Analysis of rare disruptive germline mutations in 2135 enriched BRCA-negative breast cancers excludes additional high-impact susceptibility genes.

BACKGROUND: Breast cancer has a significant heritable basis, of which ∼60% remains unexplained. Testing for BRCA1/BRCA2 offers useful discrimination of breast cancer risk within families, and identification of additional breast cancer susceptibility genes could offer clinical utility. PATIENTS AND METHODS: We included 2135 invasive breast cancer cases recruited via the Breast and Ovarian Cancer Susceptibility study, a retrospective UK study of familial breast cancer. ELIGIBILITY CRITERIA: female, BRCA-negative, white European ethnicity, and one of: (i) breast cancer family history, (ii) bilateral disease, (iii) young age of onset (<30 years), and (iv) concomitant ovarian cancer. We undertook exome sequencing of cases and carried out gene-level burden testing of rare damaging variants against those from 51 377 ethnicity-matched population controls from gnomAD. RESULTS: 159/2135 (7.4%) cases had a qualifying variant in an established breast cancer susceptibility gene, with minimal evidence of signal in other cancer susceptibility genes. Known breast cancer susceptibility genes PALB2, CHEK2, and ATM were the only genes to retain statistical significance after correcting for multiple testing. Due to the enrichment of hereditary cases in the series, we had good power (>80%) to detect a gene of BRCA1-like risk [odds ratio (OR) = 10.6] down to a population minor allele frequency of 4.6 × 10-5 (1 in 10 799, less than one-tenth that of BRCA1)and of PALB2-like risk (OR = 5.0) down to a population minor allele frequency of 2.8 × 10-4 (1 in 1779, less than half that of PALB2). Power was lower for identification of novel moderate penetrance genes (OR = 2-3) like CHEK2 and ATM. CONCLUSIONS: This is the largest case-control whole-exome analysis of enriched breast cancer published to date. Whilst additional breast cancer susceptibility genes likely exist, those of high penetrance are likely to be of very low mutational frequency. Contention exists regarding the clinical utility of such genes.

What is the value of simulated patient assessment in structured clinical examinations of undergraduate students, and how should it be used?

INTRODUCTION: The aim of this study was to explore correlations between clinical assessor and simulated patient (SP) scores drawn from summative Integrated Structured Clinical Examination (ISCE) and inform the best use of SP scores in future assessments. MATERIALS AND METHODS: This retrospective study explores summative clinical assessor and formative SP numeric scores drawn from summative ISCE assessments spanning three academic years (2017-18 to 2019-20). Analyses were carried out using R 3.5.1 (R Core Team, 2018), with the stats package. RESULTS: The sample consisted of 169 final-year BDS students across the three cohorts and included 95 females (56.2%) and 74 males (43.8%). Data from eight substations where SPs were included, were explored. Kendall's Tau, a non-parametric correlation, was used to investigate the relationships between the assessor and SP scores. Clinical assessor scores were out of a total of 20 points across various assessed domains within each substation. The formative SP assessment was out of 10 points with the same five affective domains related to communication included in each substation. Overall, the assessor and patient substation scores were not correlated (τ = 0.04, p = .272) indicating that communication skills alone, as assessed by patients, do not correlate with more holistic performance across other domains. There was significant positive correlation for two of the eight substations with the other substations showing very little correlation. CONCLUSION: This study shows that assessment of student performance by SPs does not show a correlation with examiner scores and may provide additional information relating to affective skills of students. Notwithstanding the limitations of this study, the findings underscore the need to investigate further the value of involvement of SPs in clinical assessments to explore if scores by SPs can be used to enhance the validity of assessments if used summatively.

Initiation of DNA synthesis in the prematurely condensed chromosomes of G1 cells.

