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We demonstrate the ability to load, cool and detect singly charged calcium ions in a surface electrode trap using only visible and infrared lasers for the trapped-ion control. As opposed to the standard methods of cooling using dipole-allowed transitions, we combine power broadening of a quadrupole transition at 729 nm with quenching of the upper level using a dipole allowed transition at 854 nm. By observing the resulting 393 nm fluorescence we are able to perform background-free detection of the ion. We show that this system can be used to smoothly transition between the Doppler cooling and sideband cooling regimes, and verify theoretical predictions throughout this range. We achieve scattering rates which reliably allow recooling after collision events and allow ions to be loaded from a thermal atomic beam. This work is compatible with recent advances in optical waveguides, and thus opens a path in current technologies for large-scale quantum information processing. In situations where dielectric materials are placed close to trapped ions, it carries the additional advantage of using wavelengths which do not lead to significant charging, which should facilitate high rate optical interfaces between remotely held ions.

Motivated by an open problem of validating protein identities in label-free shotgun proteomics work-flows, we present a testing procedure to validate class (protein) labels using available measurements across N instances (peptides). More generally, we present a non-parametric solution to the problem of identifying instances that are deemed as outliers relative to the subset of instances assigned to the same class. The primary assumption is that measured distances between instances within the same class are stochastically smaller than measured distances between instances from different classes. We show that the overall type I error probability across all instances within a class can be controlled by some fixed value (say α ). We also demonstrate conditions where similar results on type II error probability hold. The theoretical results are supplemented by an extensive numerical study illustrating the applicability and viability of our method. Even with up to 25% of instances initially mislabeled, our testing procedure maintains a high specificity and greatly reduces the proportion of mislabeled instances. The applicability and effectiveness of our testing procedure is further illustrated by a detailed example on a proteomics data set from children with sickle cell disease where five spike-in proteins acted as contrasting controls., Competing Interests: No potential conflict of interest was reported by the author(s)., (© 2022 Informa UK Limited, trading as Taylor & Francis Group.)

Targeted therapies have reshaped the landscape of the development of cancer therapeutics. Recent biomarker-driven, tissue-agnostic clinical trials represent a significant paradigm shift in precision cancer medicine. Despite their growth in preclinical and clinical studies, to date only a few biomarker-driven, tissue-agnostic indications have seen approval by the US Food and Drug Administration (FDA). These approvals include pembrolizumab in microsatellite instability-high or mismatch repair deficient solid tumors, as well as both larotrectinib and entrectinib in NTRK fusion-positive tumors. Complex cancer biology, clinical trial design, and identification of resistance mechanisms represent some of the challenges that future tissue-agnostic therapies have to overcome. In this Review, we present a brief history of the development of tissue-agnostic therapies, comparing the similarities in the approval of pembrolizumab, larotrectinib, and entrectinib for tissue-agnostic indications. We also explore the future of tissue-agnostic cancer therapeutics while identifying important challenges for the future that drugs targeting tissue-agnostic indications will face., (© 2020 The Authors. Clinical Pharmacology & Therapeutics published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

BCR-ABL1-positive leukemias have historically been classified as either chronic myelogenous leukemia or Ph+ acute lymphoblastic leukemia. Recent analyses suggest there may be a wider range of subtypes. We report a patient with BCR-ABL1 fusion positive T-cell ALL with a previously undescribed cell distribution of the fusion gene. The examination of sorted cells by fluorescence in situ hybridization showed the BCR-ABL1 fusion in the malignant T cells and a subpopulation of the nonmalignant B cells, but not nonmalignant T cells or myeloid or CD34+ progenitor cells providing evidence that the fusion may have occurred in an early lymphoid progenitor., (© 2019 Wiley Periodicals, Inc.)

