Efficacy and safety of pembrolizumab in metastatic urothelial carcinoma: results from KEYNOTE-045 and KEYNOTE-052 after up to 5 years of follow-up.

BACKGROUND: Immune checkpoint inhibitors are a standard therapy in metastatic urothelial carcinoma (UC). Long-term follow-up is necessary to confirm durability of response and identify further safety concerns. PATIENTS AND METHODS: In KEYNOTE-045, patients with metastatic UC that progressed on platinum-containing chemotherapy were randomly assigned 1:1 to receive pembrolizumab or investigator's choice of paclitaxel, docetaxel, or vinflunine. Primary endpoints were progression-free survival per RECIST version 1.1 by blinded independent central review (BICR) and overall survival. In KEYNOTE-052, cisplatin-ineligible patients with metastatic UC received first-line pembrolizumab. The primary endpoint was objective response rate per RECIST version 1.1 by BICR. RESULTS: A total of 542 patients (pembrolizumab, n = 270; chemotherapy, n = 272) were randomly assigned in KEYNOTE-045. The median follow-up was 62.9 months (range 58.6-70.9 months; data cut-off 1 October 2020). At 48 months, overall survival rates were 16.7% for pembrolizumab and 10.1% for chemotherapy; progression-free survival rates were 9.5% and 2.7%, respectively. The median duration of response (DOR) was 29.7 months (range 1.6+ to 60.5+ months) for pembrolizumab and 4.4 months (range 1.4+ to 63.1+ months) for chemotherapy; 36-month DOR rates were 44.4% and 28.3%, respectively. A total of 370 patients were enrolled in KEYNOTE-052. The median follow-up was 56.3 months (range 51.2-65.3 months; data cut-off 26 September 2020). The confirmed objective response rate was 28.9% (95% confidence interval 24.3-33.8), and the median DOR was 33.4 months (range 1.4+ to 60.7+ months); the 36-month DOR rate was 44.8%. Most treatment-related adverse events for pembrolizumab in either study were grade 1 or 2 and manageable, which is consistent with prior reports. CONCLUSION: With ∼5 years of follow-up, pembrolizumab monotherapy continued to demonstrate durable efficacy with no new safety signals in patients with platinum-resistant metastatic UC and as first-line therapy in cisplatin-ineligible patients. CLINICAL TRIAL REGISTRY AND ID: With ClinicalTrials.gov NCT02256436 (KEYNOTE-045); https://clinicaltrials.gov/ct2/show/NCT02256436 and NCT02335424 (KEYNOTE-052); https://clinicaltrials.gov/ct2/show/NCT02335424.

Oxidative stress-driven parvalbumin interneuron impairment as a common mechanism in models of schizophrenia.

Parvalbumin inhibitory interneurons (PVIs) are crucial for maintaining proper excitatory/inhibitory balance and high-frequency neuronal synchronization. Their activity supports critical developmental trajectories, sensory and cognitive processing, and social behavior. Despite heterogeneity in the etiology across schizophrenia and autism spectrum disorder, PVI circuits are altered in these psychiatric disorders. Identifying mechanism(s) underlying PVI deficits is essential to establish treatments targeting in particular cognition. On the basis of published and new data, we propose oxidative stress as a common pathological mechanism leading to PVI impairment in schizophrenia and some forms of autism. A series of animal models carrying genetic and/or environmental risks relevant to diverse etiological aspects of these disorders show PVI deficits to be all accompanied by oxidative stress in the anterior cingulate cortex. Specifically, oxidative stress is negatively correlated with the integrity of PVIs and the extracellular perineuronal net enwrapping these interneurons. Oxidative stress may result from dysregulation of systems typically affected in schizophrenia, including glutamatergic, dopaminergic, immune and antioxidant signaling. As convergent end point, redox dysregulation has successfully been targeted to protect PVIs with antioxidants/redox regulators across several animal models. This opens up new perspectives for the use of antioxidant treatments to be applied to at-risk individuals, in close temporal proximity to environmental impacts known to induce oxidative stress.

