Continuous versus intermittent extended adjuvant letrozole for breast cancer: final results of randomized phase III SOLE (Study of Letrozole Extension) and SOLE Estrogen Substudy.

BACKGROUND: Late recurrences in postmenopausal women with hormone receptor-positive breast cancers remain an important challenge. Avoidance or delayed development of resistance represents the main objective in extended endocrine therapy (ET). In animal models, resistance was reversed with restoration of circulating estrogen levels during interruption of letrozole treatment. This phase III, randomized, open-label Study of Letrozole Extension (SOLE) studied the effect of extended intermittent letrozole treatment in comparison with continuous letrozole. In parallel, the SOLE estrogen substudy (SOLE-EST) analyzed the levels of estrogen during the interruption of treatment. PATIENTS AND METHODS: SOLE enrolled 4884 postmenopausal women with hormone receptor-positive, lymph node-positive, operable breast cancer between December 2007 and October 2012 and among them, 104 patients were enrolled in SOLE-EST. They must have undergone local treatment and have completed 4-6 years of adjuvant ET. Patients were randomized between continuous letrozole (2.5 mg/day orally for 5 years) and intermittent letrozole treatment (2.5 mg/day for 9 months followed by a 3-month interruption in years 1-4 and then 2.5 mg/day during all of year 5). RESULTS: Intention-to-treat population included 4851 women in SOLE (n = 2425 in the intermittent and n = 2426 in the continuous letrozole groups) and 103 women in SOLE-EST (n = 78 in the intermittent and n = 25 in the continuous letrozole groups). After a median follow-up of 84 months, 7-year disease-free survival (DFS) was 81.4% in the intermittent group and 81.5% in the continuous group (hazard ratio: 1.03, 95% confidence interval: 0.91-1.17). Reported adverse events were similar in both groups. Circulating estrogen recovery was demonstrated within 6 weeks after the stop of letrozole treatment. CONCLUSIONS: Extended adjuvant ET by intermittent administration of letrozole did not improve DFS compared with continuous use, despite the recovery of circulating estrogen levels. The similar DFS coupled with previously reported quality-of-life advantages suggest intermittent extended treatment is a valid option for patients who require or prefer a treatment interruption.

Development of a conceptual model of cancer caregiver health literacy.

Caregivers play a vital role in caring for people diagnosed with cancer. However, little is understood about caregivers' capacity to find, understand, appraise and use information to improve health outcomes. The study aimed to develop a conceptual model that describes the elements of cancer caregiver health literacy. Six concept mapping workshops were conducted with 13 caregivers, 13 people with cancer and 11 healthcare providers/policymakers. An iterative, mixed methods approach was used to analyse and synthesise workshop data and to generate the conceptual model. Six major themes and 17 subthemes were identified from 279 statements generated by participants during concept mapping workshops. Major themes included: access to information, understanding of information, relationship with healthcare providers, relationship with the care recipient, managing challenges of caregiving and support systems. The study extends conceptualisations of health literacy by identifying factors specific to caregiving within the cancer context. The findings demonstrate that caregiver health literacy is multidimensional, includes a broad range of individual and interpersonal elements, and is influenced by broader healthcare system and community factors. These results provide guidance for the development of: caregiver health literacy measurement tools; strategies for improving health service delivery, and; interventions to improve caregiver health literacy.

Aromatase inhibitors associated with knee subchondral bone expansion without cartilage loss.

BACKGROUND: The profound estrogen depletion caused by aromatase inhibitors (AIs) is associated with musculoskeletal symptoms, but the underlying pathophysiology remains unclear. OBJECTIVE: To assess the effects of AI therapy on structural changes in knee cartilage and subchondral bone over 2 years in postmenopausal women. Setting and participants Thirty women with breast cancer, mean age 58.5 (standard deviation ± 5.6) years and 62 healthy controls, mean age 56.5 (standard deviation ± 4.6) years. MAIN OUTCOME MEASURES: Annualized changes in tibial cartilage volume and subchondral bone area, and worsening of tibiofemoral cartilage defects from paired knee magnetic resonance imaging 2 years apart were compared between the two groups. RESULTS: The AI-treated women had significantly greater expansion of the tibial plateau than the control group. The mean annualized differences, after adjusting for age, body mass index and baseline bone area, were 22.1 mm(2) (95% confidence interval (CI) 7.6-36.6, p = 0.003) for the medial tibial plateau and 19.1 mm(2) (95% CI 9.6-28.5, p < 0.001) for the lateral tibial plateau. The annual change in tibial cartilage volume and the worsening of cartilage defects did not differ between women taking AI therapy and controls. CONCLUSIONS: AI therapy is associated with knee subchondral bone expansion knee with no effect on knee cartilage in postmenopausal women without pre-existing joint symptoms. This suggests the effect of severe estrogen depletion on knee is on bone, with the tibial bone expansion most likely a response to mechanical load in the setting of bone loss. Whether this then results in an increased risk of knee osteoarthritis will need to be determined.

