Treatment of Chronic Lymphocytic Leukemia With Genetically Targeted Autologous T Cells: Case Report of an Unforeseen Adverse Event in a Phase I Clinical Trial (2010)

To the editor:Most patients with B-cell malignancies will die from their disease or are incurable. For this reason, innovative therapeutic approaches are direly needed. Patient T cells may be genetically modified to target antigens expressed on tumor cells through the expression of chimeric antigen receptors (CARs), which are antigen receptors designed to recognize cell surface antigens in a human leukocyte antigen–independent manner.1Sadelain M Rivière I Brentjens R Targeting tumours with genetically enhanced T lymphocytes.Nat Rev Cancer. 2003; 3: 35-45Crossref PubMed Scopus (396) Google Scholar CD19, which is expressed on most B-cell malignancies—including most non-Hodgkin's lymphomas, acute lymphoblastic leukemias, and chronic lymphocytic leukemias (CLLs)—is an attractive antigen for this approach.2Brentjens RJ Latouche JB Santos E Marti F Gong MC Lyddane C et al.Eradication of systemic B-cell tumors by genetically targeted human T lymphocytes co-stimulated by CD80 and interleukin-15.Nat Med. 2003; 9: 279-286Crossref PubMed Scopus (503) Google Scholar It is present on normal B-lineage cells from the early pre–B-cell stage until plasma cell differentiation. In a model of CD19+ acute lymphoblastic leukemia, we found that that a CAR termed 19-28z, which comprises the CD28 cytoplasmic domain in addition to that of the CD3 ζ-chain,3Maher J Brentjens R Gunset G Rivière I Sadelain M Human T-lymphocyte cytotoxicity and proliferation directed by a single chimeric TCRzeta /CD28 receptor.Nat Biotechnol. 2002; 20: 70-75Crossref PubMed Scopus (677) Google Scholar induced better responses than a ζ-chain-based receptor.4Brentjens RJ Santos E Nikhamin Y Yeh R Matsushita M La Perle K et al.Genetically targeted T cells eradicate systemic acute lymphoblastic leukemia xenografts.Clin Cancer Res. 2007; 13: 5426-5435Crossref PubMed Scopus (335) Google Scholar In preclinical in vitro studies, we demonstrated that human T cells that express CD19-specific CARs efficiently lyse human CD19+ tumor cell lines and that CLL patient–derived T cells effectively lyse autologous tumor cells.2Brentjens RJ Latouche JB Santos E Marti F Gong MC Lyddane C et al.Eradication of systemic B-cell tumors by genetically targeted human T lymphocytes co-stimulated by CD80 and interleukin-15.Nat Med. 2003; 9: 279-286Crossref PubMed Scopus (503) Google Scholar,4Brentjens RJ Santos E Nikhamin Y Yeh R Matsushita M La Perle K et al.Genetically targeted T cells eradicate systemic acute lymphoblastic leukemia xenografts.Clin Cancer Res. 2007; 13: 5426-5435Crossref PubMed Scopus (335) Google Scholar These results, and others,5Sadelain M Brentjens R Rivière I The promise and potential pitfalls of chimeric antigen receptors.Curr Opin Immunol. 2009; 21: 215-223Crossref PubMed Scopus (359) Google Scholar supported a phase I clinical trial treating refractory CLL patients with autologous T cells modified by retroviral gene transfer of the 19-28z CAR.We have thus far enrolled six patients in this clinical trial. The cohort of subjects treated with modified T cells alone at the first dose level of T cells tolerated therapy well without dose-limiting toxicities. However, the first subject (subject 4) enrolled in the second cohort of patients, in whom cyclophosphamide lymphodepleting chemotherapy was administered before infusion of the same T-cell dose, developed a syndrome of hypotension, dyspnea, and renal failure following T-cell infusion. Subject 4 died 4 days after administration of cyclophosphamide and modified T cells. Herein we describe the chronology of his treatment and report the findings of an extensive postmortem analysis.Clinical trial designSubject 4 was treated in a phase I clinical trial (IRB no. 06-138, NIH-RAC no. 0507-721, NCT00466531) designed to assess the safety of infusing autologous T cells modified to express the CD19-targeted CAR 19-28z in subjects with relapsed or purine analog–refractory CLL. For 2–3 days following T-cell infusion, the subjects are closely monitored for tumor lysis and unforeseen adverse events. If stable, subjects are discharged and subsequently closely followed in the outpatient clinic setting.This phase I clinical trial has a three-step design (Table 1). In the first step, subjects are treated with dose level 1 of modified T cells (1.2–3.0 × 107 CAR+ T cells/kg) without prior lymphodepleting chemotherapy. The subject of the current