The objective of this study was to investigate whether G1 cells could enter S phase after premature chromosome condensation resulting from fusion with mitotic cells. HeLa cell synchronized in early G1, mid-G1, late G1, and G2 and human diploid fibroblasts synchronized in G0 and G1 phases were separately fused by use of UV-inactivated Sendai virus with mitotic HeLa cells. After cell fusion and premature chromosome condensation, the fused cells were incubated in culture medium containing Colcemid (0.05 micrograms/ml) and [3H]thymidine ([3H]ThdR) (0.5 microCi/ml; sp act, 6.7 Ci/mM). At 0, 2, 4, and 6 h after fusion, cell samples were taken to determine the initation of DNA synthesis in the prematurely condensed chromosomes (PCC) on the basis of their morphology and labeling index. The results of this study indicate that PCC from G0, G1, and G2 cells reach the maximum degree of compaction or condensation at 2 h after PCC induction. In addition, the G1-PCC from normal and transformed cells initiated DNA synthesis, as indicated by their "pulverized" appearance and incorporation of [3H]ThdR. Further, the initiation of DNA synthesis in G1-PCC occurred significantly earlier than in the mononucleate G1 cells. Neither pulverization nor incorporation of label was observed in the PCC of G0 and G2 cells. These findings suggest that chromosome decondensation, although not controlling the timing of a cell's entry into S phase, is an important step for the initiation of DNA synthesis. These data also suggest that the entry of a S phase may be regulated by cell cycle phase-specific changes in the permeability of the nuclear envelope to the inducers of DNA synthesis present in the cytoplasm.

Identification of p130Cas as a mediator of focal adhesion kinase-promoted cell migration.

Previously we have demonstrated that focal adhesion kinase (FAK)-promoted migration on fibronectin (FN) by its overexpression in CHO cells is dependent on FAK autophosphorylation at Y397 and subsequent binding of Src to this site. In this report, we have examined the role of FAK association with Grb2 and p130(Cas), two downstream events of the FAK/Src complex that could mediate integrin-stimulated activation of extracellular signal-regulated kinases (Erks). We show that a Y925F FAK mutant was able to promote cell migration as efficiently as FAK and that the transfected FAK demonstrated no detectable association with Grb2 in CHO cells. In contrast, cells expressing a FAK P712/715A mutant demonstrated a level of migration comparable to that of control cells. This mutation did not affect FAK kinase activity, autophosphorylation, or Src association but did significantly reduce p130(Cas) association with FAK. Furthermore, FAK expression in CHO cells increased tyrosine phosphorylation of p130(Cas) and its subsequent binding to several SH2 domains, which depended on both the p130(Cas) binding site and the Src binding site. However, we did not detect increased activation of Erks in cells expressing FAK, and the MEK inhibitor PD98059 did not decrease FAK-promoted cell migration. Finally, we show that coexpression of p130(Cas) further increased cell migration on FN and coexpression of the p130(Cas) SH3 domain alone functioned as a dominant negative mutant and decreased cell migration. Together, these results demonstrate that p130(Cas), but not Grb2, is a mediator of FAK-promoted cell migration and suggest that FAK/ p130(Cas) complex targets downstream pathways other than Erks in mediating FAK-promoted cell migration.

Dissociation of FAK/p130(CAS)/c-Src complex during mitosis: role of mitosis-specific serine phosphorylation of FAK.

At mitosis, focal adhesions disassemble and the signal transduction from focal adhesions is inactivated. We have found that components of focal adhesions including focal adhesion kinase (FAK), paxillin, and p130(CAS) (CAS) are serine/threonine phosphorylated during mitosis when all three proteins are tyrosine dephosphorylated. Mitosis-specific phosphorylation continues past cytokinesis and is reversed during post-mitotic cell spreading. We have found two significant alterations in FAK-mediated signal transduction during mitosis. First, the association of FAK with CAS or c-Src is greatly inhibited, with levels decreasing to 16 and 13% of the interphase levels, respectively. Second, mitotic FAK shows decreased binding to a peptide mimicking the cytoplasmic domain of beta-integrin when compared with FAK of interphase cells. Mitosis-specific phosphorylation is responsible for the disruption of FAK/CAS binding because dephosphorylation of mitotic FAK in vitro by protein serine/threonine phosphatase 1 restores the ability of FAK to associate with CAS, though not with c-Src. These results suggest that mitosis-specific modification of FAK uncouples signal transduction pathways involving integrin, CAS, and c-Src, and may maintain FAK in an inactive state until post-mitotic spreading.