Cardiac safety of adjuvant pegylated liposomal doxorubicin with concurrent trastuzumab: a randomized phase II trial.

BACKGROUND: The cardiac safety of trastuzumab concurrent with pegylated liposomal doxorubicin (PLD) in an adjuvant breast cancer treatment regimen is unknown. PATIENTS AND METHODS: Women with resected node-positive or intermediate-risk node-negative HER2 overexpressing breast cancer and baseline left ventricular ejection fraction (LVEF)≥55% were randomized (1:2) to doxorubicin 60 mg/m2 (A)+cyclophosphamide 600 mg/m2 (C) every 21 days (q21d) for four cycles or PLD 35 mg/m2+C q21d+trastuzumab 2 mg/kg weekly (H) for 12 weeks. Both groups then received paclitaxel (Taxol, T) 80 mg/m2 with H for 12 weeks followed by H to complete 1 year. The primary end point was cardiac event rate or inability to administer 1 year of trastuzumab. RESULTS: Of 181 randomized patients, 179 underwent cardiac analysis. The incidence of cardiac toxicity or inability to administer trastuzumab due to cardiotoxicity was 18.6% [n=11; 95% confidence interval (CI) 9.7% to 30.9%] with A+C→T+H and 4.2% (n=5; 95% CI 1.4% to 9.5%) with PLD+C+H→T+H (P=0.0036). All events, except one, were asymptomatic systolic dysfunction or mildly symptomatic heart failure. Mean absolute LVEF reduction at cycle 8 was greater with doxorubicin (5.6% versus 2.1%; P=0.0014). CONCLUSION: PLD+C+H→T+H is feasible and results in lower early cardiotoxicity rates compared with A+C→T+H.

Renin, a secretory glycoprotein, acquires phosphomannosyl residues.

Renin is an aspartyl protease which is highly homologous to the lysosomal aspartyl protease cathepsin D. During its biosynthesis, cathepsin D acquires phosphomannosyl residues that enable it to bind to the mannose 6-phosphate (Man-6-P) receptor and to be targeted to lysosomes. The phosphorylation of lysosomal enzymes by UDP-GlcNAc:lysosomal enzyme N-acetylglucosaminylphosphotransferase (phosphotransferase) occurs by recognition of a protein domain that is thought to be present only on lysosomal enzymes. In order to determine whether renin, being structurally similar to cathepsin D, also acquires phosphomannosyl residues, human renin was expressed from cloned DNA in Xenopus oocytes and a mouse L cell line and its biosynthesis and posttranslational modifications were characterized. In Xenopus oocytes, the majority of the renin remained intracellular and underwent a proteolytic cleavage which removed the propiece. Most of the renin synthesized by oocytes was able to bind to a Man-6-P receptor affinity column (53%, 57%, and 90%, in different experiments), indicating the presence of phosphomannosyl residues. In the L cells, the majority of the renin was secreted but 5-6% of the renin molecules contained phosphomannosyl residues as demonstrated by binding of [35S]methionine-labeled renin to the Man-6-P receptor as well as direct analysis of [2-3H]mannose-labeled oligosaccharides. Although the level of renin phosphorylation differed greatly between the two cell types examined, these results demonstrate that renin is recognized by the phosphotransferase and suggest that renin contains at least part of the lysosomal protein recognition domain.

Polymorphism in the 5'-flanking region of the human insulin gene and its possible relation to type 2 diabetes.