Purpose: To determine the effect of molecular weight (MW) on the concentration of plasma-derived proteins in aqueous humor and to estimate the plasma-derived and eye-derived fractions for each protein., Methods: Aqueous humor and plasma samples were obtained during cataract surgery on an institutional review board-approved protocol. Protein concentrations were determined by ELISA and quantitative antibody microarrays. A total of 93 proteins were studied, with most proteins analyzed using 27 to 116 aqueous and 6 to 30 plasma samples., Results: Plasma proteins without evidence of intraocular expression by sequence tags were used to fit a logarithmic model relating aqueous-plasma ratio (AH:PL) to MW. The log(AH:PL) appears to be well predicted by the log(MW) (P < 0.0001), with smaller proteins such as cystatin C (13 kDa) having a higher AH:PL (1:6) than larger proteins such as albumin (66 kDa, 1:300) and complement component 5 (188 kDa, 1:2500). The logarithmic model was used to calculate the eye-derived intraocular fraction (IOF) for each protein. Based on the IOF, 66 proteins could be categorized as plasma-derived (IOF<20), whereas 10 proteins were primarily derived from eye tissue (IOF >80), and 17 proteins had contribution from both plasma and eye tissue (IOF 20-80)., Conclusions: Protein concentration of plasma-derived proteins in aqueous is nonlinearly dependent on MW in favor of smaller proteins. Our study demonstrates that for proper interpretation of results, proteomic studies evaluating changes in aqueous humor protein levels should take into account the plasma and eye-derived fractions.

Heat shock protein 90 (Hsp90) is a protein chaperone that is upregulated and released from pancreatic β cells under pro-inflammatory conditions. We hypothesized that serum Hsp90 may have utility as a biomarker of type 1 diabetes risk and exhibit elevations before the onset of clinically significant hyperglycemia. To this end, total levels of the alpha cytoplasmic isoform of Hsp90 were assayed in autoantibody-positive progressors to type 1 diabetes using banked serum samples from the TrialNet Pathway to Prevention Cohort that had been collected 12 months prior to diabetes onset, with comparison to age, sex, and BMI-category matched autoantibody-positive nonprogressors and healthy controls. Hsp90 levels were higher in autoantibody-positive progressors and nonprogressors ≤ 18 years of age compared to matched healthy controls. However, Hsp90 levels were not different between progressors and nonprogressors in any age group. Hsp90 was positively correlated with age in control subjects, but this correlation was absent in autoantibody positive individuals. In aggregate these data indicate that elevated Hsp90 levels are present in youth with β cell autoimmunity, but are not able to distinguish youth or adult type 1 diabetes progressors from nonprogressors in samples collected 12 months prior to diabetes development., Competing Interests: The authors have declared that no competing interests exist.

This retrospective audit of patients diagnosed with mature T-cell lymphoma across a 10-year period provides contemporary information on the outcomes and treatment patterns of an Australian cohort. Forty-two patients diagnosed with mature T-cell lymphoma were identified from the Tasmanian Cancer Registry and analysed using medical records and simple statistical analysis. The demographics and outcomes of patients in this cohort were comparable to large international studies with treatment patterns in line with the best available evidence., (© 2017 Royal Australasian College of Physicians.)

Background: Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) is widely used for quantitative proteomic investigations. The typical output of such studies is a list of identified and quantified peptides. The biological and clinical interest is, however, usually focused on quantitative conclusions at the protein level. Furthermore, many investigations ask complex biological questions by studying multiple interrelated experimental conditions. Therefore, there is a need in the field for generic statistical models to quantify protein levels even in complex study designs., Results: We propose a general statistical modeling approach for protein quantification in arbitrary complex experimental designs, such as time course studies, or those involving multiple experimental factors. The approach summarizes the quantitative experimental information from all the features and all the conditions that pertain to a protein. It enables both protein significance analysis between conditions, and protein quantification in individual samples or conditions. We implement the approach in an open-source R-based software package MSstats suitable for researchers with a limited statistics and programming background., Conclusions: We demonstrate, using as examples two experimental investigations with complex designs, that a simultaneous statistical modeling of all the relevant features and conditions yields a higher sensitivity of protein significance analysis and a higher accuracy of protein quantification as compared to commonly employed alternatives. The software is available at http://www.stat.purdue.edu/~ovitek/Software.html.

