The role of infiltrating lymphocytes in the neo-adjuvant treatment of women with HER2-positive breast cancer.

BACKGROUND: Pre-treatment tumour-associated lymphocytes (TILs) and stromal lymphocytes (SLs) are independent predictive markers of future pathological complete response (pCR) in HER2-positive breast cancer. Whilst studies have correlated baseline lymphocyte levels with subsequent pCR, few have studied the impact of neoadjuvant therapy on the immune environment. METHODS: We performed TIL analysis and T-cell analysis by IHC on the pretreatment and 'On-treatment' samples from patients recruited on the Phase-II TCHL (NCT01485926) clinical trial. Data were analysed using the Wilcoxon signed-rank test and the Spearman rank correlation. RESULTS: In our sample cohort (n = 66), patients who achieved a pCR at surgery, post-chemotherapy, had significantly higher counts of TILs (p = 0.05) but not SLs (p = 0.08) in their pre-treatment tumour samples. Patients who achieved a subsequent pCR after completing neo-adjuvant chemotherapy had significantly higher SLs (p = 9.09 × 10-3) but not TILs (p = 0.1) in their 'On-treatment' tumour biopsies. In a small cohort of samples (n = 16), infiltrating lymphocyte counts increased after 1 cycle of neo-adjuvant chemotherapy only in those tumours of patients who did not achieve a subsequent pCR. Finally, reduced CD3 + (p = 0.04, rho = 0.60) and CD4 + (p = 0.01, rho = 0.72) T-cell counts in 'On-treatment' biopsies were associated with decreased residual tumour content post-1 cycle of treatment; the latter being significantly associated with increased likelihood of subsequent pCR (p < 0.01). CONCLUSIONS: The immune system may be 'primed' prior to neoadjuvant treatment in those patients who subsequently achieve a pCR. In those patients who achieve a pCR, their immune response may return to baseline after only 1 cycle of treatment. However, in those who did not achieve a pCR, neo-adjuvant treatment may stimulate lymphocyte influx into the tumour.

Recombinant PAPP-A resistant insulin-like growth factor binding protein 4 (dBP4) inhibits angiogenesis and metastasis in a murine model of breast cancer.

BACKGROUND: The Insulin-like growth factor (IGF) pathway plays a role in tumour development and progression. In vivo, IGF1 activity is regulated by the IGF binding proteins (IGFBPs). IGFBP4 inhibits the activity of IGF1 but proteolytic cleavage by pregnancy-associated plasma protein-A (PAPP-A) releases active IGF1. A modified IGFBP4, dBP4, which was resistant to PAPP-A cleavage but retained IGF1 binding capacity, was engineered, expressed in Human Embryonic Kidney (HEK) 293 cells and purified. This study examined the effects of dBP4 on IGF1-induced cell migration, invasion and angiogenesis in vitro. The effect of intra-tumour injections of dBP4 on tumour angiogenesis and metastasis was examined using the 4T1.2luc orthotopic model of breast cancer. METHODS: PAPP-A resistance and IGF binding capacity of dBP4 were characterized by Western blot and surface plasmon resonance, respectively. 4T1.2luc are mouse mammary adenocarcinoma cells transfected with luciferase to allow in vivo imaging. The effect of dBP4 on IGF1-induced Akt activation in 4T1.2luc cells was assessed by Western blot. Cell migration and invasion assays were performed using 4T1.2luc cells. Angiokit™ assays and Matrigel® implants were used to assess the effects of dBP4 on angiogenesis in vitro and in vivo, respectively. An orthotopic breast cancer model - 4T1.2luc cells implanted in the mammary fat pad of BALB/c mice - was used to assess the effect of intra tumour injection of purified dBP4 on tumour angiogenesis and metastasis. Tumour growth and lung metastasis were examined by in vivo imaging and tumour angiogenesis was evaluated by CD31 immunohistochemistry. RESULTS: Our engineered, PAPP-A resistant IGFBP4 (dBP4) retained IGF1 binding capacity and inhibited IGF1 activation of Akt as well as IGF1-induced migration and invasion by 4T1.2 mammary adenocarcinoma cells. dBP4 inhibited IGF1-induced angiogenesis in vitro and in Matrigel implants in vivo. Direct intra-tumour injection of soluble dBP4 reduced angiogenesis in 4T1.2 luc mammary tumours tumour and reduced lung metastasis. CONCLUSION: A PAPP-A resistant IGFBP4, dBP4, inhibits angiogenesis and metastasis in 4T1.2 mammary fat pad tumours. This study highlights the therapeutic potential of dBP4 as an approach to block the tumour-promoting actions of IGF1.