The arrangement of the human insulin gene in DNA from 87 individuals was analyzed by the Southern blot hybridization technique with a cloned genomic human insulin probe. Insertions of 1.5 to 3.4 kilobase pairs in the 5'-flanking region of the gene were found in DNA from 38 individuals. These insertions occurred within 1.3 kilobase pairs of the transcription initiation site. In contrast, no insertions were observed in the region 3' to the coding sequence. The prevalence of these insertions in type 2 diabetes was significantly greater than in the other groups (P less than .001). The limitation of this striking length polymorphism to a potential promoter region suggests that these insertions may play a role in insulin gene expression.

Rat insulin genes: construction of plasmids containing the coding sequences.

Recombinant bacterial plasmids have been constructed that contain complementary DNA prepared from rat islets of Langerhans messenger RNA. Three plasmids contain cloned sequences representing the complete coding region of rat proinsulin I, part of the preproinsulin I prepeptide, and the untranslated 3' terminal region of the mRNA. A fourth plasmid contains sequences derived from the A chain region of rat preproinsulin II.

The impact and indications for Oncotype DX on adjuvant treatment recommendations when third-party funding is unavailable.

OBJECTIVES: Industry-supported decision impact studies demonstrate that Oncotype Dx (ODX) changes treatment recommendations (TR) in 24-40% of hormone receptor+/HER2- patients. ODX is not reimbursed by third-party payers in Australia, potentially resulting in more selective use. We sought to evaluate the impact of self-funded ODX on TRs. METHODS: Data collected included demographics, tumor characteristics, indication for ODX and pre- and post-recurrence score (RS) TR. Primary endpoint was frequency of TR change and associations with TR change were sought. RESULTS: Eighteen physicians contributed 382 patients (median age 54). A total of 232 (61%) of tumors were T1 and were grade 1, 2 and 3 in 49 (13%), 252 (66%) and 79 (21%). A total of 257 (67%) were node negative. Assay indications were: confirm need for chemotherapy (CT) (36%), confirm omission of CT (40%) and genuine equipoise (24%). RS was low (≤17) in 55%, intermediate (18-31) in 36% and high (≥32) in 9%. Thirty-eight percent of patients had TR change post-ODX. Sixty-five percent of patients recommended CT pre-ODX changed to hormone therapy alone (HT)-more likely if lower grade and if ER and/or PR > 10%. Fourteen percent of patients with pre-ODX TR for HT added CT-more likely if ER and/or PR ≤10% and if Ki67 > 15% Overall, TR for CT decreased from 47% to 24%. CONCLUSION: Patient-funded ODX changed TRs in 38% of patients, de-escalating 65% from CT to HT and adding CT to 14% of those recommended HT. These changes were greater than an industry-funded study suggesting that physicians can identify situations where the assay may influence decisions.

The effects of fasting and feeding on preproinsulin messenger RNA in rats.

The purpose of these experiments was to determine whether alterations in preproinsulin messenger (m)RNA activity could account for changes in insulin biosynthesis during fasting and refeeding. Rats were fasted 4 d and then fed for 6, 8, 24, or 48 h. With fasting, body weight decreased 25%, plasma glucose decreased from 6.1 to 2.2 mM, and pancreatic insulin content fell to 40% that of fed animals. Islet RNA decreased to 50% and protein to 55% that of control animals, while islet DNA content remained unchanged. After 6 h of refeeding, islet RNA content increased and was not significantly different from controls. Total islet and preproinsulin mRNA activity was estimated with an mRNA-dependent wheat germ cell-free protein synthesizing system. Preproinsulin and total protein synthesis was linearly dependent upon added RNA at concentrations up to 3 mug. Preproinsulin was identified by its mobility on SDS polyacrylamide gel electrophoresis and by hybrid arrested translation of preproinsulin mRNA. After an 18-h fast, islet mRNA activity decreased 33%; after 4 d mRNA activity decreased to 66% below that of control fed animals. Preproinsulin mRNA activity was decreased, but to a lesser extent, accounting for 20% of total islet protein in fed animals and 46% in the 4-d fasted animals. Total mRNA activity returned to control values after 8 h of refeeding and increased to 150% of controls at 24 and 48 h. Preproinsulin mRNA activity increased more rapidly on refeeding. By 8 h it was 160% of controls.To determine whether changes in preproinsulin mRNA activity were associated with changes in the amount of preproinsulin mRNA, nucleic acid hybridization analysis was performed. Pancreatic RNA from fed and fasted animals was electrophoresed on agarose gels, transferred to diazophenylthio paper, and hybridized to (32)P-labeled preproinsulin complementary (c)-DNA. This analysis demonstrated that changes in mRNA activity were associated with changes in the amount of hybridizable mRNA present. These studies are the first to demonstrate alterations of preproinsulin mRNA under any conditions, and the changes correlate with alterations in rates of insulin biosynthesis.