report was enrolled in cohort 1 of step 2 and treated with 1.5 g/m2 of cyclophosphamide followed 2 days later by infusion of modified T cells at dose level 1. The enrollment thus far is summarized in Table 1.Table 1Cyclophosphamide and T-cell doses in IRB protocol no. 06-138StepCyclophosphamideCAR + T-cell doseNo. of enrolled subjects101.2-3.0 × 107/kg321.5 g/m21.2-3.0 × 107/kg13.0 g/m21.2-3.0 × 107/kg03MTD0.4-1.0 × 108/kg0−11.5 g/m24.0-10 × 106/kg2CAR, chimeric antigen receptor; MTD, maximum-tolerated dose. Open table in a new tab Case reportSubject 4 was a 69-year-old man with refractory CLL who was enrolled in clinical trial IRB no. 06-138. At the time of enrollment, three previous subjects had been treated on this protocol without significant adverse events in the first planned cohort, receiving the lowest planned dose of modified T cells alone. Subject 4 was the first to receive lymphodepleting chemotherapy with cyclophosphamide (1.5 g/m2) followed 2 days later by infusion of modified T cells at the same dose tolerated earlier by the first three subjects enrolled in cohort 1 of this trial.Subject 4's CLL treatment history. Subject 4 was initially diagnosed with CLL 8 years before treatment on this protocol, when he was noted to have an elevated lymphocyte count on a routine complete blood count in the context of lymphadenopathy. The subject had a significant past medical history of myocardial infarction, coronary artery disease, hypertension, and chronic renal insufficiency. Two years after diagnosis, because of progressive symptomatic abdominal lymphadenopathy and a rapidly doubling peripheral blood lymphocyte count, he was treated per Memorial Sloan–Kettering Cancer Center (MSKCC) IRB protocol no. 98-080 with sequential fludarabine (25 mg/m2) daily for 5 days every 4 weeks for six cycles, followed by high-dose cyclophosphamide (3 g/m2) given once every 3 weeks for three cycles, followed by rituximab (375 mg/m2) given weekly for 8 weeks. He achieved a durable partial response. Five years later, he developed evidence of progressive disease as shown by increasing lymphadenopathy, increasing peripheral blood lymphocyte counts, and cytopenias. The subject was enrolled in MSKCC IRB protocol no. 05-077 and treated with six monthly cycles of combination therapy with pentostatin (4 mg/m2), cyclophosphamide (600 mg/m2), rituximab (375 mg/m2 given only on cycles 2–6), and mitoxantrone (10 mg/m2). The subject once more achieved a partial response. Two years later, the subject presented with a rapidly increasing peripheral blood lymphocyte count, worsening cytopenias, and increasing lymphadenopathy and he was enrolled in IRB protocol no. 06-138 (NIH-RAC no. 0507-721, NCT00466531). The subject was assessed and met all criteria for enrollment in this trial.Treatment course. For IRB protocol no. 06-138, the subject underwent a leukapheresis procedure, and the product was processed and frozen. Subsequently, T cells were activated and retrovirally transduced with the 19-28z retroviral vector as described.6Hollyman D Stefanski J Przybylowski M Bartido S Borquez-Ojeda O Taylor C et al.Manufacturing validation of biologically functional T cells targeted to CD19 antigen for autologous adoptive cell therapy.J Immunother. 2009; 32: 169-180Crossref PubMed Scopus (225) Google Scholar The subject was then admitted and, per protocol, received tumor lysis prophylaxis with hydration and allopurinol followed by cyclophosphamide (1.5 g/m2) infusion. He tolerated therapy well, and twice-daily serum electrolyte studies revealed no evidence of tumor lysis. On the day of T-cell infusion, the subject's tumor lysis laboratory results were unremarkable, with the exception of a mildly elevated phosphorus level (4.8 mg/dl). His creatinine was 1.3 mg/dl. The T-cell infusion was completed over 3 hours without complication. Twenty hours after modified T-cell infusion, the subject developed a fever, a transient finding observed in all three subjects treated on step 1 of this protocol; however, in contrast to previously treated subjects, subject 4's fever persisted and was associated with concomitant hypotension (Figure 1a). At the same time, the subject developed respiratory distress despite a negative chest X-ray. After bacterial blood cultures had been obtained, the patient was started on broad-spectrum antibiotics (piperacillin/tazobactam and ciprofloxacin) with pressor support. He was subsequently transferred to the intensive care unit. Laboratory studies obtained at 24 hours after T-cell infusion demonstrated an