Role for neonatal D-serine signaling: prevention of physiological and behavioral deficits in adult Pick1 knockout mice.

NMDA glutamate receptors have key roles in brain development, function and dysfunction. Regulatory roles of D-serine in NMDA receptor-mediated synaptic plasticity have been reported. Nonetheless, it is unclear whether and how neonatal deficits in NMDA-receptor-mediated neurotransmission affect adult brain functions and behavior. Likewise, the role of D-serine during development remains elusive. Here we report behavioral and electrophysiological deficits associated with the frontal cortex in Pick1 knockout mice, which show D-serine deficits in a neonatal- and forebrain-specific manner. The pathological manifestations observed in adult Pick1 mice are rescued by transient neonatal supplementation of D-serine, but not by a similar treatment in adulthood. These results indicate a role for D-serine in neurodevelopment and provide novel insights on how we interpret data of psychiatric genetics, indicating the involvement of genes associated with D-serine synthesis and degradation, as well as how we consider animal models with neonatal application of NMDA receptor antagonists.

TCC in Transplant Ureter--When and When Not to Preserve the Transplant Kidney.

We present four cases of transitional cell carcinoma of the transplant ureter (TCCtu). In three cases, localized tumor resection and a variety of reconstructive techniques were possible. Transplant nephrectomy with cystectomy was performed as a secondary treatment in one locally excised case. Transplant nephroureterectomy was performed as primary treatment in one case. The role of oncogenic viruses and genetic fingerprinting to determine the origin of TCCtu are described. Our cases and a systematic literature review illustrate the surgical, nephrological, and oncological challenges of this uncommon but important condition.

Adeno-associated virus serotypes 9 and rh10 mediate strong neuronal transduction of the dog brain.

Canine models have many advantages for evaluating therapy of human central nervous system (CNS) diseases. In contrast to nonhuman primate models, naturally occurring canine CNS diseases are common. In contrast to murine models, the dog's lifespan is long, its brain is large and the diseases affecting it commonly have the same molecular, pathological and clinical phenotype as the human diseases. We compared the ability of four intracerebrally injected adeno-associated virus vector (AAV) serotypes to transduce the dog brain with green fluorescent protein as the first step in using these vectors to evaluate both delivery and efficacy in naturally occurring canine homologs of human diseases. Quantitative measures of transduction, maximum diameter and area, identified both AAV2/9 and AAV2/rh10 as significantly more efficient than either AAV2/1 or AAV2/5 at transducing cerebral cortex, caudate nucleus, thalamus and internal capsule. Fluorescence co-labeling with cell-type-specific antibodies demonstrated that AAV2/9 and AAV2/rh10 were capable of primarily transducing neurons, although glial transduction was also identified and found to be more efficient with the AAV2/9 vector. These data are a prerequisite to evaluating the efficacy of recombinant AAV vectors carrying disease-modifying transgenes to treat naturally occurring canine models in preclinical studies of human CNS disease therapy.

Establishment of CYP2D6 reference samples by multiple validated genotyping platforms.

Cytochrome P450 2D6 (cytochrome P450, family 2, subfamily D, polypeptide 6 (CYP2D6)), a highly polymorphic drug-metabolizing enzyme, is involved in the metabolism of one-quarter of the most commonly prescribed medications. Here we have applied multiple genotyping methods and Sanger sequencing to assign precise and reproducible CYP2D6 genotypes, including copy numbers, for 48 HapMap samples. Furthermore, by analyzing a set of 50 human liver microsomes using endoxifen formation from N-desmethyl-tamoxifen as the phenotype of interest, we observed a significant positive correlation between CYP2D6 genotype-assigned activity score and endoxifen formation rate (rs = 0.68 by rank correlation test, P = 5.3 × 10(-8)), which corroborated the genotype-phenotype prediction derived from our genotyping methodologies. In the future, these 48 publicly available HapMap samples characterized by multiple substantiated CYP2D6 genotyping platforms could serve as a reference resource for assay development, validation, quality control and proficiency testing for other CYP2D6 genotyping projects and for programs pursuing clinical pharmacogenomic testing implementation.