TGF-beta signaling blockade inhibits PTHrP secretion by breast cancer cells and bone metastases development.

Breast cancer frequently metastasizes to the skeleton, and the associated bone destruction is mediated by the osteoclast. Growth factors, including transforming growth factor-beta (TGF-beta), released from bone matrix by the action of osteoclasts, may foster metastatic growth. Because TGF-beta inhibits growth of epithelial cells, and carcinoma cells are often defective in TGF-beta responses, any role of TGF-beta in metastasis is likely to be mediated by effects on the surrounding normal tissue. However, we present evidence that TGF-beta promotes breast cancer metastasis by acting directly on the tumor cells. Expression of a dominant-negative mutant (TbetaRIIDeltacyt) of the TGF-beta type II receptor rendered the human breast cancer cell line MDA-MB-231 unresponsive to TGF-beta. In a murine model of bone metastases, expression of TbetaRIIDeltacyt by MDA-MB-231 resulted in less bone destruction, less tumor with fewer associated osteoclasts, and prolonged survival compared with controls. Reversal of the dominant-negative signaling blockade by expression of a constitutively active TGF-beta type I receptor in the breast cancer cells increased tumor production of parathyroid hormone-related protein (PTHrP), enhanced osteolytic bone metastasis, and decreased survival. Transfection of MDA-MB-231 cells that expressed the dominant-negative TbetaRIIDeltacyt with the cDNA for PTHrP resulted in constitutive tumor PTHrP production and accelerated bone metastases. These data demonstrate an important role for TGF-beta in the development of breast cancer metastasis to bone, via the TGF-beta receptor-mediated signaling pathway in tumor cells, and suggest that the bone destruction is mediated by PTHrP.

RNA-mediated gene duplication: the rat preproinsulin I gene is a functional retroposon.

Rats and mice have two, equally expressed, nonallelic genes encoding preproinsulin (genes I and II). Cytological hybridization with metaphase chromosomes indicated that both genes reside on rat chromosome I but are approximately 100,000 kilobases apart. In mice the two genes reside on two different chromosomes. DNA sequence comparisons of the gene-flanking regions in rats and mice indicated that the preproinsulin gene I has lost one of the two introns present in gene II, is flanked by a long (41-base) direct repeat, and has a remnant of a polydeoxyadenylate acid tract preceding the downstream direct repeat. These structural features indicated that gene I was generated by an RNA-mediated duplication-transposition event involving a transcript of gene II which was initiated upstream from the normal capping site. Sequence divergence analysis indicated that the pair of the original gene and its retroposed, but functional, counterpart (which appeared about 35 million years ago) is maintained by strong negative selection operating primarily on the segments encoding the chains of the mature hormone, whereas the segments encoding the parts of the polypeptide that are eliminated during processing and also the introns and the flanking regions are evolving neutrally.

Rabbit phosphoglucose isomerase/neuroleukin/autocrine motility factor: cloning via interspecies identity.

Phosphoglucose isomerase is the first committed enzyme of glycolysis. The protein also has a variety of biological activities on mammalian cells. The molecular basis of these extracellular functions is unclear, and the high resolution three-dimensional structure of a mammalian enzyme has not been described. We report here the cDNA and protein sequence for phosphoglucose isomerase from rabbit muscle. The sequence was obtained directly by PCR without the need to screen clones from a cDNA library and encoded active enzyme when expressed in bacterial cells. The 558 amino acid rabbit coding sequence is the same length as and highly similar (92% residue identity) to the sequences from human and pig and less so (88%) to the mouse enzyme. Non-conservative amino acid changes between the four mammalian sequences are concentrated in the first 35 and last five residues. The rabbit protein has an additional Cys residue and amino acid changes at five positions otherwise invariant in the mammalian enzymes.

Recombinant bovine rhodanese: purification and comparison with bovine liver rhodanese.