elevated creatinine concentration (2.2 mg/dl) and rising phosphorus (7.4 mg/dl), potassium (5.0 mEq/L), and uric acid (8.3 mg/dl) concentrations.The subject became anuric, consistent with acute renal failure. In the intensive care unit, his blood pressure responded to inotropic support, from which he was successfully and fully weaned over the course of the day. However, he remained anuric with increasing serum potassium and phosphorus concentrations (Figure 1b). By the early evening, he once more became hypotensive, and inotropic support was restarted. A worsening respiratory status led to intubation and mechanical ventilation. Supportive care was withdrawn shortly thereafter at the request of the subject's health-care proxy. The subject expired 44 hours after infusion of modified T cells. Laboratory studies just before his death demonstrated an increasing serum creatinine concentration at 3.7 mg/dl, as well as markedly elevated potassium and phosphorus concentrations at 9.1 mEq/L and 14.2 mg/dl, respectively (Figure 1b). The subject's peripheral blood lymphocytosis remained generally stable over time, beginning with initial chemotherapy and following modified T-cell infusion (data not shown).Postmortem pathology report. Both gross and histologic postmortem analyses of tissues from subject 4 revealed extensive CLL with diffuse bulky adenopathy, including a large abdominal tumor (2.5 kg) and associated increased lymphadenopathy. revealed diffuse CLL in including the and as well as the tissues were generally than to support a of tumor lysis syndrome as the of renal failure. of the and tissues no significant initial blood as well as all cultures obtained after were The cell product was at the time of infusion, a we after the of the adverse of serum in the protocol, serum were obtained from all subjects before and after stage of the treatment of serum revealed a significant in the concentrations of and following cyclophosphamide chemotherapy as with the serum obtained days earlier (Figure the cyclophosphamide chemotherapy in this in serum is by the time the and serum the of may have been to a prior that was subsequently by in the syndrome in this serum tumor and were before and after T-cell infusion (Figure concentrations in subject were obtained days before cyclophosphamide 2 hours before T-cell infusion and 4 and hours after T-cell infusion The is infusion. tumor serum were and in subjects and of Subject a 69-year-old patient with bulky was the first to receive T cells following prior lymphodepleting chemotherapy (IRB protocol no. 06-138, step cohort 1). In the first cohort in which three subjects were treated with the lowest planned modified T-cell dose all transient following T-cell infusion tolerated therapy These subjects had no evidence of hypotension, tumor or acute renal failure. In contrast to subjects treated in the first cohort, subject 4 developed following T-cell infusion, became hypotensive, and developed acute renal all consistent with a clinical of acute renal failure developed in the of clinical evidence of tumor lysis A later in serum and uric acid concentrations may be of a tumor lysis. of events thus that the patient developed renal failure to hypotension as a of a supported by the postmortem of renal consistent with an the subject's blood cultures as well as postmortem cultures to bacterial that the may have been by broad-spectrum revealed markedly elevated of the and and following cyclophosphamide chemotherapy and the T-cell infusion. The of this elevated because the serum was obtained days before chemotherapy. of the this to T-cell and may for the tumor lysis in subject 4 not in subjects this evidence with the and this despite negative blood is consistent with to to hypotension, to acute renal failure and, is consistent with as a and of and in patients with of clinical trial findings to the in subject 4 (IRB protocol no. to the modified T cells. because of the of the autologous T-cell infusion to this we infusion of modified T cells as a to this in the protocol, we the CAR+ T-cell dose in the cohort of patients to the dose × 107 modified T Table 1). a to patient we modified the protocol by T cells as a infusion, infusing of the dose on day 2 following cyclophosphamide therapy and, in the of evidence of tumor hypotension, or renal the of the planned T-cell dose on day 3 following cyclophosphamide the subject treated on this trial modified not evidence of a following cyclophosphamide chemotherapy and tolerated infusion of modified T cells without toxicities. will to