The protein kinase family: conserved features and deduced phylogeny of the catalytic domains.

In recent years, members of the protein kinase family have been discovered at an accelerated pace. Most were first described, not through the traditional biochemical approach of protein purification and enzyme assay, but as putative protein kinase amino acid sequences deduced from the nucleotide sequences of molecularly cloned genes or complementary DNAs. Phylogenetic mapping of the conserved protein kinase catalytic domains can serve as a useful first step in the functional characterization of these newly identified family members.

Tyrosine phosphorylation of focal adhesion kinase at sites in the catalytic domain regulates kinase activity: a role for Src family kinases.

Focal adhesion kinase (FAK) is a widely expressed nonreceptor protein-tyrosine kinase implicated in integrin-mediated signal transduction pathways and in the process of oncogenic transformation by v-Src. Elevation of FAK's phosphotyrosine content, following both cell adhesion to extracellular matrix substrata and cell transformation by Rous sarcoma virus, correlates directly with an increased kinase activity. To help elucidate the role of FAK phosphorylation in signal transduction events, we used a tryptic phosphopeptide mapping approach to identify tyrosine sites of phosphorylation responsive to both cell adhesion and Src transformation. We have identified four tyrosines, 397, 407, 576, and 577, which are phosphorylated in mouse BALB/3T3 fibroblasts in an adhesion-dependent manner. Tyrosine 397 has been previously recognized as the major site of FAK autophosphorylation. Phosphorylation of tyrosines 407, 576, and 577, which are previously unrecognized sites, is significantly elevated in the presence of c-Src in vitro and v-Src in vivo. Tyrosines 576 and 577 lie within catalytic subdomain VIII--a region recognized as a target for phosphorylation-mediated regulation of protein kinase activity. We found that maximal kinase activity of FAK immune complexes requires phosphorylation of both tyrosines 576 and 577. Our results indicate that phosphorylation of FAK by Src (or other Src family kinases) is an important step in the formation of an active signaling complex.

Induced focal adhesion kinase (FAK) expression in FAK-null cells enhances cell spreading and migration requiring both auto- and activation loop phosphorylation sites and inhibits adhesion-dependent tyrosine phosphorylation of Pyk2.

Focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase involved in integrin-mediated control of cell behavior. Following cell adhesion to components of the extracellular matrix, FAK becomes phosphorylated at multiple sites, including tyrosines 397, 576, and 577. Tyr-397 is an autophosphorylation site that promotes interaction with c-Src or Fyn. Tyr-576 and Tyr-577 lie in the putative activation loop of the kinase domain, and FAK catalytic activity may be elevated through phosphorylation of these residues by associated Src family kinase. Recent studies have implicated FAK as a positive regulator of cell spreading and migration. To further study the mechanism of adhesion-induced FAK activation and the possible role and signaling requirements for FAK in cell spreading and migration, we utilized the tetracycline repression system to achieve inducible expression of either wild-type FAK or phosphorylation site mutants in fibroblasts derived from FAK-null mouse embryos. Using these Tet-FAK cells, we demonstrated that both the FAK autophosphorylation and activation loop sites are critical for maximum adhesion-induced FAK activation and FAK-enhanced cell spreading and migration responses. Negative effects on cell spreading and migration, as well as decreased phosphorylation of the substrate p130(Cas), were observed upon induced expression of the FAK autophosphorylation site mutant. These negative effects appear to result from an inhibition of integrin-mediated signaling by the FAK-related kinase Pyk2/CAKbeta/RAFTK/CadTK.

Mechanisms of CAS substrate domain tyrosine phosphorylation by FAK and Src.