Motivation: Nuclear magnetic resonance (NMR) spectroscopy is widely used for high-throughput characterization of metabolites in complex biological mixtures. However, accurate interpretation of the spectra in terms of identities and abundances of metabolites can be challenging, in particular in crowded regions with heavy peak overlap. Although a number of computational approaches for this task have recently been proposed, they are not entirely satisfactory in either accuracy or extent of automation., Results: We introduce a probabilistic approach Bayesian Quantification (BQuant), for fully automated database-based identification and quantification of metabolites in local regions of (1)H NMR spectra. The approach represents the spectra as mixtures of reference profiles from a database, and infers the identities and the abundances of metabolites by Bayesian model selection. We show using a simulated dataset, a spike-in experiment and a metabolomic investigation of plasma samples that BQuant outperforms the available automated alternatives in accuracy for both identification and quantification., Availability: The R package BQuant is available at: http://www.stat.purdue.edu/~ovitek/BQuant-Web/.

This chapter presents a case study, which applies statistical design and analysis to an LC-MS-based -investigation of subjects with coronary artery disease. First, we discuss the principles of statistical -experimental design, and the specification of an Analysis of Variance (ANOVA) model that describes the major sources of variation in the data. Second, we discuss procedures for detecting differentially abundant proteins, estimating protein abundance in individual samples, testing predefined groups of proteins for enrichment in differential abundance, and calculating sample size for a future experiment. The discussion is accompanied by examples of computer code implemented in the open-source statistical software R, which can be followed for an independent implementation of a similar investigation.

The goal of many LC-MS proteomic investigations is to quantify and compare the abundance of proteins in complex biological mixtures. However, the output of an LC-MS experiment is not a list of proteins, but a list of quantified spectral features. To make protein-level conclusions, researchers typically apply ad hoc rules, or take an average of feature abundance to obtain a single protein-level quantity for each sample. We argue that these two approaches are inadequate. We discuss two statistical models, namely, fixed and mixed effects Analysis of Variance (ANOVA), which views individual features as replicate measurements of a protein's abundance, and explicitly account for this redundancy. We demonstrate, using a spike-in and a clinical data set, that the proposed models improve the sensitivity and specificity of testing, improve the accuracy of patient-specific protein quantifications, and are more robust in the presence of missing data.

We present the architecture and approach of an evolving campus-wide information service for tissues with clinical and data annotations to be used and contributed to by clinical researchers across the campus. The services provided include specimen tracking, long term data storage, and computational analysis services. The project is conceived and sustained by collaboration among researchers on the campus as well as participation in standards organizations and national collaboratives.

A technique that combines ion mobility spectrometry (IMS) with reversed-phase liquid chromatography (LC), collision-induced dissociation (CID) and mass spectrometry (MS) has been developed. The approach is described as a high throughput means of analysing complex mixtures of peptides that arise from enzymatic digestion of protein mixtures. In this approach, peptides are separated by LC and, as they elute from the column, they are introduced into the gas phase and ionised by electrospray ionisation. The beam of ions is accumulated in an ion trap and then the concentrated ion packet is injected into a drift tube where the ions are separated again in the gas phase by IMS, a technique that differentiates ions based on their mobilities through a buffer gas. As ions exit the drift tube, they can be subjected to collisional activation to produce fragments prior to being introduced into a mass spectrometer for detection. The IMS separation can be carried out in only a few milliseconds and offers a number of advantages compared with LC-MS alone. An example of a single 21-minute LC-IMS-(CID)-MS analysis of the human plasma proteome reveals approximately 20,000 parent ions and approximately 600,000 fragment ions and evidence for 227 unique protein assignments.