Genome-wide meta-analysis identifies variants associated with platinating agent susceptibility across populations.

Platinating agents are used in the treatment of many cancers, yet they can induce toxicities and resistance that limit their utility. Using previously published and additional world population panels of diverse ancestry totaling 608 lymphoblastoid cell lines (LCLs), we performed meta-analyses of over 3 million single-nucleotide polymorphisms (SNPs) for both carboplatin- and cisplatin-induced cytotoxicity. The most significant SNP in the carboplatin meta-analysis is located in an intron of NBAS (neuroblastoma amplified sequence; P=5.1 × 10(-7)). The most significant SNP in the cisplatin meta-analysis is upstream of KRT16P2 (P=5.8 × 10(-7)). We also show that cisplatin-susceptibility SNPs are enriched for carboplatin-susceptibility SNPs. Most of the variants that associate with platinum-induced cytotoxicity are polymorphic across multiple world populations; therefore, they could be tested in follow-up studies in diverse clinical populations. Seven genes previously implicated in platinating agent response, including BCL2 (B-cell CLL/lymphoma 2), GSTM1 (glutathione S-transferase mu 1), GSTT1, ERCC2 and ERCC6, were also implicated in our meta-analyses.

Transfusion of 28-day-old leucoreduced or non-leucoreduced stored red blood cells induces an inflammatory response in healthy dogs.

BACKGROUND AND OBJECTIVES: Studies in mice suggest that rapid transfusions of red blood cells (RBCs), refrigerator stored for longer durations, induce a pro-inflammatory cytokine response. Studies in human neonates confirm these findings; however, to date, adult human studies have failed to replicate these findings. We used healthy research dogs to begin to examine the factors affecting the cytokine response to transfusion. MATERIALS AND METHODS: In a prospective study, healthy dogs were randomized for two autologous packed RBC transfusions after 7 (i.e. 'fresh') and 28 (i.e. 'old') days of storage, or after 28 and 7 days of storage, with or without prestorage leucoreduction (LR). RESULTS: No significant differences were observed between LR and non-LR transfusions for all circulating analytes measured following transfusion. A pro-inflammatory cytokine response, exemplified by monocyte chemoattractant protein-1, was observed 6 h after only old RBC transfusions, irrespective of infusion rate (P < 0·001). This response was accompanied by increased neutrophil counts (P < 0·001) and decreased platelet counts (P < 0·001). CONCLUSION: In healthy dogs, old RBC transfusions induce inflammation, which is unaffected by infusion rate.

Validation of a suite of ERP and QEEG biomarkers in a pre-competitive, industry-led study in subjects with schizophrenia and healthy volunteers.

OBJECTIVE: Complexity and lack of standardization have mostly limited the use of event-related potentials (ERPs) and quantitative EEG (QEEG) biomarkers in drug development to small early phase trials. We present results from a clinical study on healthy volunteers (HV) and patients with schizophrenia (SZ) that assessed test-retest, group differences, variance, and correlation with functional assessments for ERP and QEEG measures collected at clinical and commercial trial sites with standardized instrumentation and methods, and analyzed through an automated data analysis pipeline. METHODS: 81 HV and 80 SZ were tested at one of four study sites. Subjects were administered two ERP/EEG testing sessions on separate visits. Sessions included a mismatch negativity paradigm, a 40 Hz auditory steady-state response paradigm, an eyes-closed resting state EEG, and an active auditory oddball paradigm. SZ subjects were also tested on the Brief Assessment of Cognition (BAC), Positive and Negative Syndrome Scale (PANSS), and Virtual Reality Functional Capacity Assessment Tool (VRFCAT). RESULTS: Standardized ERP/EEG instrumentation and methods ensured few test failures. The automated data analysis pipeline allowed for near real-time analysis with no human intervention. Test-retest reliability was fair-to-excellent for most of the outcome measures. SZ subjects showed significant deficits in ERP and QEEG measures consistent with published academic literature. A subset of ERP and QEEG measures correlated with functional assessments administered to the SZ subjects. CONCLUSIONS: With standardized instrumentation and methods, complex ERP/EEG testing sessions can be reliably performed at clinical and commercial trial sites to produce high-quality data in near real-time.