Recombinant bovine rhodanese (thiosulfate: cyanide sulfurtransferase, EC 2.8.1.1) has been purified to homogeneity from Escherichia coli BL21(DE3) by cation-exchange chromatography. Recombinant and bovine liver rhodanese coelectrophorese under denaturing conditions, with an apparent subunit molecular weight of 33,000. The amino terminal seven residues of the recombinant protein are identical to those of the bovine enzyme, indicating that E. coli also removes the N-terminal methionine. The Km for thiosulfate is the same for the two proteins. The specific activity of the recombinant enzyme is 12% higher (816 IU/mg) than that of the bovine enzyme (730 IU/mg). The two proteins are indistinguishable as to their ultraviolet absorbance and their intrinsic fluorescence. The ability of the two proteins to refold from 8 M urea to enzymatically active species was similar both for unassisted refolding, and when folding was assisted either by the detergent, lauryl maltoside or by the E. coli chaperonin system composed of cpn60 and cpn10. Bovine rhodanese is known to have multiple electrophoretic forms under native conditions. In contrast, the recombinant protein has only one form, which comigrates with the least negatively charged of the bovine liver isoforms. This is consistent with the retention of the carboxy terminal residues in the recombinant protein that are frequently removed from the bovine liver protein.

Effects of glucose on proinsulin messenger RNA in rats in vivo.

The purpose of these studies was to determine whether glucose, the principal regulator of insulin biosynthesis in mammals, controls synthesis through alterations in levels of proinsulin mRNA in whole animals. Rats were starved for 3 days and then either refed or injected with glucose or saline for 24 h. Glucose injection raised plasma glucose levels equivalent to levels seen with refeeding but provided less than 20% of caloric replacement. Pancreatic RNA was extracted and the relative concentration of proinsulin mRNA was determined by blot hybridization with a cloned rat proinsulin cDNA probe. In starved animals proinsulin mRNA levels were 15-20% that of fed controls. Glucose injection produced a specific three- to fourfold increase in proinsulin mRNA levels relative to total pancreatic RNA, within 24 h. The effect was measurable 2 h after glucose injection and appeared largely complete by 12 h. Actinomycin D blocked the glucose-induced increase in proinsulin mRNA. These studies demonstrate effects of changes of plasma glucose on levels of proinsulin mRNA. Their rapidity of onset and large magnitude are comparable to effects of glucose on rates of insulin biosynthesis in isolated islets and suggest that insulin biosynthesis is regulated at least in part by levels of proinsulin mRNA.

Analysis of insulin gene expression in human pancreas.

The purpose of these studies was to determine whether insulin gene expression at the level of proinsulin mRNA could be studied in human pancreas. RNA was isolated from autopsy specimens and analyzed by RNA-blot hybridization with various 32P-human insulin gene probes spanning either the entire gene or the second intervening sequence. A major band 0.62 kilobases (kb) in length accounted for over 95% of the mRNA, consistent in size with presumed mature proinsulin mRNA. In addition, minor bands of 1.5 and 1.3 kb were seen, consistent with an initial gene transcript containing both intervening sequences and with a processed intermediate. The 1.5- and 1.3-kb RNA were confirmed to be proinsulin mRNA precursors by hybridization specifically with the IVS II probe. Total RNA and polyadenylated RNA from five normal pancreata and two insulinomas revealed the same pattern. This method provides a means of determining whether altered insulin gene expression is one cause of diabetes.

The crystal structure of human phosphoglucose isomerase at 1.6 A resolution: implications for catalytic mechanism, cytokine activity and haemolytic anaemia.

Phosphoglucose isomerase (PGI) is a multifunctional protein, which, inside the cell, functions as a housekeeping enzyme of glycolysis and gluconeogenesis and, outside the cell, exerts wholly unrelated cytokine properties. We have determined the structure of human PGI to a resolution of 1.6 A using X-ray crystallography. The structure is highly similar to other PGIs, especially the architecture of the active site. Fortuitous binding of a sulphate molecule from the crystallisation solution has facilitated an accurate description of the substrate phosphate-binding site. Comparison with both native and inhibitor-bound rabbit PGI structures shows that two loops move closer to the active site upon binding inhibitor. Interestingly, the human structure most closely resembles the inhibitor-bound structure, suggesting that binding of the phosphate moiety of the substrate may trigger this conformational change. We suggest a new mechanism for catalysis that uses Glu357 as the base catalyst for the isomerase reaction rather than His388 as proposed previously. The human PGI structure has also provided a detailed framework with which to map mutations associated with non-spherocytic haemolytic anaemia.

