Ado-trastuzumab emtansine (T-DM1) in patients with HER2-amplified tumors excluding breast and gastric/gastroesophageal junction (GEJ) adenocarcinomas: results from the NCI-MATCH trial (EAY131) subprotocol Q.

BACKGROUND: The National Cancer Institute-Molecular Analysis for Therapy Choice (NCI-MATCH) is a national precision medicine study incorporating centralized genomic testing to direct refractory cancer patients to molecularly targeted treatment subprotocols. This treatment subprotocol was designed to screen for potential signals of efficacy of ado-trastuzumab emtansine (T-DM1) in HER2-amplified histologies other than breast and gastroesophageal tumors. METHODS: Eligible patients had HER2 amplification at a copy number (CN) >7 based on targeted next-generation sequencing (NGS) with a custom Oncomine AmpliSeq™ (ThermoFisher Scientific) panel. Patients with prior trastuzumab, pertuzumab or T-DM1 treatment were excluded. Patients received T-DM1 at 3.6 mg/kg i.v. every 3 weeks until toxicity or disease progression. Tumor assessments occurred every three cycles. The primary end point was centrally assessed objective response rate (ORR). Exploratory end points included correlating response with HER2 CN by NGS. The impact of co-occurring genomic alterations and PTEN loss by immunohistochemistry were also assessed. RESULTS: Thirty-eight patients were enrolled and 36 included in efficacy analysis. Median prior therapies in the metastatic setting was 3 (range 0-9; unknown in one patient). Median HER2 CN was 17 (range 7-139). Partial responses were observed in two (5.6%) patients: one mucoepidermoid carcinoma of parotid gland and one parotid gland squamous cell cancer. Seventeen patients (47%) had stable disease including 8/10 (80%) with ovarian and uterine carcinomas, with median duration of 4.6 months. The 6-month progression-free survival rate was 23.6% [90% confidence interval 14.2% to 39.2%]. Common toxicities included fatigue, anemia, fever and thrombocytopenia with no new safety signals. There was a trend for tumor shrinkage with higher levels of gene CN as determined by the NGS assay. CONCLUSION: T-DM1 was well tolerated. While this subprotocol did not meet the primary end point for ORR in this heavily pre-treated diverse patient population, clinical activity was seen in salivary gland tumors warranting further study in this tumor type in dedicated trials.

Impact + resistance training improves bone health and body composition in prematurely menopausal breast cancer survivors: a randomized controlled trial.

UNLABELLED: Our randomized controlled trial in prematurely menopausal breast cancer survivors showed that impact + resistance training prevented increases in percentage of body fat compared with controls and also improved BMD at the hip and prevented BMD loss at the spine among exercise-trained women who were menopausal for >1 year. INTRODUCTION: Cancer treatment-related menopause worsens bone health and body composition in breast cancer survivors (BCS). We investigated whether impact + resistance training could improve bone mineral density (BMD), reduce bone turnover, build muscle, and decrease fat mass in BCS with premature menopause. METHODS: We conducted a randomized controlled trial in 71 BCS (mean age, 46.5 years) within 5 years of treatment-related menopause. Women were randomly assigned to one of two groups: (1) impact + resistance training (prevent osteoporosis with impact + resistance (POWIR)) or (2) exercise placebo (FLEX) 3×/week for 1 year. Outcomes were hip and spine BMD (in grams per square centimeter) and body composition (percent body fat (%BF) and lean and fat mass (in kilograms)) by DXA and bone turnover markers (serum osteocalcin (in nanograms per milliliter) and urinary deoxypryrodinoline (in nanomoles per milliliter). RESULTS: There were no significant group × time interactions for bone outcomes when using an intent-to-treat approach on the full sample. In analyses restricted to BCS who were menopausal for ≥1 year, POWIR increased BMD at the hip and slowed BMD loss at the spine compared with FLEX (femoral neck-POWIR, 0.004 ± 0.093 g/cm(2) vs. FLEX, -0.010 ± 0.089 g/cm(2); p < 0.01; spine-POWIR, -0.003 ± 0.114 g/cm(2) vs. FLEX, -0.020 ± 0.110 g/cm(2); p = 0.03). POWIR prevented increases in %BF (POWIR, 0.01 % vs. FLEX, 1.3 %; p < 0.04). Women with attendance to POWIR at ≥64 % had better improvements in %BF than women attending less often (p < 0.03). CONCLUSION: Impact + resistance training may effectively combat bone loss and worsening body composition from premature menopause in BCS.