on analyses of serum studies before and following cyclophosphamide chemotherapy and modified T-cell infusion, with to the and to infusion the of this we have treated a subject at the treatment dose Table 1). subject a transient 24 hours after T-cell infusion that responded to increased by a transient rapidly with no evidence of or renal serum analyses were To the patients with B-cell malignancies will die from their disease or are incurable. For this reason, innovative therapeutic approaches are direly needed. Patient T cells may be genetically modified to target antigens expressed on tumor cells through the expression of chimeric antigen receptors (CARs), which are antigen receptors designed to recognize cell surface antigens in a human leukocyte antigen–independent manner.1Sadelain M Rivière I Brentjens R Targeting tumours with genetically enhanced T lymphocytes.Nat Rev Cancer. 2003; 3: 35-45Crossref PubMed Scopus (396) Google Scholar CD19, which is expressed on most B-cell malignancies—including most non-Hodgkin's lymphomas, acute lymphoblastic leukemias, and chronic lymphocytic leukemias (CLLs)—is an attractive antigen for this approach.2Brentjens RJ Latouche JB Santos E Marti F Gong MC Lyddane C et al.Eradication of systemic B-cell tumors by genetically targeted human T lymphocytes co-stimulated by CD80 and interleukin-15.Nat Med. 2003; 9: 279-286Crossref PubMed Scopus (503) Google Scholar It is present on normal B-lineage cells from the early pre–B-cell stage until plasma cell differentiation. In a model of CD19+ acute lymphoblastic leukemia, we found that that a CAR termed 19-28z, which comprises the CD28 cytoplasmic domain in addition to that of the CD3 ζ-chain,3Maher J Brentjens R Gunset G Rivière I Sadelain M Human T-lymphocyte cytotoxicity and proliferation directed by a single chimeric TCRzeta /CD28 receptor.Nat Biotechnol. 2002; 20: 70-75Crossref PubMed Scopus (677) Google Scholar induced better responses than a ζ-chain-based receptor.4Brentjens RJ Santos E Nikhamin Y Yeh R Matsushita M La Perle K et al.Genetically targeted T cells eradicate systemic acute lymphoblastic leukemia xenografts.Clin Cancer Res. 2007; 13: 5426-5435Crossref PubMed Scopus (335) Google Scholar In preclinical in vitro studies, we demonstrated that human T cells that express CD19-specific CARs efficiently lyse human CD19+ tumor cell lines and that CLL patient–derived T cells effectively lyse autologous tumor cells.2Brentjens RJ Latouche JB Santos E Marti F Gong MC Lyddane C et al.Eradication of systemic B-cell tumors by genetically targeted human T lymphocytes co-stimulated by CD80 and interleukin-15.Nat Med. 2003; 9: 279-286Crossref PubMed Scopus (503) Google Scholar,4Brentjens RJ Santos E Nikhamin Y Yeh R Matsushita M La Perle K et al.Genetically targeted T cells eradicate systemic acute lymphoblastic leukemia xenografts.Clin Cancer Res. 2007; 13: 5426-5435Crossref PubMed Scopus (335) Google Scholar These results, and others,5Sadelain M Brentjens R Rivière I The promise and potential pitfalls of chimeric antigen receptors.Curr Opin Immunol. 2009; 21: 215-223Crossref PubMed Scopus (359) Google Scholar supported a phase I clinical trial treating refractory CLL patients with autologous T cells modified by retroviral gene transfer of the 19-28z have thus far enrolled six patients in this clinical trial. The cohort of subjects treated with modified T cells alone at the first dose level of T cells tolerated therapy well without dose-limiting toxicities. However, the first subject (subject 4) enrolled in the second cohort of patients, in whom cyclophosphamide lymphodepleting chemotherapy was administered before infusion of the same T-cell dose, developed a syndrome of hypotension, dyspnea, and renal failure following T-cell infusion. Subject 4 died 4 days after administration of cyclophosphamide and modified T cells. Herein we describe the chronology of his treatment and report the findings of an extensive postmortem trial design Subject 4 was treated in a phase I clinical trial (IRB no. 06-138, NIH-RAC no. 0507-721, NCT00466531) designed to assess the safety of infusing autologous T cells modified to express the CD19-targeted CAR 19-28z in subjects with relapsed or purine analog–refractory CLL. For 2–3 days following T-cell infusion, the subjects are closely monitored for tumor lysis and unforeseen adverse events. If stable, subjects are discharged and subsequently closely followed in the outpatient clinic phase I clinical trial has a three-step design (Table 1). In the first step, subjects are treated