Toward a real liquid biopsy in metastatic breast and prostate cancer: Diagnostic LeukApheresis increases CTC yields in a European prospective multicenter study (CTCTrap).

Frequently, the number of circulating tumor cells (CTC) isolated in 7.5 mL of blood is too small to reliably determine tumor heterogeneity and to be representative as a "liquid biopsy". In the EU FP7 program CTCTrap, we aimed to validate and optimize the recently introduced Diagnostic LeukApheresis (DLA) to screen liters of blood. Here we present the results obtained from 34 metastatic cancer patients subjected to DLA in the participating institutions. About 7.5 mL blood processed with CellSearch® was used as "gold standard" reference. DLAs were obtained from 22 metastatic prostate and 12 metastatic breast cancer patients at four different institutions without any noticeable side effects. DLA samples were prepared and processed with different analysis techniques. Processing DLA using CellSearch resulted in a 0-32 fold increase in CTC yield compared to processing 7.5 mL blood. Filtration of DLA through 5 µm pores microsieves was accompanied by large CTC losses. Leukocyte depletion of 18 mL followed by CellSearch yielded an increase of the number of CTC but a relative decrease in yield (37%) versus CellSearch DLA. In four out of seven patients with 0 CTC detected in 7.5 mL of blood, CTC were detected in DLA (range 1-4 CTC). The CTC obtained through DLA enables molecular characterization of the tumor. CTC enrichment technologies however still need to be improved to isolate all the CTC present in the DLA.

Activity of nonmuscle myosin II isoforms determines localization at the cleavage furrow of megakaryocytes.

Megakaryocyte polyploidy is characterized by cytokinesis failure resulting from defects in contractile forces at the cleavage furrow. Although immature megakaryocytes express 2 nonmuscle myosin II isoforms (MYH9 [NMIIA] and MYH10 [NMIIB]), only NMIIB localizes at the cleavage furrow, and its subsequent absence contributes to polyploidy. In this study, we tried to understand why the abundant NMIIA does not localize at the furrow by focusing on the RhoA/ROCK pathway that has a low activity in polyploid megakaryocytes. We observed that under low RhoA activity, NMII isoforms presented different activity that determined their localization. Inhibition of RhoA/ROCK signaling abolished the localization of NMIIB, whereas constitutively active RhoA induced NMIIA at the cleavage furrow. Thus, although high RhoA activity favored the localization of both the isoforms, only NMIIB could localize at the furrow at low RhoA activity. This was further confirmed in erythroblasts that have a higher basal RhoA activity than megakaryocytes and express both NMIIA and NMIIB at the cleavage furrow. Decreased RhoA activity in erythroblasts abolished localization of NMIIA but not of NMIIB from the furrow. This differential localization was related to differences in actin turnover. Megakaryocytes had a higher actin turnover compared with erythroblasts. Strikingly, inhibition of actin polymerization was found to be sufficient to recapitulate the effects of inhibition of RhoA/ROCK pathway on NMII isoform localization; thus, cytokinesis failure in megakaryocytes is the consequence of both the absence of NMIIB and a low RhoA activity that impairs NMIIA localization at the cleavage furrow through increased actin turnover.

Uncoupling of the Hippo and Rho pathways allows megakaryocytes to escape the tetraploid checkpoint.