Cyclophosphamide-based in vivo T-cell depletion for HLA-haploidentical transplantation in Fanconi anemia.

Allogeneic hematopoietic cell transplantation (HCT) is the only known cure for patients with Fanconi anemia (FA) who develop aplasia or leukemia. However, transplant regimens typically contain high-dose alkylators, which are poorly tolerated in FA patients. Furthermore, as many patients lack human leukocyte antigen (HLA)-matched family donors, alternative donors are used, which can increase the risk of both graft rejection and graft-versus-host disease (GVHD). To improve on these three concerns, we developed a multi-institutional clinical trial using a fludarabine (FLU)-based conditioning regimen with limited alkylators/low-dose radiation, HLA-haploidentical marrow, followed by reduced-dose cyclophosphamide (CY) to treat three FA patients with aplasia. All three patients engrafted with 100% donor CD3 chimerism at 1 month. One patient died early from disseminated toxoplasmosis infection. Of the two survivors, one had significant pretransplant co-morbidities and inadequate immunosuppression, and developed severe acute GVHD. The other patient had only mild acute and no chronic GVHD. With a follow-up of 2 and 3 years, respectively, both patients are doing well, are transfusion-independent, and maintain full donor chimerism. The patient with severe GVHD has resolving oral GVHD and good quality of life. We conclude that using low-intensity conditioning, HLA-haploidentical marrow, and reduced-dose CY for in vivo T-cell depletion can correct life-threatening aplasia in FA patients.

Stages in development of mink cell focus-inducing (MCF) virus-accelerated leukemia in AKR mice.

Flow cytometric techniques involving correlated dual parameter analysis of fluorescence and light scatter and transplantation bioassays were used to describe a series of cellular changes in thymus of young (1-4 mo old) AKR mice during development of mink cell focus-inducing (MCF) virus-accelerated leukemia. Three stages of leukemogenesis were defined before appearance of frankly leukemic mice. Stage 1, apparent 28-40 d after injection of MCF 69L1 virus, represented steady-state infection of thymocytes by MCF virus without apparent change in light scatter properties of the cells or in expression of alloantigens Thy-1, Lyt-1, Lyt-2, L3T4a, B2A2, or H-2K on the major thymocyte subpopulations. Expression of MCF virus was highest in the population of small cortical thymocytes. Stage II was observed at highest frequency 50-60 d postinjection and represented the emergence of a clonal population of cells with transformed properties which could be resolved from normal thymocytes by light scatter and expression of B2A2, H-2K, and gp70 antigens. Stage III was observed at highest frequency at 70 d postinjection, when considerable enlargement of thymus had occurred, and appeared to represent the outgrowth of fully transformed cells that replaced the normal thymocyte subpopulations. The alloantigen phenotype of blast cells from frankly leukemic mice did not differ qualitatively from that of stage II or stage III cells but displayed considerable heterogeneity with respect to quantitative expression of alloantigens and gp70. At least two populations of leukemic blasts could be resolved in the majority of primary thymomas analyzed. It is unclear whether these populations represent the outgrowth of independent clones of transformed cells or if they are related in some way. Our data are consistent with MCF virus-induced transformation of cells in the lineage to small peanut agglutinin-positive, cortisone-sensitive thymocytes, a subpopulation that predominates in the thymus and which is thought to be destined for cell death in situ.