Inhibition of baroreflex by angiotensin II via Fos expression in nucleus tractus solitarii of the rat.

We evaluated the modulatory action of angiotensin II at the nucleus tractus solitarii on spontaneous baroreceptor reflex response, the angiotensin subtype receptors involved, and the role of Fos protein in this process, using Sprague-Dawley rats anesthetized with pentobarbital sodium. Microinjection bilaterally of angiotensin (Ang ) II (5, 10, 20, or 40 pmol) into the nucleus tractus solitarii significantly suppressed the spontaneous baroreceptor reflex, as represented by the magnitude of transfer function between systemic arterial pressure and heart rate signals. There also was a concomitant increase in Fos-like immunoreactivity in the nucleus tractus solitarii. Both the suppression of spontaneous baroreceptor reflex and Fos expression in nucleus tractus solitarii neurons elicited by Ang II were discernibly attenuated by pretreatment with or comicroinjection into the bilateral nucleus tractus solitarii of a 15-mer antisense c-fos oligonucleotide that targets against the initiation codon of c-fos mRNA. In addition, those 2 actions of Ang II were reversed by the coadministration of the nonpeptide Ang II type 1 (AT(1)) receptor antagonist losartan (1.6 nmol) but not by the nonpeptide AT(2) receptor antagonist PD 123,319 (1.6 nmol). Control treatments with artificial cerebrospinal fluid, sense cDNA, or antisense oligonucleotide with a scrambled sequence were ineffective. We conclude that under minimal cardiovascular perturbation, Fos expression mediated via activation of AT(1) subtype receptors may underlie the inhibitory modulation of beat-to-beat baroreflex control of blood pressure by Ang II at the nucleus tractus solitarii.

Direct genetic demonstration of G alpha 13 coupling to the orphan G protein-coupled receptor G2A leading to RhoA-dependent actin rearrangement.

G2A is an orphan G protein-coupled receptor (GPCR), expressed predominantly in T and B cells and homologous to a small group of GPCRs of unknown function expressed in lymphoid tissues. G2A is transcriptionally induced in response to diverse stimuli, and its ectopic expression suppresses transformation of B lymphoid precursors by BCR-ABL. G2A induces morphological transformation of NIH 3T3 fibroblasts. Microinjection of constructs encoding G2A into Swiss 3T3 fibroblasts induces actin reorganization into stress fibers that depends on RhoA, but not CDC42 or RAC. G2A elicits RhoA-dependent transcriptional activation of serum response factor. Direct evaluation of RhoA activity demonstrates elevated levels of RhoA-GTP in G2A-expressing cells. Microinjection of embryonic fibroblasts derived from various G alpha knockout mice establishes a requirement for G alpha 13 but not G alpha 12 or G alpha q/11 in G2A-induced actin rearrangement. In conclusion, G2A represents a family of GPCRs expressed in lymphocytes that may link diverse stimuli to cytoskeletal reorganization and transcriptional activation through a pathway involving G alpha 13 and RhoA.

Role of the distal half of the c-Mpl intracellular domain in control of platelet production by thrombopoietin in vivo.