Megakaryocytes are naturally polyploid cells that increase their ploidy by endomitosis. However, very little is known regarding the mechanism by which they escape the tetraploid checkpoint to become polyploid. Recently, it has been shown that the tetraploid checkpoint was regulated by the Hippo-p53 pathway in response to a downregulation of Rho activity. We therefore analyzed the role of Hippo-p53 pathway in the regulation of human megakaryocyte polyploidy. Our results revealed that Hippo-p53 signaling pathway proteins are present and are functional in megakaryocytes. Although this pathway responds to the genotoxic stress agent etoposide, it is not activated in tetraploid or polyploid megakaryocytes. Furthermore, Hippo pathway was observed to be uncoupled from Rho activity. Additionally, polyploid megakaryocytes showed increased expression of YAP target genes when compared to diploid and tetraploid megakaryocytes. Although p53 knockdown increased both modal ploidy and proplatelet formation in megakaryocytes, YAP knockdown caused no significant change in ploidy while moderately affecting proplatelet formation. Interestingly, YAP knockdown reduced the mitochondrial mass in polyploid megakaryocytes and decreased expression of PGC1α, an important mitochondrial biogenesis regulator. Thus, the Hippo pathway is functional in megakaryocytes, but is not induced by tetraploidy. Additionally, YAP regulates the mitochondrial mass in polyploid megakaryocytes.

Impact of rituximab on stem cell mobilization following ACVBP regimen in poor-risk patients with diffuse large B-cell lymphoma: results from a large cohort of patients.

BACKGROUND: The ACVBP regimen is an efficient induction regimen for poor-risk patients with diffuse large B-cell lymphoma (DLBCL) before consolidative autologous stem cell transplantation. Adjunction of the monoclonal anti-CD20 antibody rituximab (R-ACVBP) was recently found to be superior to ACVBP alone. This study assessed the impact of rituximab on stem cell mobilization in two similar consecutive groups of patients treated with ACVBP in two prospective, controlled trials. STUDY DESIGN AND METHODS: The first trial (LNH-98B-3) involved 137 patients treated with ACVBP alone. In the second trial (LNH-03-3B), 91 patients received an R-ACVBP regimen. Stem cell mobilization was performed after a course of (R)-ACVBP. RESULTS: The median peak numbers of blood CD34+ cell counts recorded before the first apheresis procedure in the ACVBP and R-ACVBP groups were 69×10(6) and 63×10(6) /L, respectively (p=0.55). The median numbers of CD34+ cells collected were 7.1×10(6) and 6.0×10(6) CD34+ cells/kg for the ACVBP and R-ACVBP groups, respectively (p=0.13). The median number of apheresis procedures required for gathering the minimum amount of CD34+ cells (2×10(6) /kg) was the same in the two groups. CONCLUSION: When compared with ACVBP alone, adjunction of rituximab does not impair stem cell mobilization.

Clinicians' perceptions of factors contributing to complexity and intensity of care of outpatients with traumatic brain injury.

PRIMARY OBJECTIVE: This study investigated clinicians' perceptions on factors linked to patient complexity in traumatic brain injury (TBI) outpatient rehabilitation. METHOD: Twelve clinicians from various disciplines, working in TBI outpatient programmes from three rehabilitation institutions in Montreal, Quebec, were recruited using convenience and snowball sampling. Data was collected through focus groups and individual interviews and thematic analysis was used to identify themes. MAIN OUTCOMES AND RESULTS: Participants identified complexity factors falling under the following themes: sequelae of TBI (cognitive/behavioural/psychological impacts), personal factors (personality traits, pre-medical state, lifestyle and age), patients' environment (architectural, social, language, cultural and financial) and therapeutic relationship (mismatch, misunderstanding and personality clashes). Clinicians also reported facilitators to optimal treatment delivery such as quality of services and working in an interdisciplinary team. Limited time, training and resources were identified as barriers to treatment. CONCLUSION: A substantial proportion of patients in outpatient TBI programmes seem to follow an atypical evolution and exhibit added complexity. In order to optimize quality of care, clinicians recommended increased community awareness about TBI, increased resources for rehabilitation clinicians and specialized services post-discharge. These findings are insightful for stakeholders; providing a basis for discussions on policy changes that can better meet this population's needs.

Donor Lymphocyte Infusions After Allogeneic Transplantation: A Single-Center Experience.