Tyrosine phosphorylation of CAS (Crk-associated substrate, p130(Cas)) has been implicated as a key signaling step in integrin control of normal cellular behaviors, including motility, proliferation, and survival. Aberrant CAS tyrosine phosphorylation may contribute to cell transformation by certain oncoproteins, including v-Crk and v-Src, and to tumor growth and metastasis. The CAS substrate domain (SD) contains 15 Tyr-X-X-Pro motifs, which are thought to represent the major tyrosine phosphorylation sites and to function by recruiting downstream signaling effectors, including c-Crk and Nck. CAS makes multiple interactions, direct and indirect, with the tyrosine kinases Src and focal adhesion kinase (FAK), and as a result of this complexity, several plausible models have been proposed for the mechanism of CAS-SD phosphorylation. The objective of this study was to provide experimental tests of these models in order to determine the most likely mechanism(s) of CAS-SD tyrosine phosphorylation by FAK and Src. In vitro kinase assays indicated that FAK has a very poor capacity to phosphorylate CAS-SD, relative to Src. However, FAK expression along with Src was found to be important for achieving high levels of CAS tyrosine phosphorylation in COS-7 cells, as well as recovery of CAS-associated Src activity toward the SD. Structure-functional studies for both FAK and CAS further indicated that FAK plays a major role in regulating CAS-SD phosphorylation by acting as a docking or scaffolding protein to recruit Src to phosphorylate CAS, while a secondary FAK-independent mechanism involves Src directly bound to the CAS Src-binding domain (SBD). Our results do not support models in which FAK either phosphorylates CAS-SD directly or phosphorylates CAS-SBD to promote Src binding to this site.

Reflections on learning and enhancing communication skills through community engagement: a student perspective.

Students in Peninsula School of Dentistry (PSD), Plymouth, undertake community engagement projects during the first two years of their undergraduate curriculum. These projects involve interaction with a variety of specific community groups and the planning and delivery of an appropriate and meaningful oral health intervention. Many of the project outcomes are based on enhancing communication skills and encouraging students to transfer these into their patient treatment sessions. This report draws on the experience of students who undertook two specific projects to demonstrate how they feel this is achieved.

Phosphorylation of E2F-1 by cyclin A-cdk2.

Transcription factor E2F-1 has a putative consensus sequence for phosphorylation by cyclin dependent kinase (Ser-Pro-X-Lys/Arg). Therefore, we studied the phosphorylation of E2F-1 in vivo and in vitro and its biological functions. E2F-1 was prepared by immunoprecipitation with anti-E2F-1 antibody from IMR32 lysates and was effectively phosphorylated by human cyclin A-cdk2 which was expressed in insect cells using baculovirus system. GST-E2F-1 was phosphorylated by cyclin A-cdk2 more efficiently than by cyclin E-cdk2. Cyclin D1-cdk4 phosphorylated pRB but scarcely phosphorylated GST-E2F-1 or H1 histone. The 60 kd protein precipitated with anti-E2F-1 antibody was phosphorylated in vivo. Phospho-peptide mapping indicated that its cleavage profile was identical with that of E2F-1 phosphorylated by cyclin A-cdk2 in vitro. This 60 kd protein, which is likely to be E2F-1, was not phosphorylated during the G0 and early G1 phase. Phosphorylation of E2F-1 began from the S phase while phosphorylation of pRB started nearly at G1/S. The in vivo phosphorylation of E2F-1 was inhibited by butyrolactone I, a cyclin-dependent kinase inhibitor (Kitagawa et al., 1993, Oncogene, 8, 2425-2432). The binding of E2F-1 to E2 promoter was found to be reduced by phosphorylation of E2F-1 by cyclin A-cdk2, suggesting that phosphorylation of E2F-1 may induce shut off of gene expression at the transcriptional level. These results suggest that E2F-1 is phosphorylated by cyclin A-cdk2 in the S phase in vivo as well as in vitro and that its phosphorylation by cyclin A-cdk2 may modulate its activity.