HER2-family signalling mechanisms, clinical implications and targeting in breast cancer.

Approximately 20 % of human breast cancers (BC) overexpress HER2 protein, and HER2-positivity is associated with a worse prognosis. Although HER2-targeted therapies have significantly improved outcomes for HER2-positive BC patients, resistance to trastuzumab-based therapy remains a clinical problem. In order to better understand resistance to HER2-targeted therapies in HER2-positive BC, it is necessary to examine HER family signalling as a whole. An extensive literature search was carried out to critically assess the current knowledge of HER family signalling in HER2-positive BC and response to HER2-targeted therapy. Known mechanisms of trastuzumab resistance include reduced receptor-antibody binding (MUC4, p95HER2), increased signalling through alternative HER family receptor tyrosine kinases (RTK), altered intracellular signalling involving loss of PTEN, reduced p27kip1, or increased PI3K/AKT activity and altered signalling via non-HER family RTKs such as IGF1R. Emerging strategies to circumvent resistance to HER2-targeted therapies in HER2-positive BC include co-targeting HER2/PI3K, pan-HER family inhibition, and novel therapies such as T-DM1. There is evidence that immunity plays a key role in the efficacy of HER-targeted therapy, and efforts are being made to exploit the immune system in order to improve the efficacy of current anti-HER therapies. With our rapidly expanding understanding of HER2 signalling mechanisms along with the repertoire of HER family and other targeted therapies, it is likely that the near future holds further dramatic improvements to the prognosis of women with HER2-positive BC.

A preclinical evaluation of the PI3K alpha/delta dominant inhibitor BAY 80-6946 in HER2-positive breast cancer models with acquired resistance to the HER2-targeted therapies trastuzumab and lapatinib.

The PI3K pathway is a key mechanism of trastuzumab resistance, but early attempts to indirectly target this pathway with mTOR inhibitors have had limited success. We present the results of a preclinical study of the selective alpha/delta isoform dominant PI3K inhibitor BAY 80-6946 tested alone and in combination with HER2-targeted therapies in HER2-positive cell lines, including models with acquired resistance to trastuzumab and/or lapatinib. A panel of HER2-positive breast cancer cells were profiled for their mutational status using Sequenom MassARRAY, PTEN status by Western blot, and anti-proliferative response to BAY 80-6946 alone and in combination with the HER2-targeted therapies trastuzumab, lapatinib and afatinib. Reverse phase protein array was used to determine the effect of BAY 80-6946 on expression and phosphorylation of 68 proteins including members of the PI3K and MAPK pathways. The Boyden chamber method was used to determine if BAY 80-6946 affected cellular invasion and migration. BAY 80-6946 has anti-proliferative and anti-invasive effects when used alone in our panel of cell lines (IC50s 3.9-29.4 nM). BAY 80-6946 inhibited PI3K signalling and was effective in cells regardless of their PI3K, P53 or PTEN status. The combination of HER2-targeted therapies and BAY 80-6946 inhibited growth more effectively than either therapy used alone (with clear synergism in many cases), and can restore sensitivity to trastuzumab and lapatinib in cells with acquired resistance to either trastuzumab and/or lapatinib. The addition of BAY 80-6946 to HER2-targeted therapy could represent an improved treatment strategy for patients with refractory metastatic HER2-positive breast cancer, and should be considered for clinical trial evaluation.

BCL2 protein signalling determines acute responses to neoadjuvant chemoradiotherapy in rectal cancer.