Allogeneic hematopoietic cell transplantation (AHCT) represents the only curative therapy for many hematological malignancies. The graft versus leukemia effect, driven by donor T cells, plays a major role in its curative potential. This effect is sometimes very evident when patients with acute myeloid leukemia and myelodysplasia relapse after AHCT and are treated with donor lymphocyte infusions (DLIs). We retrospectively reviewed the charts of 64 patients who received DLI between 2012 and 2017 in our center. The mean age of the patients was 59 years (range, 34-79). Fifty percent were male (n = 32). The mean follow-up time after AHCT was 50.17 months (range, 8-174). The indication for DLI were disease progression, mixed chimerism, minimal residual disease, and other etiologies in 43.8%, 40.7%, 14%, and 1.5% of patients, respectively. The most common diagnosis was acute leukemia, followed by multiple myeloma. Of all patients, 59.4% received a transplant from a related donor, 39% received a transplant from an unrelated donor, and 1.6% received a transplant from a haploidentical donor. Reduced-intensity conditioning AHCT was the most frequent regimen used (53%). DLI was given alone in 79.7% of patients. Prophylactic DLI was given at 30 days after transplantation in patients who received human leukocyte antigen (HLA)-matched related human stem cell transplantation (HSCT) or 45 to 60 days post-transplant in patients receiving haploidentical HSCT or HLA-matched unrelated HSCT. Patients were treated without graft versus host disease (GVHD) prophylaxis. The use of DLI after transplantation remains a feasible procedure with rates of response >60%. Moreover, DLIs are well tolerated with a GVHD rate <10% in our series. We can hypothesize that in our experience the efficacy of this strategy does not rely on the induction of GVHD.

Genetic characterization of a unique neuroendocrine transdifferentiation prostate circulating tumor cell-derived eXplant model.

Transformation of castration-resistant prostate cancer (CRPC) into an aggressive neuroendocrine disease (CRPC-NE) represents a major clinical challenge and experimental models are lacking. A CTC-derived eXplant (CDX) and a CDX-derived cell line are established using circulating tumor cells (CTCs) obtained by diagnostic leukapheresis from a CRPC patient resistant to enzalutamide. The CDX and the derived-cell line conserve 16% of primary tumor (PT) and 56% of CTC mutations, as well as 83% of PT copy-number aberrations including clonal TMPRSS2-ERG fusion and NKX3.1 loss. Both harbor an androgen receptor-null neuroendocrine phenotype, TP53, PTEN and RB1 loss. While PTEN and RB1 loss are acquired in CTCs, evolutionary analysis suggest that a PT subclone harboring TP53 loss is the driver of the metastatic event leading to the CDX. This CDX model provides insights on the sequential acquisition of key drivers of neuroendocrine transdifferentiation and offers a unique tool for effective drug screening in CRPC-NE management.

A retrospective, matched paired analysis comparing bendamustine containing BeEAM versus BEAM conditioning regimen: results from a single center experience.

The combination of carmustine, etoposide, aracytin, and melphalan(BEAM) conditioning regimen in autologous stem-cell transplantation (ASCT) is widely used in patients with relapsed/refractory non-Hodgkin lymphoma (NHL) and Hodgkin lymphoma. It is also an option in patients with very-high risk aggressive NHL in first complete remission (CR). Recently, a phase Ib-II feasibility study using bendamustine replacing carmustine (BCNU) was reported. We report herein a safety and efficacy analysis of bendamustine-EAM (BeEAM) with a control BEAM counterpart paired cohort (1/2). One hundred and two patients were analyzed. Overall survival (OS) and progression-free survival (PFS) were not reached and seemed to be comparable between both groups. However, grade III or greater diarrhea was significantly higher in BeEAM patients (44 vs. 15%, p = .002). The median number of days with fever >38 °C was significantly higher in BeEAM group (5.5 vs. 2, p < .001). This case-control study suggests that BeEAM followed by ASCT using bendamustine at 100 mg/m2/d is effective but has a different toxicity profile than the BEAM regimen.