Selective insolubility of active hsp70 gene chromatin.

A gentle chromatin fractionation procedure was used to investigate solubility properties of Drosophila hsp70 heat-shock genes. After a brief digestion of isolated nuclei with micrococcal nuclease, most DNA is readily solubilized under low-ionic-strength conditions that maintain native nucleosomal organization. Actively transcribing hsp70 genes, however, are found to be enriched in the insoluble nuclear residue. Inactive genes are not resistant to solubilization, showing a fractionation pattern similar to that of bulk DNA. The insolubility characteristic correlates well with two other structural features of active hsp70 chromatin: increased sensitivity to endonuclease attack and disruption of the nucleosomal repeat pattern. The 5'-flanking regulatory region of active hsp70 genes is particularly resistant to solubilization, suggesting a role for binding of transcription factors in mediating this effect.

The tubulogenic activity associated with an activated form of Flt-1 kinase is dependent on focal adhesion kinase.

Focal adhesion kinase (FAK) is known to be located at the intersection between extracellular matrix and growth factor signaling pathways to regulate cell motility. We have shown previously that an activated form (BCR-FLTm1) of Flt-1 kinase, a receptor for vascular endothelial growth factor, had a tubulogenic activity not only in endothelial cells but also in fibroblastic cells. Here we show that tubulogenesis by BCR-FLTm1 depends on FAK and that FAK tyrosine phosphorylation and association with an activated Flt-1 receptor complex is increased after vascular endothelial growth factor stimulation of NIH3T3 cells overexpressing Flt-1.

Messenger ribonucleic acid encoding an apparent isoform of phosphorylase kinase catalytic subunit is abundant in the adult testis.

The complete amino acid sequence for a novel member of the protein kinase family was deduced from the nucleotide sequence of a cloned human cDNA. This putative protein kinase, given the preliminary designation "PSK-C3," is similar in primary structure to phosphorylase kinase catalytic subunit (PhK-gamma) isolated from rabbit skeletal muscle. The level of similarity does not appear sufficient, however, to suggest that PSK-C3 represents the human homolog of skeletal muscle PhK-gamma. Rather, it seems likely that PSK-C3 is a novel PhK-gamma isoform. From a cross-species Northern hybridization experiment using adult rat tissue RNA, a transcript homologous to PSK-C3 was found to be abundant in the testis but could not be detected in any of 12 other tissues tested, including skeletal muscle, liver, and ovary. Increasing levels of PSK-C3 mRNA in the testis correlate with postnatal testicular development, suggesting possible hormonal regulation of gene transcription. Energy released by glycogeneolysis in the testis may help fuel the process of spermatogenesis.

A marrow harvest procedure under local anesthesia.

This report details a bone marrow harvest procedure performed outside the hospital setting under local anesthesia, thereby avoiding many of the risks associated with the traditional surgical procedure. In approximately 30 minutes, 450 milliliters of marrow can be collected from eight bone punctures, containing a median of 4.18 x 10(9) cells and 33 x 10(6) progenitor cells as defined by CD34 expression. Reinfusion of a median 1.2 x 10(6) CD34+ cells/kg in 10 breast cancer and lung cancer patients after dose-intensive chemotherapy resulted in the recovery of granulocytes > 100/mm3 by day 14 and platelets > 20,000 by day 21. Without progenitor cell support, such recoveries could take 30 and 40 days, respectively. Collection of marrow using this protocol does not compromise the engraftment capability of the progenitor cells, seldom necessitates blood product support, is safer for the patient, and reduces the cost of harvesting by 75% compared to inpatient or day surgery procedures.

Cloning of a Xenopus laevis cDNA encoding focal adhesion kinase (FAK) and expression during early development.