with dose level 1 of modified T cells (1.2–3.0 × 107 CAR+ T cells/kg) without prior lymphodepleting chemotherapy. The subject of the current report was enrolled in cohort 1 of step 2 and treated with 1.5 g/m2 of cyclophosphamide followed 2 days later by infusion of modified T cells at dose level 1. The enrollment thus far is summarized in Table 1. chimeric antigen receptor; MTD, maximum-tolerated dose. Case report Subject 4 was a 69-year-old man with refractory CLL who was enrolled in clinical trial IRB no. 06-138. At the time of enrollment, three previous subjects had been treated on this protocol without significant adverse events in the first planned cohort, receiving the lowest planned dose of modified T cells alone. Subject 4 was the first to receive lymphodepleting chemotherapy with cyclophosphamide (1.5 g/m2) followed 2 days later by infusion of modified T cells at the same dose tolerated earlier by the first three subjects enrolled in cohort 1 of this trial. Subject 4's CLL treatment history. Subject 4 was initially diagnosed with CLL 8 years before treatment on this protocol, when he was noted to have an elevated lymphocyte count on a routine complete blood count in the context of lymphadenopathy. The subject had a significant past medical history of myocardial infarction, coronary artery disease, hypertension, and chronic renal insufficiency. Two years after diagnosis, because of progressive symptomatic abdominal lymphadenopathy and a rapidly doubling peripheral blood lymphocyte count, he was treated per Memorial Sloan–Kettering Cancer Center (MSKCC) IRB protocol no. 98-080 with sequential fludarabine (25 mg/m2) daily for 5 days every 4 weeks for six cycles, followed by high-dose cyclophosphamide (3 g/m2) given once every 3 weeks for three cycles, followed by rituximab (375 mg/m2) given weekly for 8 weeks. He achieved a durable partial response. Five years later, he developed evidence of progressive disease as shown by increasing lymphadenopathy, increasing peripheral blood lymphocyte counts, and cytopenias. The subject was enrolled in MSKCC IRB protocol no. 05-077 and treated with six monthly cycles of combination therapy with pentostatin (4 mg/m2), cyclophosphamide (600 mg/m2), rituximab (375 mg/m2 given only on cycles 2–6), and mitoxantrone (10 mg/m2). The subject once more achieved a partial response. Two years later, the subject presented with a rapidly increasing peripheral blood lymphocyte count, worsening cytopenias, and increasing lymphadenopathy and he was enrolled in IRB protocol no. 06-138 (NIH-RAC no. 0507-721, NCT00466531). The subject was assessed and met all criteria for enrollment in this trial. course. For IRB protocol no. 06-138, the subject underwent a leukapheresis procedure, and the product was processed and frozen. Subsequently, T cells were activated and retrovirally transduced with the 19-28z retroviral vector as described.6Hollyman D Stefanski J Przybylowski M Bartido S Borquez-Ojeda O Taylor C et al.Manufacturing validation of biologically functional T cells targeted to CD19 antigen for autologous adoptive cell therapy.J Immunother. 2009; 32: 169-180Crossref PubMed Scopus (225) Google Scholar The subject was then admitted and, per protocol, received tumor lysis prophylaxis with hydration and allopurinol followed by cyclophosphamide (1.5 g/m2) infusion. He tolerated therapy well, and twice-daily serum electrolyte studies revealed no evidence of tumor lysis. On the day of T-cell infusion, the subject's tumor lysis laboratory results were unremarkable, with the exception of a mildly elevated phosphorus level (4.8 mg/dl). His creatinine was 1.3 mg/dl. The T-cell infusion was completed over 3 hours without complication. Twenty hours after modified T-cell infusion, the subject developed a fever, a transient finding observed in all three subjects treated on step 1 of this protocol; however, in contrast to previously treated subjects, subject 4's fever persisted and was associated with concomitant hypotension (Figure 1a). At the same time, the subject developed respiratory distress despite a negative chest X-ray. After bacterial blood cultures had been obtained, the patient was started on broad-spectrum antibiotics (piperacillin/tazobactam and ciprofloxacin) with pressor support. He was subsequently transferred to the intensive care unit. Laboratory studies obtained at 24 hours after T-cell infusion demonstrated an elevated creatinine concentration (2.2 mg/dl) and rising phosphorus (7.4 mg/dl), potassium (5.0 mEq/L), and uric acid (8.3 mg/dl) The subject