MUB40 Binds to Lactoferrin and Stands as a Specific Neutrophil Marker.

Neutrophils represent the most abundant immune cells recruited to inflamed tissues. A lack of dedicated tools has hampered their detection and study. We show that a synthesized peptide, MUB40, binds to lactoferrin, the most abundant protein stored in neutrophil-specific and tertiary granules. Lactoferrin is specifically produced by neutrophils among other leukocytes, making MUB40 a specific neutrophil marker. Naive mammalian neutrophils (human, guinea pig, mouse, rabbit) were labeled by fluorescent MUB40 conjugates (-Cy5, Dylight405). A peptidase-resistant retro-inverso MUB40 (RI-MUB40) was synthesized and its lactoferrin-binding property validated. Neutrophil lactoferrin secretion during in vitro Shigella infection was assessed with RI-MUB40-Cy5 using live cell microscopy. Systemically administered RI-MUB40-Cy5 accumulated at sites of inflammation in a mouse arthritis inflammation model in vivo and showed usefulness as a potential tool for inflammation detection using non-invasive imaging. Improving neutrophil detection with the universal and specific MUB40 marker will aid the study of broad ranges of inflammatory diseases.

Engraftment of chronic myelomonocytic leukemia cells in immunocompromised mice supports disease dependency on cytokines.

Chronic myelomonocytic leukemia (CMML) is a clonal hematopoietic stem cell disorder that typically associates with mutations in epigenetic, splicing, and signaling genes. Genetically modified mouse models only partially recapitulate the disease phenotype, whereas xenotransplantation of CMML cells in immunocompromised mice has been rarely successful so far. Here, CMML CD34+ cells sorted from patient bone marrow (BM) or peripheral blood (PB) were injected intravenously into NSG (NOD/LtSz-scid IL2rγnull) mice and NSG mice engineered to express human granulo-monocyte colony-stimulating factor, stem cell factor, and interleukin-3 (NSGS mice). Fifteen out of 16 patient samples (94%) successfully engrafted into NSG or NSGS or both mouse strains. The expansion of human cells, predominant in the BM, was also observed in the spleen and the PB and was greatly enhanced in mice producing the 3 human cytokines. Gene mutations identified in engrafted cells were mostly similar to those identified in patient cells before injection. Successful secondary engraftment was obtained in NSGS mice in 3 out of 10 attempts. Thus, primary CMML leukemic cells expand much better in NSGS compared with NSG mice with limited efficacy of secondary transplant.

Critical role of the HDAC6-cortactin axis in human megakaryocyte maturation leading to a proplatelet-formation defect.

Thrombocytopenia is a major side effect of a new class of anticancer agents that target histone deacetylase (HDAC). Their mechanism is poorly understood. Here, we show that HDAC6 inhibition and genetic knockdown lead to a strong decrease in human proplatelet formation (PPF). Unexpectedly, HDAC6 inhibition-induced tubulin hyperacetylation has no effect on PPF. The PPF decrease induced by HDAC6 inhibition is related to cortactin (CTTN) hyperacetylation associated with actin disorganization inducing important changes in the distribution of megakaryocyte (MK) organelles. CTTN silencing in human MKs phenocopies HDAC6 inactivation and knockdown leads to a strong PPF defect. This is rescued by forced expression of a deacetylated CTTN mimetic. Unexpectedly, unlike human-derived MKs, HDAC6 and CTTN are shown to be dispensable for mouse PPF in vitro and platelet production in vivo. Our results highlight an unexpected function of HDAC6-CTTN axis as a positive regulator of human but not mouse MK maturation.

Dendritic cell-derived exosomes as maintenance immunotherapy after first line chemotherapy in NSCLC.