The cytokine thrombopoietin (TPO) controls the formation of megakaryocytes and platelets from hematopoietic stem cells. TPO exerts its effect through activation of the c-Mpl receptor and of multiple downstream signal transduction pathways. While the membrane-proximal half of the cytoplasmic domain appears to be required for the activation of signaling molecules that drive proliferation, the distal half and activation of the mitogen-activated protein kinase pathway have been implicated in mediating megakaryocyte maturation in vitro. To investigate the contribution of these two regions of c-Mpl and the signaling pathways they direct in mediating the function of TPO in vivo, we used a knock-in (KI) approach to delete the carboxy-terminal 60 amino acids of the c-Mpl receptor intracellular domain. Mice lacking the C-terminal 60 amino acids of c-Mpl (Delta60 mice) have normal platelet and megakaryocyte counts compared to wild-type mice. Furthermore, platelets in the KI mice are functionally normal, indicating that activation of signaling pathways connected to the C-terminal half of the receptor is not required for megakaryocyte differentiation or platelet production. However, Delta60 mice have an impaired response to exogenous TPO stimulation and display slower recovery from myelosuppressive treatment, suggesting that combinatorial signaling by both ends of the receptor intracellular domain is necessary for an appropriate acute response to TPO.

Characterization of the human suppressor of fused, a negative regulator of the zinc-finger transcription factor Gli.

Drosophila Suppressor of fused (Su(fu)) encodes a novel 468-amino-acid cytoplasmic protein which, by genetic analysis, functions as a negative regulator of the Hedgehog segment polarity pathway. Here we describe the primary structure, tissue distribution, biochemical and functional analyses of a human Su(fu) (hSu(fu)). Two alternatively spliced isoforms of hSu(fu) were identified, predicting proteins of 433 and 484 amino acids, with a calculated molecular mass of 48 and 54 kDa, respectively. The two proteins differ only by the inclusion or exclusion of a 52-amino-acid extension at the carboxy terminus. Both isoforms were expressed in multiple embryonic and adult tissues, and exhibited a developmental profile consistent with a role in Hedgehog signaling. The hSu(fu) contains a high-scoring PEST-domain, and exhibits an overall 37% sequence identity (63% similarity) with the Drosophila protein and 97% sequence identity with the mouse Su(fu). The hSu(fu) locus mapped to chromosome 10q24-q25, a region which is deleted in glioblastomas, prostate cancer, malignant melanoma and endometrial cancer. HSu(fu) was found to repress activity of the zinc-finger transcription factor Gli, which mediates Hedgehog signaling in vertebrates, and to physically interact with Gli, Gli2 and Gli3 as well as with Slimb, an F-box containing protein which, in the fly, suppresses the Hedgehog response, in part by stimulating the degradation of the fly Gli homologue. Coexpression of Slimb with Su(fu) potentiated the Su(fu)-mediated repression of Gli. Taken together, our data provide biochemical and functional evidence for the hypothesis that Su(fu) is a key negative regulator in the vertebrate Hedgehog signaling pathway. The data further suggest that Su(fu) can act by binding to Gli and inhibiting Gli-mediated transactivation as well as by serving as an adaptor protein, which links Gli to the Slimb-dependent proteasomal degradation pathway.

Activating Smoothened mutations in sporadic basal-cell carcinoma.

Basal-cell carcinomas (BCCs) are the commonest human cancer. Insight into their genesis came from identification of mutations in the PATCHED gene (PTCH) in patients with the basal-cell nevus syndrome, a hereditary disease characterized by multiple BCCs and by developmental abnormalities. The binding of Sonic hedgehog (SHH) to its receptor, PTCH, is thought to prevent normal inhibition by PTCH of Smoothened (SMO), a seven-span transmembrane protein. According to this model, the inhibition of SMO signalling is relieved following mutational inactivation of PTCH in basal-cell nevus syndrome. We report here the identification of activating somatic missense mutations in the SMO gene itself in sporadic BCCs from three patients. Mutant SMO, unlike wild type, can cooperate with adenovirus E1A to transform rat embryonic fibroblast cells in culture. Furthermore, skin abnormalities similar to BCCs developed in transgenic murine skin overexpressing mutant SMO. These findings support the role of SMO as a signalling component of the SHH-receptor complex and provide direct evidence that mutated SMO can function as an oncogene in BCCs.

Zfx mutation results in small animal size and reduced germ cell number in male and female mice.