Focal adhesion kinase (FAK) is a widely produced nonreceptor protein-tyrosine kinase thought to participate in signalling pathways activated in response to cell interaction with the extracellular matrix. Fibronectin-dependent cell adhesion mediated by integrin receptors plays a critical role in mesodermal cell migration during amphibian gastrulation in early development. As a first step toward understanding the role of FAK in Xenopus laevis (Xl) early development, we isolated cDNAs encoding Xl FAK and deduced the entire amino acid (aa) sequence. Xl FAK has 89-91% overall identity to the homologs previously described from mouse, human and chicken sources. Within the catalytic domain, the aa identity is about 97%. Northern blot analysis revealed that abundant maternal FAK transcript is present in Xl eggs, with levels decreasing slightly through cleavage and early blastula stages. At early gastrulation, the FAK mRNA level becomes modestly elevated, followed by a steady decline through late gastrulation. The mRNA level undergoes a further drop at the neurula stage, then begins a steady increase through the tailbud and tadpole stages. These data indicate that the steady-state level of FAK mRNA is regulated during Xl early development, and are consistent with a proposed role for FAK in the process of gastrulation.

The acute effects of postoperative radiation therapy on the transverse rectus abdominis myocutaneous flap used in immediate breast reconstruction.

PURPOSE: To analyze the acute effects of postoperative radiation therapy on the transverse rectus abdominis myocutaneous (TRAM) flap reconstruction following modified radical mastectomy for breast cancer. METHODS AND MATERIALS: Twenty-five consecutive patients were treated with postoperative radiation therapy after TRAM flap reconstruction between 1985 and 1999. The radiation records for these patients were retrospectively reviewed. Information regarding treatment techniques, timing, and dose was obtained and correlated with the extent of erythema, desquamation, and the need for treatment break. RESULTS: The median age was 48 years. The median dose of chest wall radiation was 5040 cGy. Additional boost doses were delivered in 13 patients. Twelve patients (48%) developed mild erythema in the treatment field during the course of treatment and 13 patients (52%) developed moderate (40%) or brisk (12%) erythema. Only 10 patients (40%) developed any kind of desquamation; 5 patients (20%) developed dry desquamation and another 5 patients (20%) developed moist desquamation. No patients required a break in the course of treatment because of acute side effects. None of the parameters evaluated (the use of chemotherapy prior to radiation, the interval between surgery and radiation, smoking, prior incidence of fat necrosis, the use of bolus during radiation, and the use of a boost) were predictive of an increased incidence of either the extent of erythema or the development of desquamation in the treatment field. CONCLUSION: Postmastectomy radiation for TRAM flap reconstruction is well tolerated and is not associated with an increased incidence of acute side effects. Radiation technique and the use of preradiation chemotherapy do not appear to be correlated with an increased incidence of acute side effects.

Selective T cell depletion with CD8-conjugated magnetic beads in the prevention of graft-versus-host disease after allogeneic bone marrow transplantation.

The effects of a new immunomagnetic method of selectively depleting CD8+ lymphocytes from donor bone marrow were studied in 29 patients undergoing transplantation from HLA-identical sibling (n = 20) or alternative (n = 9) donors. The direct immunomagnetic depletion method consistently removed > 95% of CD8+ cells and the non-specific loss of other cell subsets was only about 15%. Recovery of CFU-GM and BFU-e was on average > 100%. The final graft contained 0.9 +/- 0.6 x 10(8)/kg nucleated cells and 1.4 +/- 2.7 x 10(5)/kg CD8+ cells. Patients also received cyclosporine starting day -1. Engraftment occurred in 28 patients (97%), including three patients who received a non-TBI conditioning regimen. One patient receiving an unrelated transplant failed to engraft. Median time to ANC > 500 x 10(6)/L was 17 (12-23) days. Four of 20 patients receiving grafts from HLA-identical siblings (20%) developed acute GVHD grade > or = II. However, five of eight patients with grafts from alternative donors (63%) had grade > or = II GVHD. Nearly all patients developed fever around day 7, accompanied by fluid overload, mild skin rash and shortness of breath. This syndrome necessitated treatment with steroids. Immunomagnetic CD8 depletion is a simple and reproducible method of selective T cell depletion. In combination with cyclosporine it appears to be effective in the prevention of severe acute GVHD in HLA-identical sibling transplants, but not in transplants from less perfectly matched donors.