Dendritic cell-derived exosomes (Dex) are small extracellular vesicles secreted by viable dendritic cells. In the two phase-I trials that we conducted using the first generation of Dex (IFN-γ-free) in end-stage cancer, we reported that Dex exerted natural killer (NK) cell effector functions in patients. A second generation of Dex (IFN-γ-Dex) was manufactured with the aim of boosting NK and T cell immune responses. We carried out a phase II clinical trial testing the clinical benefit of IFN-γ-Dex loaded with MHC class I- and class II-restricted cancer antigens as maintenance immunotherapy after induction chemotherapy in patients bearing inoperable non-small cell lung cancer (NSCLC) without tumor progression. The primary endpoint was to observe at least 50% of patients with progression-free survival (PFS) at 4 mo after chemotherapy cessation. Twenty-two patients received IFN-γ-Dex. One patient exhibited a grade three hepatotoxicity. The median time to progression was 2.2 mo and median overall survival (OS) was 15 mo. Seven patients (32%) experienced stabilization of >4 mo. The primary endpoint was not reached. An increase in NKp30-dependent NK cell functions were evidenced in a fraction of these NSCLC patients presenting with defective NKp30 expression. Importantly, MHC class II expression levels of the final IFN-γ-Dex product correlated with expression levels of the NKp30 ligand BAG6 on Dex, and with NKp30-dependent NK functions, the latter being associated with longer progression-free survival. This phase II trial confirmed the capacity of Dex to boost the NK cell arm of antitumor immunity in patients with advanced NSCLC.

Platelet transfusion containing ABO-incompatible plasma and hepatic veno-occlusive disease after hematopoietic transplantation in young children.

BACKGROUND: Hepatic veno-occlusive disease is a major limiting factor of high-dose chemotherapy in children. The cells lining the hepatic vascular endothelium express blood group A and/or B antigens according to the patient's blood group. We designed a study evaluating the impact of platelet concentrates containing ABO-incompatible plasma transfused to young children with a high risk of hepatic veno-occlusive disease. METHODS: In all, 186 consecutive children (median age: 4 years, range: 0.75-17 years), treated with high-dose chemotherapy containing busulfan followed by hematopoietic stem cell transplantation for neuroblastoma (n=112) or brain tumor (n=74) between 1988 and 1998, were investigated. The main endpoint was the occurrence of hepatic veno-occlusive disease. Multivariate analysis was performed using a Cox's regression model with transfusion of platelet concentrates containing ABO-incompatible plasma as a time-dependent covariate. RESULTS: We found that 73 out of 186 (39%) children developed hepatic veno-occlusive disease after transplantation. Multivariate analysis demonstrated that two factors significantly increased the risk of hepatic veno-occlusive disease occurrence: transfusion of platelet concentrates containing ABO-incompatible plasma (P=0.003) and use of melphalan in the conditioning regimen (P=0.006). Conversely, the number of platelet concentrates transfusions per week, child's age, weight, sex, and use of cyclophosphamide in the conditioning regimen had no effect. CONCLUSIONS: Transfusion of platelet concentrates containing ABO-incompatible plasma increases the risk of hepatic veno-occlusive disease in young children treated with a busulfan-containing regimen. Binding of A and/or B antigens expressed on the surface of hepatic endothelial cells may promote this complication. Transfusion of platelet concentrates containing ABO-incompatible plasma should be avoided in these children.

Clinical impact of the NKp30/B7-H6 axis in high-risk neuroblastoma patients.