G(AKSL2): a new cell surface antigen of the mouse related to the dualtropic mink cell focus-inducing class of murine leukemia virus detected by naturally occurring antibody.

Normal mouse sera were tested for cytotoxic antibody to surface antigens of cultured monolayer cells infected with AKR-derived ecotropic MuLV, xentropic MuLV, or dualtropic MCF 247 MuLV. Antibody to ecotropic MuLV-infected cells was found in a proportion of C57BL/6, C3Hf/Bi, AKR-Fv-1b, and (C3Hf/Bi X AKR)F1 mice, but not AKR or (AKR X C3Hf/Bi)F1 mice. Antibody to xenotropic MuLV-infected cells was virtually restricted to C57BL/6 mice. Antibody to MCF 247-infected cells was found in all strains tested, including AKR mice. Absorption analysis of (C3Hf/Bi x akr)f1 and AKR-Fv-1b sera with selective reactivity for MCF 247-infected cells showed that these sera recognize distinctive antigens on MCF 247-infected cells that are not present on ecotropic or xenotropic MuLV-infected cells. The transplantable AKR spontaneous leukemia AKSL2 was found to be uniquely sensitive to the cytotoxic action of naturally occurring antibody to MCF 247-related antigens and absorption tests with AKSL2 as the target cell and sera from a single AKR-Fv-1b mouse have permitted the definition of a new MuLV-related cell surface antigen, which has been designated G(AKSL2). Thymocytes from young mice of high leukemia-incidence strains (AKR, C58, and PL) express G(AKSL2), whereas thymocytes from 12 other strains do not. In AKR mice, the antigen is expressed in higher amounts on cells from thymus and bone marrow than on spleen cells. All AKR spontaneous leukemias tested express G(AKSL2), as did three MuLV-induced leukemias arising in G(AKSL2)- strains. Five X-ray-induced leukemias of G(AKSL2)- strains were G(AKSL2)-, as were MuLV+ and MuLV- chemically induced sarcomas. In the limited survey conducted to date, natural antibody to G(AKSL2) has been restricted to strains expressing G(AKSL2) in their normal tissues: AKR, AKR congenic mice AKR-Fv-1b and AKR hybrid mice (C3Hf/Bi x akr)f1 and (C57BL/6 X AKR)F1. In vitro G(AKSL2) induction tests involving MuLV infection of cultured monolayer cells showed that 8 of 12 newly isolated dualtropic MuLV shared the property of G(AKSL2) induction with the prototype MCF MuLV, MCF 247. Of the 12 ecotropic MuLV tested, only the N-tropic MuLV isolated from a leukemia originally induced by Passage A Gross virus induced G(AKSL2). The xenotropic and amphotropic MuLV isolates tested lacked G(AKSL2) inducing activity. Recognition of the g(aksl2) system provides a way to trace the origin and natural history of a class of dualtropic MCF MuLV in the mouse and to determine whether natural antibody to G(AKSL2) plays a role in AKR leukemogenesis.

Relationships of gp70 of MuLV envelopes to gp70 components of mouse lymphocyte plasma membranes.

The family of glycoproteins called gp70 includes molecules that are the main constituent of murine C-type viral envelopes, and some that are expressed as mendelian constituents of thymocyte plasma membranes in the absence of virions. To investigate further the relation of viral gp70s to plasma- membrane gp70s we compared peptide maps of gp70s derived by immunoprecipitation from cells infected with chosen viruses and from various thymocytes and leukemiacells known to express one or more of three immunogenetically defined gp70 types: Glx-gp70, X-gp70, and O-gp70. Maps of gp70 from cultured cells infected with ecotropic and xenotropic viruses were distinguishable from one another, and in general resembled gp70 maps prepared directly from ecotropic and xenotropic virions respectively. Maps of gp70s immunoprecipitated from thymocytes of five mouse strains and from two A strain T-cell leukemias also fell into two distinguishable and generally corresponding patterns. Thus peptide-mapping substantiates earlier conclusions that viral gp70s and plasma-membrane gp70s inherited independently of virus-production are highly related or identical molecules. The gp70 maps of thymocytes from B6, B6-G(+IX), 129, and A mice formed a group resembling the map from cultured cells infected with xenotropic virus. Thymocytes from AKR mice, and the two A strain leukemias, gave gp70 maps conforming more to the second pattern, that of cultured cells infected with ecotropic virus. This second pattern probably comprises at least two gp70 types, one of which is X-gp70. Our data indicate that the G(IX)-gp70 and O-gp70 sub-species of gp70 expressed in the cell populations we have studied are coded by xenotropic viral genomes, and X-gp70 by ecotropic viral genomes.