Proinsulin I and II gene expression in inbred mouse strains.

Mice and rats express two nonidentical insulins from a pair of unlinked genes. We have applied a nuclease protection assay, which can sensitively quantify each of the mouse insulin mRNAs, to the resolution of the following questions concerning their expression. First, it has not been established whether alterations in expression of one or both of these genes cause differing total insulin biosynthetic capacity noted between several inbred mouse strains. These studies showed that the relative abundance of mRNAs encoding mouse insulins I and II was identical in four separate mouse strains. In spontaneously obese, hyperinsulinemic (db/db)C57BL/KsJ mice, both proinsulin I and proinsulin II mRNAs were increased relative to the levels in normal (+/db) C57BL/KsJ mice, but again the ratio of the two mRNAs did not differ. The ratio was nearly identical to that for the orthologous mRNAs in rats, indicating that the mechanisms which regulate insulin mRNAs in rodents are conserved in both genes in several mouse strains and between rodent species. This finding suggests that differences between mouse strains in insulin biosynthetic capacity result from differences in the glucose sensing/signalling mechanism at a point before coordinate gene transcription. Second, low levels of insulin synthesis have been suggested as an explanation for relatively high levels of insulin in several nonpancreatic tissues. We showed that the ribonuclease protection assay, sufficiently sensitive to measure 1/2000th the amount of insulin mRNA present in pancreas, was unable to detect insulin mRNA in salivary gland. This result indicates that the high levels of radioimmunoassayable insulin detected in salivary glands are not the result of insulin synthesis in situ.

Molecular biology for nonmolecular biologists.

Molecular biology offers various powerful tools for understanding the etiology of diabetes mellitus. These methods can be understood by nonmolecular biologists. Topics briefly introduced include 1) control of gene expression, 2) cloning of genes, 3) how gene promoters work, 4) transgenic mice, 5) mutagenesis in vitro, 6) human molecular genetics, and 7) the new powerful polymerase chain-reaction technique, which greatly simplifies many of the other methods.

Insulin gene structure and function: a review of studies using recombinant DNA methodology.

This review focuses on recent advances in molecular biology as they pertain to the insulin gene and diabetes mellitus. The structure of the human insulin gene is examined, and factors related to its normal functioning in the beta cells of the pancreas are explored. DNA polymorphisms near the insulin locus and their relationship with certain types of diabetes are considered, as are recently characterized human insulin gene mutations. Events in animal models for diabetes that reflect altered insulin gene expression are discussed and the potential application of gene therapy in human diabetes is examined. Recombinant DNA methodology holds great promise as a tool for providing better understanding of the causes of diabetes and potential curative treatment.

Unmet needs and depression among carers of people newly diagnosed with cancer.

AIMS: The aims of this analysis were to examine levels of unmet needs and depression among carers of people newly diagnosed with cancer and to identify groups who may be at higher risk, by examining relationships with demographic characteristics. METHODS: One hundred and fifty dyads of people newly diagnosed with cancer and their carers, aged 18 years and older, were recruited from four Australian hospitals. People with cancer receiving adjuvant cancer treatment with curative intent, were eligible to participate. Carers completed the Supportive Care Needs Survey-Partners & Caregivers (SCNS-P&C45), and both carers and patients completed the Centre of Epidemiologic-Depression Scale (CES-D). RESULTS: Overall, 57% of carers reported at least one, 37% at least three, 31% at least five, and 15% at least 10 unmet needs; the most commonly endorsed unmet needs were in the domains of information and health care service needs. Thirty percent of carers and 36% of patients were at risk of clinical depression. A weak to moderate positive relationship was observed between unmet needs and carer depression (r=0.30, p<0.001). Carer levels of unmet needs were significantly associated with carer age, hospital type, treatment type, cancer type, living situation, relationship status (in both uni- and multi-factor analysis); person with cancer age and carer level of education (in unifactor analysis only); but not with carer gender or patient gender (in both uni- and multi-factor analyses). CONCLUSION: Findings highlight the importance of developing tailored programmes to systematically assist carers who are supporting patients through the early stages of cancer treatment.