The immunosurveillance mechanisms governing high-risk neuroblastoma (HR-NB), a major pediatric malignancy, have been elusive. We identify a potential role for natural killer (NK) cells, in particular the interaction between the NK receptor NKp30 and its ligand, B7-H6, in the metastatic progression and survival of HR-NB after myeloablative multimodal chemotherapy and stem cell transplantation. NB cells expressing the NKp30 ligand B7-H6 stimulated NK cells in an NKp30-dependent manner. Serum concentration of soluble B7-H6 correlated with the down-regulation of NKp30, bone marrow metastases, and chemoresistance, and soluble B7-H6 contained in the serum of HR-NB patients inhibited NK cell functions in vitro. The expression of distinct NKp30 isoforms affecting the polarization of NK cell functions correlated with 10-year event-free survival in three independent cohorts of HR-NB in remission from metastases after induction chemotherapy (n = 196, P < 0.001), adding prognostic value to known risk factors such as N-Myc amplification and age >18 months. We conclude that the interaction between NKp30 and B7-H6 may contribute to the fate of NB patients and that both the expression of NKp30 isoforms on circulating NK cells and the concentration of soluble B7-H6 in the serum may be clinically useful as biomarkers for risk stratification.

Bacterial contamination of a cornea tissue bank: implications for the safety of graft engineering.

PURPOSE: To analyze the difficulties involved in managing an episode of bacterial contamination in a cornea bank. We describe (1) the circumstances of bacterial contamination discovery, (2) the methods used to investigate the outbreak, (3) the corrective measures adopted, and (4) the method introduced to improve the reaction capacity in case of bacterial contamination. METHODS: All the samples collected were cultured in an attempt to identify the environmental reservoir of the contaminated epidemic clone. Bacteria were identified by Gram stain, oxidase test, and biochemical characteristics. The clonality of the strains was assessed by pulsed-field gel electrophoresis. RESULTS: The bacterial contamination was confirmed for 28 corneas, and 70 additional corneas were discarded. The source of the contamination was identified 17 days after the beginning of the episode. It consisted of a clonal bacterial strain that was found in trypan blue, the dye, used to examine all the tissues. The contaminating bacterium was Burkholderia cepacia, a well-known nosocomial pathogen. A total of 169 grafted corneas had been checked with the contaminated reagent. No cases of post-graft infection were recorded. CONCLUSION: Trypan blue played a major role in this outbreak. The mode and chronology of contamination remain unresolved. This exceptional event emphasizes the risk of bacterial contamination in tissue/cell banks, the necessity to improve methods for its prevention, and procedures to limit its consequences.

Blood versus marrow hematopoietic allogeneic graft.

Allogeneic G-CSF-mobilized blood cell transplantation (BCT), an alternative to allogeneic bone marrow transplantation (BMT), is associated with enhanced engraftment and accelerated hematopoietic recovery. In addition, immune reconstitution and overall alloreactivity after BCT versus BMT differ significantly. Indeed, despite an increased number of donor T cells infused, the incidence of acute graft-versus-host disease (GvHD) after BCT appears to remain identical or lesser than after BMT. On the other hand, a higher risk of chronic GvHD has been reported after BCT. In a SFGM phase III trial, 101 patients with early leukemia and an HLA-matched sibling donor randomly received a BCT or BMT. BCT was associated with a higher number of infused CD34+ cells, accelerated platelet and neutrophil reconstitution, fewer platelet transfusions and similar acute GvHD incidence. However, chronic GvHD occurred more frequently after BCT. With a median follow-up of 20 months, relapse, survival and leukemia-free survival were not different. In the course of this study, immune parameters related to the graft as well as to early reconstitution were prospectively examined. T cells subsets, B cells, NK cells and monocytes numbers were significantly higher in BC grafts (versus BM). T cells in BC grafts were less activated than in BM grafts. Frequency of IFN-gamma, IL-2- and TNF-alpha-secreting cells and single-cell IFN-gamma production potential was reduced in BC graft. One month after BCT, blood T-cell counts were 3-fold higher than after BMT. Moreover, post-BCT T cells were less activated and counts correlated with the number of T cells infused with the graft, which was not the case after BMT. Several acute hemolysis episodes, resulting from anti-A and/or -B donor-derived Ab directed at Ag present on recipient red blood cells (minor ABO mismatch), have been described after BCT. Recipients indeed exhibited significantly increased anti-A and/or -B Ab titers after BCT, particularly in the setting of a "minor" ABO mismatch. Furthermore, the frequency of anti-HLA Ab early after BCT was significantly increased (despite the reduction in platelet transfusion requirements). The higher number of activated B cells and/or CD4 T cells and monocytes in a BCT graft and/or the higher number of circulating CD4 T- and B-cells after BCT could be associated with the enhanced alloAb production. G-CSF-induced TH2 cytokine profile of the T cells present in the graft could also be contributive. Recent studies have determined that BC grafts contained a higher number of type 2 dendritic cells (DC2), themselves associated with high frequencies of TH2 CD4+ cells. Since chronic GvHD is associated with the occurrence of Ab-mediated auto-immune-like syndromes, it is tempting to speculate that a higher incidence of chronic GvHD may result from these findings. In conclusion, BCT results in clinically relevant distinct hematopoietic and immune reconstitution patterns.