The zinc-finger proteins ZFX and ZFY, encoded by genes on the mammalian X and Y chromosomes, have been speculated to function in sex differentiation, spermatogenesis, and Turner syndrome. We derived Zfx mutant mice by targeted mutagenesis. Mutant mice (both males and females) were smaller, less viable, and had fewer germ cells than wild-type mice, features also found in human females with an XO karyotype (Turner syndrome). Mutant XY animals were fully masculinized, with testes and male genitalia, and were fertile, but sperm counts were reduced by one half. Homozygous mutant XX animals were fully feminized, with ovaries and female genitalia, but showed a shortage of oocytes resulting in diminished fertility and shortened reproductive lifespan, as in premature ovarian failure in humans. The number of primordial germ cells was reduced in both XX and XY mutant animals at embryonic day 11.5, prior to gonadal sex differentiation. Zfx mutant animals exhibited a growth deficit evident at embryonic day 12.5, which persisted throughout postnatal life and was not complemented by the Zfy genes. These phenotypes provide the first direct evidence for a role of Zfx in growth and reproductive development.

Cloning and characterization of a human leptin receptor using a biologically active leptin immunoadhesin.

Leptin, the product of the ob gene, is a hormone secreted by fat cells which is primarily involved in the regulation of body weight. We have generated a leptin immunoadhesin (leptin-IgG) which was more potent than leptin alone at reducing body weight and food intake when injected into ob/ob mice. This molecule was used to identify high affinity binding sites on human embryonic 293 kidney cells and subsequently to isolate a cDNA encoding the leptin receptor from this cell line by expression cloning. This receptor corresponds to the short form of the recently isolated leptin receptor. Analysis of the expression pattern of the two forms of receptor by Northern blot, in situ hybridization and quantitative PCR showed that the receptor is expressed in most tissues but that the long form is prevalent in the hypothalamus.

Low levels of erythroid and myeloid progenitors in thrombopoietin-and c-mpl-deficient mice.

Thrombopoietin (TPO), the ligand for the c-mpl receptor, has been shown to be the major regulator of platelet production. Mice deficient in either c-mpl or TPO generated by homologous recombination show a dramatic decrease in platelet counts, but other blood cell counts are normal. Because TPO treatment of myelosuppressed mice not only enhances the recovery of platelets but also accelerates erythroid recovery, we investigated the levels of myeloid and erythroid progenitor cells in TPO-or c-mpl-deficient mice. Our results show that the number of megakaryocyte, granulocyte-macrophage, erythroid, and multilineage progenitors are significantly reduced in the bone marrow, spleen, and peripheral blood of either TPO-or c-mpl-deficient mice. Administration of recombinant murine TPO to TPO-deficient mice and control littermate mice significantly increased the absolute number of myeloid, erythroid, and mixed progenitors in bone marrow and spleen. This increase was especially apparent in TPO-deficient mice where numbers were increased to a level greater than in diluent-treated control mice and approached or equaled that in the TPO-treated control mice. Moreover, TPO-administration greatly increased the number of circulating progenitors as well as platelets in both TPO-deficient and control mice. Furthermore, the megakaryocytopoietic activity of other cytokines in the absence of a functional TPO or c-mpl gene was shown both in vitro and in vivo.

The bglX gene located at 47.8 min on the Escherichia coli chromosome encodes a periplasmic beta-glucosidase.

A new Escherichia coli gene, bgIX, encoding a beta-D-glucosidase (EC 3.2.1.21) has been characterized. The bgIX gene is located adjacent to the dld gene at 47.8 min or 2225 kb on the E. coli chromosome. The sequence of a 2.6 kb DNA fragment from this region revealed a large open reading frame encoding a protein of 765 amino acids. The BgIX protein was purified from the periplasm, and amino-terminal sequencing suggests that the mature protein is derived from cleavage of a 20 residue signal peptide. A search of the sequence databases revealed that BgIX is a member of a large family of beta-glucosidases from a variety of bacteria and fungi, and the plant Arabidopsis thaliana. It differs from the other four E. coli phospho-beta-glucosidases in sequence and in its periplasmic rather than cytoplasmic location. The BgIX enzyme has a Km of 18 +/- 1 mM and a Vmax of 3 +/- 0.7 mumol min-1 for the colorimetric substrate o-nitrophenyl beta-D-glucopyranoside.