UNLABELLED: In locally advanced rectal cancer, neoadjuvant chemoradiotherapy is performed prior to surgery to downstage the tumour. Thirty to 40 % of patients do not respond. Defects in apoptotic machinery lead to therapy resistance; however, to date, no study quantitatively assessed whether B cell lymphoma 2 (BCL2)-dependent regulation of mitochondrial apoptosis, effector caspase activation downstream of mitochondria or a combination of both predicts patient responses. In a cohort of 20 rectal cancer patients, we performed protein profiling of tumour tissue and employed validated ordinary differential equation-based systems models of apoptosis signalling to calculate the ability of cancer cells to undergo apoptosis. Model outputs were compared to clinical responses. Systems modelling of BCL2-signalling predicted patients in the poor response group (p = 0.0049). Systems modelling also demonstrated that rectal cancers depended on BCL2 rather than B cell lymphoma-extra large (BCL(X)L) or myeloid cell leukemia 1 (MCL1) for survival, suggesting that poor responders may benefit from therapy with selective BCL2 antagonists. Dynamic modelling of effector caspase activation could not stratify patients with poor response and did not further improve predictive power. We deliver a powerful patient stratification tool identifying patients who will likely not benefit from neoadjuvant chemoradiotherapy and should be prioritised for surgical resection or treatment with BCL2 antagonists. KEY MESSAGES: Modelling BCL2-family proteins identifies patients unresponsive to therapy. Caspase activation downstream of mitochondria cannot identify these patients. Rectal tumours of poor responders are BCL2- but not BCL-XL-dependent. DR_MOMP allows clinicians to identify patients who would not benefit from therapy. DR_MOMP is also a useful patient stratification tool for BCL2 antagonists.

Is obesity associated with reduced health-related quality of life in Latino, Black and White children in the community?

OBJECTIVE: Few studies have examined the impact of obesity on health-related quality of life (HRQOL) in non-clinical community samples of children, and methodological limitations have hindered drawing firm conclusions, especially whether the impact is similar across racial/ethnic groups. The present aims were to examine at what levels of non-normal weight, school-aged children experience lower HRQOL and whether this differs among racial/ethnic groups, when controlling for socioeconomic status (SES) differences. DESIGN: Cross-sectional community cohort survey. SUBJECTS AND METHODS: Data are from the Healthy Passages, reporting on 4824 Latino, black and white 5th graders in a population-based survey conducted in three United States metropolitan areas. Children's weight status was classified from measured weight and height into underweight (1%), normal weight (52%), overweight (19%), obese (13%) and extremely obese (14%). Children reported their own HRQOL using the Pediatric Quality of Life Inventory and additional scales addressing global self-worth, physical appearance and body satisfaction. Parents reported children's overall health status. RESULTS: Each increment in higher non-healthy weight class-overweight to obese to extremely obese-was associated with significantly lower scores in more domains of psychosocial HRQOL compared with that in normal weight. However, only extremely obese children reported significantly lower physical HRQOL. Differences among weight classes remained when adjusting for SES and were independent of race/ethnicity. Underweight children generally reported HRQOL that was not significantly different from normal weight children. CONCLUSIONS: Overweight, obese and extremely obese 5th graders on average experience worse HRQOL than normal weight children, especially in psychosocial domains including self-worth and peer relationships, regardless of race/ethnicity. If messages can be conveyed in a sensitive and supportive manner, the desire to improve HRQOL could provide additional motivation for children and their parents in addressing unhealthy weight.

The influence of task environment and health literacy on the quality of parent-reported ADHD data.

OBJECTIVES: To determine 1) the extent to which paper-based and computer-based environments influence the sufficiency of parents' report of child behaviors and the accuracy of data on current medications, and 2) the impact of parents' health literacy on the quality of information produced. METHODS: We completed a randomized controlled trial of data entry tasks with parents of children with Attention Deficit Hyperactivity Disorder (ADHD). Parents completed the NICHQ Vanderbilt ADHD screen and a report of current ADHD medications on paper or using a computer application designed to facilitate data entry. Literacy was assessed by the Test of Functional Health Literacy in Adults (TOFHLA). Primary outcomes included sufficient data to screen for ADHD subtypes and accurate report of total daily dose of prescribed ADHD medications. RESULTS: Of 271 parents screened, 194/271 were eligible and 182 were randomized. Data from 180 parents were analyzed. 5.6% parents had inadequate/marginal TOFHLA scores. Using the computer, parents provided more sufficient and accurate data compared to paper (sufficiency for ADHD screening, paper vs. computer: 87.8% vs. 93.3%, P = 0.20; accuracy of medication report: 14.3% vs. 69.4%; p<0.0001). Parents with adequate literacy had increased odds of reporting sufficient and accurate data (sufficiency for ADHD screening: OR 8.0, 95% CI 2.0-32.1; accuracy of medication report: OR 4.4, 95% CI 0.5-37.4). In adjusted models, the computer task environment remained a significant predictor of accurate medication report (OR 18.7, 95% CI 7.5-46.9). CONCLUSIONS: Structured, computer-based data entry by parents may improve the quality of specific types of information needed for ADHD care. Health literacy affects parents' ability to share valid information.