G(RADA1): a new cell surface antigen of mouse leukemia defined by naturally occurring antibody and its relationship to murine leukemia virus.

A new cell surface antigenic system of the mouse, designated G(RADA1), is described. The antigen is defined by cytotoxic tests with the A strain X-ray-induced leukemia RADA1 and naturally occurring antibody from random-bred Swiss mice and can be distinguished from all other serologically detected cell surface antigens of the mouse. Absorption tests indicate that G(RADA1) is present in the normal lymphatic tissue and leukemias of mouse strains with high spontaneous leukemia-incidence, e.g., AKR, C58, and C3H/Figge. Low leukemia-incidence strains, e.g., C57BL/6, BALB/c, and A lack G(RADA1) in their normal tissues, but a proportion of leukemias and solid tumors arising in these strains are G(RADA1)+. The relation of G(RADA1) to MuLV is shown by G(RADA1) appearance after MuLV infection of permissive cells in vitro; four of five N-tropic MuLV isolates, one of four B-tropic MuLV, and none of four xenotropic MuLV induce G(RADA1). Two MCF MuLV, thought to represent recombinants between N-ecotropic and xenotropic MuLV, also induce G(RADA1). Serological and biochemical characterization indicates that G(RADA1) is a type-specific determinant of the gp70 component of certain MuLV. The presence of natural antibody to RADA1 in various mouse strains and the emergence of G(RADA1)+ leukemias and solid tumors in mice of G(RADA1)- phenotype suggest widespread occurrence of genetic information coding for this antigen.

A cell surface antigen of the mouse related to xenotropic MuLv defined by naturally occurring antibody and monoclonal antibody. Relation to Gix G(rada1), G(aksl2) systems of MuLV-related antigens.

A new cell surface antigen of the mouse related to xenotropic murine leukemia virus (MuLV) is described. The antigen, designated G(erld), is defined by cytotoxic tests with the B6-x-ray-induced ERLD and naturally occurring antibody. G(erld) is distinct from all previously defined cell surface antigens. Monoclonal antibody with the same specificity has been developed. Inbred mouse strains are classified as G(erld)+ or G(erld)- according to the presence of absence of the antigen on lymphoid cells. G(erld)+ strains differ with regard to quantitative expression of G(erld) on normal thymocytes. The emergence of G(erld)+ tumors in G(erld)- strains indicates the presence of genes coding for the antigen even in strains not normally expressing the antigen. G(erld) has the characteristic of a differentiation antigen in normal mice. In G(erld)+ strains, high levels of the antigen are found on thymocytes with lower levels being detected on cells of spleen, lymph nodes and bone marrow. No G(erld) was detected in brain or kidney or on erythrocytes. The segregation ratios for G(erld) expression on thymocytes in backcross and F2 mice of crosses between G(erld)+ (B6, 129, and B6-Gix+) and G(erld)- (BALB/c) strains suggest that G(erld) expression is controlled by a single locus in B6, by two unlinked loci in 129, and by three unlinked loci in B6-Gix+ mice. Induction of the antigen by MuLV infection of permissive cells in vitro indicates that G(erld) is closely related to xenotropic and dualtropic MuLV; all xenotropic and dualtropic MuLV tested induced the antigen, whereas the majority of ecotropic and the two amphotropic MuLV failed to do so. As dualtropic MuLV are thought to be recombinants between ecotropic and xenotropic MuLV sequences, G(erld) coding by dualtropic MuLV may signify the contribution of the xenotropic part in the recombinational event. Serological and biochemical characterization indicates that G(erld) is related to the gp 70 component of the MuLV envelope. The relation of G(erld) to the previously defined gp 70-related cell surface antigens (Gix, G(rada), and G(aksl2) is discussed, particularly with regard to their characteristics as differentiation antigens, the genetic origin of dualtropic MuLV, and the leukemogenicity of MuLV.

