapped reads; q < 0.05; n = 117) and in the University of Washington/Fred Hutchinson Cancer Research Center cohort (9.2 v 0.99 fragments per kilobase of transcript per million mapped reads; P < .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 (P < .05). DKK1 hypomethylation was associated with increased DKK1 mRNA expression (Pearson r = -0.66; P < .0001) in a rapid autopsy cohort (n = 7). DKK1-high mCRPC biopsies are infiltrated with significantly higher numbers of quiescent natural killer (NK) cells (P < .005) and lower numbers of activated NK cells (P < .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).

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

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.

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.

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.

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

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.

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

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.

Evaluation of CisBio ELISA for Chromogranin A Measurement.

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.

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

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.

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

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.

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

CONTEXT: Challenging clinical scenario in which elevated ß-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.