became anuric, consistent with acute renal failure. In the intensive care unit, his blood pressure responded to inotropic support, from which he was successfully and fully weaned over the course of the day. However, he remained anuric with increasing serum potassium and phosphorus concentrations (Figure 1b). By the early evening, he once more became hypotensive, and inotropic support was restarted. A worsening respiratory status led to intubation and mechanical ventilation. Supportive care was withdrawn shortly thereafter at the request of the subject's health-care proxy. The subject expired 44 hours after infusion of modified T cells. Laboratory studies just before his death demonstrated an increasing serum creatinine concentration at 3.7 mg/dl, as well as markedly elevated potassium and phosphorus concentrations at 9.1 mEq/L and 14.2 mg/dl, respectively (Figure 1b). The subject's peripheral blood lymphocytosis remained generally stable over time, beginning with initial chemotherapy and following modified T-cell infusion (data not pathology report. Both gross and histologic postmortem analyses of tissues from subject 4 revealed extensive CLL with diffuse bulky adenopathy, including a large abdominal tumor (2.5 kg) and associated increased lymphadenopathy. revealed diffuse CLL in including the and as well as the tissues were generally than to support a of tumor lysis syndrome as the of renal failure. of the and tissues no significant initial blood as well as all cultures obtained after were The cell product was at the time of infusion, a we after the of the adverse of serum in the protocol, serum were obtained from all subjects before and after stage of the treatment of serum revealed a significant in the concentrations of and following cyclophosphamide chemotherapy as with the serum obtained days earlier (Figure the cyclophosphamide chemotherapy in this in serum is by the time the and serum the of may have been to a prior that was subsequently by in the syndrome in this serum tumor and were before and after T-cell infusion (Figure of Subject a 69-year-old patient with bulky was the first to receive T cells following prior lymphodepleting chemotherapy (IRB protocol no. 06-138, step cohort 1). In the first cohort in which three subjects were treated with the lowest planned modified T-cell dose all transient following T-cell infusion tolerated therapy These subjects had no evidence of hypotension, tumor or acute renal failure. In contrast to subjects treated in the first cohort, subject 4 developed following T-cell infusion, became hypotensive, and developed acute renal all consistent with a clinical of acute renal failure developed in the of clinical evidence of tumor lysis A later in serum and uric acid concentrations may be of a tumor lysis. of events thus that the patient developed renal failure to hypotension as a of a supported by the postmortem of renal consistent with an the subject's blood cultures as well as postmortem cultures to bacterial that the may have been by broad-spectrum revealed markedly elevated of the and and following cyclophosphamide chemotherapy and the T-cell infusion. The of this elevated because the serum was obtained days before chemotherapy. of the this to T-cell and may for the tumor lysis in subject 4 not in subjects this evidence with the and this despite negative blood is consistent with to to hypotension, to acute renal failure and, is consistent with as a and of and in patients with CLL. of clinical trial findings to the in subject 4 (IRB protocol no. to the modified T cells. because of the of the autologous T-cell infusion to this we infusion of modified T cells as a to this in the protocol, we the CAR+ T-cell dose in the cohort of patients to the dose × 107 modified T Table 1). a to patient we modified the protocol by T cells as a infusion, infusing of the dose on day 2 following cyclophosphamide therapy and, in the of evidence of tumor hypotension, or renal the of the planned T-cell dose on day 3 following cyclophosphamide the subject treated on this trial modified not evidence of a following cyclophosphamide chemotherapy and tolerated infusion of modified T cells without toxicities. will to on analyses of serum studies before and following cyclophosphamide chemotherapy and modified T-cell infusion, with to the and to infusion the of this we have treated a subject at the treatment dose Table 1). subject a transient 24 hours after T-cell infusion that responded to increased by a transient rapidly with no evidence of or renal serum analyses were