Strategies that increase the ability of human hematopoietic stem and progenitor cells to repair alkylator-induced DNA damage may prevent the severe hematopoietic toxicity in patients with cancer undergoing high-dose alkylator therapy. In the context of genetic diseases, this approach may allow for selection of small numbers of cells that would not otherwise have a favorable growth advantage. No studies have tested this approach in vivo using human hematopoietic stem and progenitor cells. Human CD34(+) cells were transduced with a bicistronic oncoretrovirus vector that coexpresses a mutant form of O(6)-methylguanine DNA methyltransferase (MGMT(P140K)) and the enhanced green fluorescent protein (EGFP) and transplanted into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. Mice were either not treated or treated with O(6)-benzylguanine (6BG) and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). At 8-weeks postinjection, a 2- to 8-fold increase in the percentage of human CD45(+)EGFP(+) cells in 6BG/BCNU-treated versus nontreated mice was observed in the bone marrow and was associated with increased MGMT(P140K)-repair activity. Functionally, 6BG/BCNU-treated mice demonstrated multilineage differentiation in vivo, although some skewing in the maturation of myeloid and B cells was observed in mice transplanted with granulocyte-colony stimulating factor (G-CSF)-mobilized peripheral blood compared to umbilical cord blood. Expansion of human cells in 6BG/BCNU-treated mice was observed in the majority of mice previously transplanted with transduced umbilical cord blood cells. In addition, a significant increase in the number of EGFP(+) progenitor colonies in treated versus nontreated mice were observed in highly engrafted mice indicating that selection and maintenance of human progenitor cells can be accomplished by expression of MGMT(P140K) and treatment with 6BG/BCNU.

The overexpression of mutant forms of O(6)-methylguanine-DNA-methyltransferase (MGMT), resistant to the MGMT inhibitor O(6)-benzylguanine (BG), protects hematopoietic cells from the toxicity of combined BG plus O(6)-alkylating agent chemotherapy. To evaluate the feasibility of this approach for clinically relevant O(6)-alkylating agents, combined therapy with BG and two chloroethylnitrosourea-type drugs, ACNU or BCNU, or the triazene derivative temozolomide (TMZ) was investigated in a murine bone marrow transplant model allowing transgenic expression of the highly BG-resistant MGMT(P140K) mutant. Whereas 20/20 control animals transplanted with nontransduced cells died of progressive myelosuppression during therapy, nearly all animals transplanted with MGMT(P140K)-transduced cells survived treatment with BG/ACNU (12/15), BG/TMZ (10/10), or BG/BCNU (5/5). In surviving animals, hematological parameters improved during chemotherapy and pretreatment levels were reestablished during or shortly after therapy. All animals showed enrichment of transgenic granulocytes (range: 15- to 101-fold) and lymphocytes (range: 16- to 55-fold) in peripheral blood, bone marrow, and spleen. No significant differences were observed between individual treatment groups. Serial transplants demonstrated protection in secondary recipients and confirmed the transduction of transplantable stem cells. Thus, these data demonstrate efficient protection from hematotoxicity and substantial enrichment of transgenic cells following MGMT(P140K) gene transfer and treatment with different O(6)-alkylating drugs.

We studied interleukin-6 (IL-6) levels on the day of transplantation in 31 patients undergoing autologous haemopoietic stem cell transplantation (SCT) (either peripheral blood stem cell transplantation (PBSCT) or bone marrow transplantation (BMT)) for neoplastic diseases to determine if there was a relationship between IL-6 level and rate of haemopoietic recovery, length of stay in hospital, and survival. There was no apparent delay in post-transplant recovery associated with elevated IL-6 levels. However, increased values of IL-6 tended to be associated with an increased length of stay in hospital (P = 0.083). There was a highly significant adverse association between higher IL-6 levels and survival following transplantation (P = 0.0001). This association remained significant (P = 0.013) in the uniform subgroup of patients with malignant lymphoma with chemosensitive disease who had undergone BMT (that is, excluding patients who had undergone PBSCT) (n = 13). Knowledge of IL-6 levels on the day of transplant has the potential to provide valuable prognostic information in patients undergoing autologous haemopoietic SCT.