Hematopoietic growth factor after autologous peripheral blood transplantation: comparison of G-CSF and GM-CSF.

Autologous peripheral blood stem cell (PBSC) transplantation results in rapid hematologic recovery when sufficient numbers of CD34+ cells/kg are infused. Recent studies suggest that filgrastim (G-CSF) administration following transplantation leads to more rapid neutrophil recovery and lower total transplant costs. This study compares the use of G-CSF (5 microg/kg/day) with sargramostim (GM-CSF) 500 microg/day from day 0 until neutrophil recovery (ANC >1500/mm3) in patients with breast cancer or myeloma who had PBSC mobilized with the combination of cyclophosphamide, etoposide, and G-CSF. Twenty patients (13 breast cancer and seven myeloma) received GM-CSF and 26 patients (14 breast cancer and 12 myeloma) received G-CSF. The patients were comparable for age and stage of disease, and received stem cell grafts that were not significantly different (CD34+ x 10(6)/kg was 12.5 +/- 11.1 (mean +/- s.d.) for GM-CSF and 19.8 +/- 18.5 for G-CSF; P = 0.10). The use of red cells (2.8 vs 2.3 units), and platelet transfusions (2.5 vs 3.1) was similar for the two groups, as was the use of intravenous antibiotics (4.3 vs 4.6 days) and the number of days with temperature >38.3 degrees C (2.3 vs 1.8). Platelet recovery was also similar in both groups (platelets >50,000/mm3 reached after 11.8 vs 14.9 days). The recovery of neutrophils, however, was faster using G-CSF. ANC >500/mm3 and >1000/mm3 were reached in the GM-CSF group at 10.5 +/- 1.5 and 11.0 +/- 1.7 days, respectively, whereas with G-CSF only 8.8 +/- 1.2 and 8.9 +/- 2.2 days were required (P < 0.001). As a result, patients given G-CSF received fewer injections than the GM-CSF patients (10.9 vs 12.3). Resource utilization immediately attributable to the use of growth factors and the duration of pancytopenia, excluding hospitalization, were similar for the two groups. This study suggests that neutrophil recovery occurs more quickly following autologous PBSC transplant using G-CSF in comparison to GM-CSF, but the difference is not extensive enough to result in lower total cost.

Immunomagnetic CD4+ and CD8+ cell depletion for patients at high risk for severe acute GVHD.

Acute GVHD remains a major problem in allogeneic BMT, in particular when donors other than HLA-identical siblings are used. To determine the efficacy of an immunomagnetic method for depletion of CD4+ and CD8+ lymphocytes from the marrow graft, a series of 15 patients was studied. Thirteen patients had matched unrelated donors, and two patients had related donors. Cyclosporine was used as GVHD prophylaxis in combination with CD4+ and CD8+ depletion, which removed 94.1 +/- 3.2%, 97.0 +/- 5.1%, and 96.7 +/- 3.1% of CD3+, CD4+ and CD8+ cells, respectively. All patients engrafted promptly with AGC > 500/mm3 after a median of 16 days post-BMT. Acute GVHD grade II-IV developed in 0/2 related transplants and 4/13 MUD transplants; only one patient had grade III-IV acute GVHD. No late graft failure was observed. Three patients relapsed; two had advanced disease at the time of BMT. Seven patients are alive and in CCR after a median of 497 days; actuarial survival is 39% at 24 months. The fever syndrome observed with selective CD8+ cell depletion was not seen with the combined CD4+ and CD8+ cell depletion. Immunomagnetic CD4+ and CD8+ cell depletion of marrow grafts, in combination with in vivo cyclosporine, is a simple, reproducible and effective method to decrease the incidence and severity of acute GVHD in patients at high risk for this complication after allogeneic BMT.