Heritable and non-genetic factors as variables of pharmacologic phenotypes in lymphoblastoid cell lines.

Publicly available genetic and expression data on lymphoblastoid cell lines (LCLs) make them a unique resource for understanding the genetic underpinnings of pharmacological outcomes and disease. LCLs have been used for pharmacogenomic discovery and validation of clinical findings associated with drug response. However, variation in cellular growth rate, baseline Epstein-Barr virus (EBV) copy number and ATP levels can all be confounders in such studies. Our objective is to better define confounding variables that affect pharmacological end points in LCLs. To this end, we evaluated the effect of these three variables on drug-induced cytotoxicity in LCLs. The drugs evaluated included daunorubicin, etoposide, carboplatin, cisplatin, cytarabine, pemetrexed, 5'-deoxyfluorouridine, vorinostat, methotrexate, 6-mercaptopurine, and 5-fluorouracil. Baseline ATP or EBV copy number were not significantly correlated with cellular growth rate or drug-induced cytotoxicity. In contrast, cellular growth rate and drug-induced cytotoxicity were significantly, directly related for all drugs except vorinostat. Importantly, cellular growth rate is under appreciable genetic influence (h²=0.30-0.39) with five suggestive linkage regions across the genome. Not surprisingly, a percentage of SNPs that significantly associate with drug-induced cytotoxicity also associate with cellular growth rate (P ≤ 0.0001). Studies using LCLs for pharmacologic outcomes should therefore consider that a portion of the genetic variation explaining drug-induced cytotoxicity is mediated via heritable effects on growth rate.

GNAS1 mutations occur more commonly than previously thought in intramuscular myxoma.

Mutation detection plays an important role in diagnostic pathology, not only in providing a tissue diagnosis, but also in predicting response to antitumourigenic agents. However, mutation detection strategies are often hampered by masking of mutant alleles by wild-type sequences. Coamplification at lower denaturation temperature PCR (COLD-PCR) reportedly increases the proportion of rare variant sequences in a wild-type background by using PCR cycles in which the denaturation temperature is reduced to favour product formation with lower melt temperatures and heteroduplexes arising from minor variants. Intramuscular myxoma is a rare benign soft tissue neoplasm that occurs sporadically and less commonly in association with fibrous dysplasia (Mazabraud's syndrome). Fibrous dysplasia results from activating GNAS1 mutations, and the same mutations have been identified in small numbers of intramuscular myxoma. The aim of the study was primarily to establish whether COLD-PCR is more sensitive than conventional PCR; this was achieved by testing for GNAS1 mutations in intramuscular myxomas using the two methodologies. Mutations were detected in 8 of 28 (29%) cases of intramuscular myxomas using conventional PCR followed by mutation-specific restriction enzyme digestion (PCR-MSRED) whereas 17 of 28 (61%) mutations were detected using COLD-PCR/MSRED. Mutations were detected in two cases where a diagnosis of low-grade myxofibrosarcoma had been favoured over intramuscular myxoma. No mutations were detected in an additional 9 low-grade and 19 high-grade myxofibrosarcomas, and another 40 control samples. This study shows the power of COLD-PCR compared with conventional PCR in mutation detection, and shows that GNAS1 mutation detection increases diagnostic accuracy when distinguishing between intramuscular myxoma and low-grade myxofibrosarcoma.