By identifying the sequence of retro- and lentiviral integration sites in peripheral blood leukocytes, the clonal composition and fate of genetically modified hematopoietic progenitor and stem cells could be mapped in vitro and in vivo. Previously available methods have been limited to the analysis of mono- or oligoclonal integration sites present in high copy numbers. Here, we perform characterization of multiple rare retroviral and lentiviral integration sites in highly complex DNA samples. The reliability of this method results from nontarget DNA removal via magnetic extension primer tag selection (EPTS) preceding solid-phase ligation-mediated PCR. EPTS/LM-PCR allowed the simultaneous direct genomic sequencing of multiple proviral LTR-flanking sequences of retro- and lentiviral vectors even if only 1 per 100 to 1000 cells contained the provirus. A primer walking "around" the integration locus demonstrated the adaptability of EPTS/LM-PCR to study unknown flanking DNA regions unrelated to proviruses. The technique is fast, inexpensive, and sensitive in minimal samples. It enables studies of retro- and lentiviral integration, viral vector tracking in gene therapy, insertional mutagenesis, transgene integration, and direct genomic sequencing that until now have been difficult or impossible to perform.

Direct reversal of O6 adducts caused by chemotherapy agents is accomplished in mammalian cells by the protein O6-methylguanine DNA methyltransferase (MGMT). Some tumors overexpress MGMT and are resistant to alkylator therapy. One future approach to treatment of these tumors may rely on concurrent pharmacological depletion of tumor MGMT with O6-benzylguanine (6-BG) and protection of sensitive tissues, such as hematopoietic stem and progenitor cells, using genetic modification with 6-BG-resistant MGMT mutants. We have used retroviral-mediated gene transfer to transduce murine hematopoietic bone marrow cells with MGMT point mutants showing resistance to 6-BG depletion in vitro. These mutants include proline to alanine and proline to lysine substitutions at the 140 position (P140A and P140K, respectively), which show 40- and 1000-fold resistance to 6-BG compared with wild-type (WT) MGMT. Lethally irradiated mice were reconstituted with murine stem cells transduced with murine stem cell virus retrovirus expressing each mutant, WT MGMT, or mock-infected cells and then treated with a combination of 30 mg/kg 6-BG and 10 mg/kg 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) or with 40 mg/kg BCNU alone. Compared with mice treated with BCNU alone, significant myeloid toxicity and death occurred in mice reconstituted with mock-infected or WT MGMT (<0.1 probability of survival) or the P140A mutant (0.13 probability of survival) MGMT cDNAs. In contrast, after an initial period of mild cytopenia, mice reconstituted with the P140K mutant (0.83 probability of survival) recovered nearly normal blood counts, even during continued treatment. Comparison of peripheral blood neutrophils after completion of 5 weekly treatments in these animals showed a direct correlation between the treatment and in vivo selection for progeny of transduced cells (pretreatment, approximately 8-12% transduced cells; no treatment, approximately 6% transduced cells; BCNU only, 51% transduced cells; 6-BG/BCNU, 93% transduced cells). To determine whether this selection occurred at the stem cell level, bone marrow from each treatment group was infused into secondary recipients. Whereas animals that received bone marrow from untreated animals reconstituted with 2% transduced cells, animals receiving marrow from 6-BG/BCNU-treated animals reconstituted with 94% transduced cells, demonstrating nearly complete selection for stem cells in the primary animals. Mice reconstituted with marrow from animals treated with BCNU only demonstrated 23% transduced cells, consistent with partial selection of stem cells in the primary mice. The levels of transduced cells also correlated with survival during a second round of intensive combination chemotherapy (probability of survival: 6-BG/BCNU, 1.0; BCNU alone, >0.70; no treatment, <0.1). These data demonstrate that mutant MGMT expressed in the bone marrow can protect mice from time- and dose-intensive chemotherapy and that the combination of 6-BG and BCNU leads to uniform selection of transduced stem cells in vivo in mice.