Vascular endothelial growth factor-related protein: a ligand and specific activator of the tyrosine kinase receptor Flt4.

The tyrosine kinases Flt4, Flt1, and Flk1 (or KDR) constitute a family of endothelial cell-specific receptors with seven immunoglobulin-like domains and a split kinase domain. Flt1 and Flk1 have been shown to play key roles in vascular development; these two receptors bind and are activated by vascular endothelial growth factor (VEGF). No ligand has been identified for Flt4, whose expression becomes restricted during development to the lymphatic endothelium. We have identified cDNA clones from a human glioma cell line that encode a secreted protein with 32% amino acid identity to VEGF. This protein, designated VEGF-related protein (VRP), specifically binds to the extracellular domain of Flt4, stimulates the tyrosine phosphorylation of Flt4 expressed in mammalian cells, and promotes the mitogenesis of human lung endothelial cells. VRP fails to bind appreciably to the extracellular domain of Flt1 or Flk1. The protein contains a C-terminal, cysteine-rich region of about 180 amino acids that is not found in VEGF. A 2.4-kb VRP mRNA is found in several human tissues including adult heart, placenta, ovary, and small intestine and in fetal lung and kidney.

Physiological regulation of early and late stages of megakaryocytopoiesis by thrombopoietin.

Thrombopoietin (TPO) has recently been cloned and shown to regulate megakaryocyte and platelet production by activating the cytokine receptor c-mpl. To determine whether TPO is the only ligand for c-mpl and the major regulator of megakaryocytopoiesis, TPO deficient mice were generated by gene targeting. TPO-/- mice have a >80% decrease in their platelets and megakaryocytes but have normal levels of all the other hematopoietic cell types. A gene dosage effect observed in heterozygous mice suggests that the TPO gene is constitutively expressed and that the circulating TPO level is directly regulated by the platelet mass. Bone marrow from TPO-/- mice have decreased numbers of megakaryocyte-committed progenitors as well as lower ploidy in the megakaryocytes that are present. These results demonstrate that TPO alone is the major physiological regulator of both proliferation and differentiation of hematopoietic progenitor cells into mature megakaryocytes but that TPO is not critical to the final step of platelet production.

Mutations of the growth hormone receptor in children with idiopathic short stature. The Growth Hormone Insensitivity Study Group.

BACKGROUND: Short stature in children who are not deficient in growth hormone (GH) is probably caused by a variety of defects. Some children with idiopathic short stature have low serum concentrations of GH-binding protein, which is derived from the GH receptor. The possibility that low serum concentrations of GH-binding protein might indicate partial insensitivity to GH led us to investigate possible defects in the gene for the GH receptor in children with idiopathic short stature and low serum concentrations of GH-binding protein. METHODS: We studied 14 children with idiopathic short stature who were selected on the basis of normal GH secretion and low serum concentrations of GH-binding protein. Analysis of single-strand conformation polymorphisms and DNA sequencing were both used to identify mutations in the GH-receptor gene. RESULTS: Mutations in the region of the GH-receptor gene that codes for the extracellular domain of the receptor were found in 4 of the 14 children, but in none of 24 normal subjects. One of the four children with mutations was a compound heterozygote, with one mutation that reduced the affinity of the receptor for GH and a second mutation that may affect a function other than ligand binding. The remaining three children had single mutations in one allele of the gene. One mutation introduced a premature termination codon, and two caused substitutions of single amino acids in a structurally conserved domain of the receptor. CONCLUSIONS: Some children with idiopathic short stature may have partial insensitivity to GH due to mutations in the GH-receptor gene.

CpG islands in human ZFX and ZFY and mouse Zfx genes: sequence similarities and methylation differences.