Updated technology to produce highly immunogenic dendritic cell-derived exosomes of clinical grade: a critical role of interferon-γ.

Dendritic cell-derived exosomes (Dex) are nanovesicles bearing major histocompatibility complexes promoting T-cell-dependent antitumor effects in mice. Two phase I clinical trials aimed at vaccinating cancer patients with peptide-pulsed Dex have shown the feasibility and safety of inoculating clinical-grade Dex, but have failed to show their immunizing capacity. These low immunogenic capacities have led us to develop second-generation Dex with enhanced immunostimulatory properties. Here, we show that interferon-γ is a key cytokine conditioning the dendritic cell to induce the expression of CD40, CD80, CD86, and CD54 on Dex, endowing them with direct and potent peptide-dependent CD8(+) T-cell-triggering potential in vitro and in vivo. In this study, we describe the clinical grade process to manufacture large-scale interferon-γ-Dex vaccines and their quality control parameters currently used in a phase II trial.

Results of cryopreserved parathyroid autografts: a retrospective multicenter study.

BACKGROUND: The functionality of cryopreserved parathyroid autotransplantation (CPAT) has been evaluated in few studies, mostly conducted by experienced single-institution centers that have reported different success rates ranging from 17% to 83%. In France, CPAT are rare and their functionality has never been evaluated. Moreover, French tissue banks are facing an accumulation of ungrafted samples. The aim of our work was to evaluate the implantation rate of cryopreserved parathyroid samples and the functionality of CPAT in a multicenter study. METHODS: Data from 9 French tissue banks were analyzed. CPAT functionality was defined as fully functional (normal parathyroid hormone [PTH] and calcium levels without treatment), partially functional (normal PTH levels but need for treatment to maintain normocalcemia), and nonfunctional (low PTH levels and need for treatment). For dialyzed patients, CPAT was considered nonfunctional if the PTH level in the nongrafted arm was less than 20 pg/mL, partially functional if the PTH level was between 20 and 50 pg/mL, and fully functional if the PTH level was between 50 and 300 pg/mL. RESULTS: The 9 centers had cryopreserved 1376 samples of parathyroid tissue and only 22 (1.6%) had been autografted in 20 patients (65% renal hyperparathyroidism, 20% multiple endocrine neoplasia type 1, 15% "other") by 12 different surgical teams. The median duration of storage was 11.1 months (range, 0.4-28.5). Only 2 autografts (10%) were fully functional, 2 (10%) were partially functional, and 17 (80%) were nonfunctional at 26 months median follow-up. CONCLUSION: The reimplantation rate is low, and the functionality of CPAT is less than those published by experienced centers. Logistical and technical problems occurring in less experienced centers are probably the main reasons for nonfunctioning implants. Considering the results of this study, we suggest that cryopreservation of parathyroid glands should be abandoned when not performed in very large experimented centers, that CPAT should be used only for patients with hyperplasic parathyroid tissue, and that tissue samples should be systematically destroyed when patients do not have hypoparathyroidism or after 1 year of storage.