Background: PBSC collected following G-CSF priming lead to more rapid hemopoietic reconstitution (HR) after autologous transplantation than do unprimed BMstem cells. However, PBSC have a number of disadvantages compared with BM cells, including the need for an extended collection period and requirement for good venous access., Methods: We retrospectively analysed our experience with an alternative source of hemopoietic stem cells, G-CSF primed BM. Fortyfour patients who underwent BM harvesting after 6 days' administration of G-CSF, at a dose of 5 microg/kg per day, were compared with an equal number who underwent standard (unprimed) BM harvesting. We also analysed HR after autologous transplantation in 16 patients who received unprimed BM, 18 who received G-CSF-primed BM and 14 who received PBSC., Results: G-CSF-primed BM was collected more quickly (p<0.00005) and yielded a larger number of cells (p<0.0001) than unprimed BM. Consequently, larger numbers of cells were available for administration following transplantation with G-CSF-primed BM. The results of HR after transplantation with G-CSF primed BM were intermediate between those of unprimed BM and PBSC. For example, platelet independence (unsupported platelet count > or = 20 2 10(9)/L) occurred after 22 days with unprimed BM, 14 days with G-CSF-printed BM and 10 days with PBSC (p for trend <0.0001) and the mean number of days when platelet transfusions were given was 10, 6 and 3 respectively (p for trend <0.005). These results reflected transplant cell doses., Conclusion: G-CSF-primed BM is a valuable source of hemopoietic stem cells for autologous transplantation and a useful alternative to PBSC to certain circumstances.

CD28, a T cell costimulatory receptor, provides a signal that induces both optimal proliferation and the production of IL-2 by TCR-activated T cells. We show that the stimulation of CD28 leads to the activation of p21-activated kinase and MEK kinase 1. The same pathway was also stimulated in T cells treated with the cell-permeable ceramide analogue, C2-ceramide. The combined stimulation of either CD3 and CD28 or CD3 concurrently with C2-ceramide largely enhanced the activity of p21-activated kinase and MEK kinase 1. Therefore the Rac1/CDC42-coupled pathway(s) is a candidate that transduces and facilitates cross-talk between the CD28 costimulatory signal and the TCR signal.

Unless a costimulatory signal is provided, TCR recognition of Ag bound to the MHC is insufficient to induce optimal T cell proliferation or the production of IL-2. Here we show that the stimulation of CD28, a T cell costimulatory receptor, by a specific Ab increases F-actin contents in T cells. The interaction between T cells and B7-2-transfected Chinese hamster ovary cells expressing the CD28 ligand leads to the rearrangement of the actin cytoskelton in the region of cell-cell contact. Within the Rho family of G proteins, Rac1, but not Rho, translocates to the sites of cell-cell contact where Tailin also accumulates. These results indicate that the interaction between B7-2 and CD28 establishes a focal adhesion-like cell contact between T cell and APCs. The results also suggest that CD28 signaling is primarily transduced by a cytoskeletal rearrangement/signaling pathway mediated by the Rho family G proteins.