The human ZFX, human ZFY, and mouse Zfx genes have CpG islands near their 5; ends. These islands are typical in that they span about 1.5 kb, contain transcription initiation sites, and encompass some 5' untranslated exons and introns. However, comparitive nucleotide sequencing of these human and mouse islands provided evidence of evolutionary conservation to a degree unprecedented among mammalian 5' CpG islands. In one stretch of 165 nucleotides containing 19 CpGs, mouse Zfx and human ZFX are identical to each other and differ from human ZFY at only 9 nucleotides. In contrast, we found no evidence of homologous CpG islands in the mouse Zfy genes, whose transcription is more circumscribed than that of human ZFX, human ZFY, and mouse Zfx. Using the isoschizomers HpaII and MspI to examine a highly conserved segment of the ZFX CpG island, we detected methylation on inactive mouse X chromosomes but not on inactive human X chromosomes. These observations parallel the previous findings that mouse Zfx undergoes X inactivation while human ZFX escapes it.

Isolation of cDNA clones encoding the mouse protein V-1.

The amino acid (aa) sequence of rat V-1, a developmentally regulated brain protein, was used to isolate clones encoding mouse V-1, a protein of 118 aa, from an embryoid body cDNA library. The aa sequences of rat and mouse V-1 are identical. V-1 shares several properties (including a 23 of 24 aa match of a tryptic peptide) with myotrophin, a protein that induces cardiac myocyte hypertrophy. Attempts to show that V-1 produced in Escherichia coli could induce the cardiac myocyte hypertrophy ascribed to myotrophin were unsuccessful.

Expression cloning of cardiotrophin 1, a cytokine that induces cardiac myocyte hypertrophy.

Heart failure continues to be a leading cause of mortality worldwide. A hallmark of this disease is dilated cardiac hypertrophy, which is accompanied by a reactivation of genes expressed in fetal heart development. Reasoning that fetal or embryonic growth factors may mediate the onset of cardiac hypertrophy, we have coupled expression cloning with an embryonic stem cell-based model of cardiogenesis to isolate a 21.5-kDa protein, cardiotrophin 1, that potently induces cardiac myocyte hypertrophy in vitro. Amino acid similarity data indicate that cardiotrophin 1 is a member of the leukemia inhibitory factor/ciliary neurotrophic factor/oncostatin M/interleukin 6/interleukin 11 family of cytokines. Several members of this family that are known to signal through the transmembrane protein gp130 stimulate cardiac myocyte hypertrophy, like cardiotrophin 1, suggesting that the gp130 signaling pathway may play a role in cardiac hypertrophy. A 1.4-kb cardiotrophin 1 mRNA is expressed in the heart and several other mouse tissues.

The structure of the Zfx gene on the mouse X chromosome.

Genes homologous to mouse Zfx have been identified on the X and Y chromosomes of all placental mammals examined. The genes of this ZFX/ZFY family appear to encode proteins comprising an amino-terminal acidic domain, a putative nuclear localizing signal, and a carboxy-terminal domain of 13 zinc fingers. These proteins likely function as transcription activators. Although roles for these proteins in sex determination, Turner syndrome, and spermatogenesis have been proposed, the biological processes in which these proteins function are not known. No comprehensive studies of gene structure have been reported for any member of the ZFX/ZFY family. Here, we report that mouse Zfx spans 50 kb and contains at least 11 exons. Exons 1 through 4 contain 5' untranslated sequences, exons 5 through 10 encode the acidic domain, exon 10 also encodes the putative nuclear localizing signal, and exon 11 encodes 13 zinc fingers and contains the 3' untranslated sequences. The 5' untranslated exons exhibit complex patterns of differential splicing. At the 5' end of this widely expressed gene, a 1.5-kb CpG island encompasses multiple transcription initiation sites as well as the first and second exons. The 5' portion of the CpG island displays promoter activity. This knowledge of the Zfx gene structure allowed us to reconstruct the splicing and retroposition events by which the Zfa gene on mouse chromosome 10 arose from a Zfx transcript.