Molecular and ultrastructural defects in a Drosophila myosin heavy chain mutant: differential effects on muscle function produced by similar thick filament abnormalities.

We have determined the molecular defect of the Drosophila melanogaster myosin heavy chain (MHC) mutation Mhc and the mutation's effect on indirect flight muscle, jump muscle, and larval intersegmental muscle. We show that the Mhc1 mutation is essentially a null allele which results in the dominant-flightless and recessive-lethal phenotypes associated with this mutant (Mogami, K., P. T. O'Donnell, S. I. Bernstein, T. R. F. Wright, C. P. Emerson, Jr. 1986. Proc. Natl. Acad. Sci. USA. 83:1393-1397). The mutation is a 101-bp deletion in the MHC gene which removes most of exon 5 and the intron that precedes it. S1 nuclease mapping indicates that mutant transcripts follow two alternative processing pathways. Both pathways result in the production of mature transcripts with altered reading frames, apparently yielding unstable, truncated MHC proteins. Interestingly, the preferred splicing pathway uses the more distal of two available splice donor sites. We present the first ultrastrutural characterization of a completely MHC-null muscle and show that it lacks any discernable thick filaments. Sarcomeres in these muscles are completely disorganized suggesting that thick filaments play a critical role in sarcomere assembly. To understand why the Mhc1 mutation severely disrupts indirect flight muscle and jump muscle function in heterozygotes, but does not seriously affect the function of other muscle types, we examined the muscle ultrastructure of Mhc1/+ heterozygotes. We find that these organisms have a nearly 50% reduction in the number of thick filaments in indirect flight muscle, jump muscle, and larval intersegmental muscle. In addition, aberrantly shaped thick filaments are common in the jump muscle and larval intersegmental muscle. We suggest that the differential sensitivity of muscle function to the Mhc1 mutation is a consequence of the unique myofilament arrays in each of these muscles. The highly variable myofilament array of larval intersegmental muscle makes its function relatively insensitive to changes in thick filament number and morphology. Conversely, the rigid double hexagonal lattice of the indirect flight muscle, and the organized lattice of the jump muscle cannot be perturbed without interfering with the specialized and evolutionarily more complex functions they perform.

Analysis of Drosophila paramyosin: identification of a novel isoform which is restricted to a subset of adult muscles.

In this report we show that Drosophila melanogaster muscles contain the standard form of the thick filament protein paramyosin, as well as a novel paramyosin isoform, which we call miniparamyosin. We have isolated Drosophila paramyosin using previously established methods. This protein is approximately 105 kD and cross-reacts with polyclonal antibodies made against Caenorhabditis elegans or Heliocopris dilloni paramyosin. The Heliocopris antibody also cross-reacts with a approximately 55-kD protein which may be miniparamyosin. We have cloned and sequenced cDNA's encoding both Drosophila isoforms. Standard paramyosin has short nonhelical regions at each terminus flanking the expected alpha-helical heptad repeat seen in other paramyosins and in myosin heavy chains. The COOH-terminal 363 amino acids are identical in standard and miniparamyosin. However, the smaller isoform has 114 residues at the NH2 terminus that are unique as compared to the current protein sequence data base. The paramyosin gene is located at chromosome position 66E1. It appears to use two promoters to generate mRNA's that have either of two different 5' coding sequences joined to common 3' exons. Each protein isoform is encoded by two transcripts that differ only in the usage of polyadenylation signals. This results in four size classes of paramyosin mRNA which are expressed in a developmentally regulated pattern consistent with that observed for other muscle-specific RNA's in Drosophila. In situ hybridization to Drosophila tissue sections shows that standard paramyosin is expressed in all larval and adult muscle tissues whereas miniparamyosin is restricted to a subset of the adult musculature. Thus miniparamyosin is a novel muscle-specific protein that likely plays a role in thick filament structure or function in some adult muscles of Drosophila.