This retrospective study compared the overall survival, the event-free survival, and the timing of chemotherapy in patients with advanced Burkitt lymphoma with and without laparotomy. Thirty-five patients with advanced abdominal Burkitt lymphoma treated at least partially at the Centre Léon Bérard between 1981 and 1992 were included in this study. The diagnosis was obtained by laparotomy (LAP group) in 21 patients (17 stage III, 4 stage IV) and by other methods (non-LAP group) in 14 patients (5 stage III, 9 stage IV). The overall survival (71 and 93%) and the event-free survival (66 and 79%) were similar in the LAP and non-LAP groups, and the relapse rate was five (three local) in the LAP group compared with three (none local) in the non-LAP group. The local complication rate (9 of 21 versus 2 of 14) and the toxic death rate (2 of 21 versus 1 of 14) were slightly higher in the LAP group. Laparotomy also caused delays in therapy and increased the overall hospital stay. The mean interval from diagnosis to the start of the fourth course of chemotherapy was 57 days compared with 48 days and the average hospital stay was 44.4 days compared with 39 days for the LAP and non-LAP groups, respectively. Because advanced Burkitt lymphoma can be diagnosed by fine-needle aspiration, and chemotherapy cures more than 80% of the patients, there is no need for initial surgery, apart from acute emergencies. Furthermore, laparotomy delay chemotherapy and might reduce the survival rate.

45,X/47,XYY mosaicism is a rare condition with scarce information on the tissue-specific distribution of the different cell lines. Fluorescence in situ hybridization with repetitive, chromosome-specific DNA probes was applied to gain insight into the tissue-specific distribution of the two cell lines in biopsies of the streak gonad and dysgenetic testis of a 2-year-old individual exhibiting 45,X/47,XYY mosaicism. The distribution of the 45,X/47,XYY cells within different tissues was found to be nonrandom. The coelomic epithelium, the vascular endothelium, and the prepuberal germ cells exhibited predominantly a 47,XYY karyotype. In contrast, Sertoli cells exhibited both karyotypes, and the remaining tissue was predominantly composed of 45,X cells. The contribution of the two cell lines to gonadal development is discussed.

We have localized the human CD59 gene encoding a membrane protein that inhibits cell lysis by the membrane-attack complex of the homologous complement system. Using chromosomal in situ suppression hybridization and pulsed-field gel electrophoresis, we mapped this gene to chromosome 11p13, distal to the breakpoint of acute T-cell leukemia and proximal to the locus of the Wilms' tumor gene on a 30-kb SacII fragment.

In order to search for small tumor-specific deletions in 11p13 we analysed DNA isolated from 30 fresh Wilms' tumor (WT) samples with pulsed field gel electrophoresis. For these studies we have isolated new probes from the ends of several Notl fragments. Using these and previously described probes from 11p13 we first completed and extended the existing map of the 11p13 region. The analysis of the tumor material showed that (I) tumor-specific deletions were very rare: one homozygous deletion out of 30 tumors analysed, (2) hemizygous deletions were not observed in any of the tumors. The homozygous deletion in one patient spans 220 kb and is composed of a tumor-specific translocation associated with a deletion on one chromosome and a deletion of about 220 kb on the other chromosome at the same site. The WT-33 Wilms' tumor candidate gene maps to this deleted segment. A small constitutional deletion of 1,300 kb was identified in a patient with WT and genital tract malformations. These results suggest that in the majority of sporadic WT loss of gene function is due to subtle alterations in the gene, e.g., point mutations or very small deletions.

Using chromosome walking techniques, overlapping lambda and cosmid clones from the T cell receptor alpha (TCR alpha) region have been isolated; these span the entire J alpha region and parts of the TCR delta gene. Molecular analysis of the acute childhood leukemia cells (T-ALL) 8511 revealed a rearrangement on one chromosome 14 in J alpha 58 kb 5' of C alpha; this does not result in production of alpha message. The translocation was identified 90 kb 5' of C alpha at the previously identified J delta 2 element. A probe derived from the 5' region of the translocation breakpoint hybridized to DNA from a mouse-human cell hybrid containing chromosome 11 as the only human chromosome. This probe was used to isolate cosmid clones from chromosome 11. Several rare cutting restriction enzyme sites were found in close vicinity to the translocation breakpoint, and a long-range map spanning 1000 kb of chromosome region 11p13 was established. Analysis of the DNA from 15 cases of sporadic and familial Wilms' tumor did not reveal any changes, indicating that the translocation breakpoint does not reside in this gene.