Previously unidentified complex cytogenetic changes found in a pediatric case of solid-pseudopapillary neoplasm of the pancreas.

Solid pseudopapillary neoplasm of the pancreas (SPNP) is a rare tumor with low malignant potential found in adolescent girls and young women. The pathogenesis of SPNP remains uncertain and its management is controversial. Genetic changes associated with SPNP have seldom been reported. We describe here the cytogenetic investigation of a case of SPNP in a 13-year-old girl whose tumor cells revealed two unrelated clones: one clone characterized by complex karyotypic changes, including breakpoints in two common fragile sites at chromosome 2, band q33, and chromosome 4, band q31, and the second clone defined by partial monosomy for chromosome X. Loss of heterozygosity for HRAS was also identified by array comparative genomic hybridization (a-CGH). These cumulative changes seem insufficient for activation of cell transformation, but could possibly play a role in priming the cell for future mutagenic events.

Regression of pre-established atherosclerosis in the apoE-/- mouse by conjugated linoleic acid.

Conjugated linoleic acid (CLA) refers to a group of positional and geometric isomers of linoleic acid that has been shown to suppress the development of atherosclerosis in a rabbit model. We investigated whether CLA acts as a cyclo-oxygenase (COX) inhibitor or as an agonist of the peroxisome-proliferator-activator receptor (PPAR) gamma in the ApoE(-/-) mouse model. In vitro, a 9-cis, 11-trans isomer of CLA inhibited prostaglandin formation and oxygen consumption by both isoforms of COX, with no evidence by MS of alternative products being generated. In vivo, supplementation with CLA was found to induce resolution of atherosclerosis. The effect of CLA in vivo could not be explained by COX inhibition alone, as urinary prostaglandin levels were unchanged in animals receiving CLA supplementation, and administration of selective COX inhibitors did not induce lesion regression. There was however induction of PPAR gamma, a known response to agonists of this nuclear orphan receptor.

Reactivity and isotype profiling of monoclonal antibodies using multiple antigenic peptides.

The characterisation of monoclonal antibodies (MAbs) is essential for the development of assay systems particularly where antigens have been developed using synthetic peptides. Indeed some peptide-carrier conjugates fail to induce immune responses and may not generate antibodies that bind to native protein. As an alternative to peptide-carrier conjugates, multiple antigenic peptides (MAPs) have been used for immunization strategies, but with little regard to the characteristics of the MAbs produced. In this study, we used 3 MAPs of Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) to immunise BALB/c mice. Overall, the polyclonal antibody responses from tail bleeds showed that MAPs evoked B-cell responses. However, on screening 144 hybridomas, 24 MAb supernatants exhibited weak to moderate reactivity in enzyme-linked immunosorbant assay (ELISA) and against cell cytospin preparations (B95.8 and AG876 LCL), respectively. Isotype profiling of hybridoma supernatants also showed that 11 out of 24 were IgM. Further characterization of 6 MAbs in Western blotting showed reactivity to recombinant LMP1 and only one MAb (B28D) showed weak reactivity to the malignant cells (Hodgkin/Reed-Sternberg; HRS cells) of an EBV+ Hodgkin's lymphoma using paraffin-embedded tissue. It is probable that these MAPs failed to augment T-cell help and contributed to the production of low affinity (IgM) antibodies. These observations may be of importance to future immunization strategies, where MAPs are used in the production of monoclonal reagents.

Modular component exchange for treatment of recurrent dislocation of a total hip replacement in selected patients.

BACKGROUND: Exchange of modular components is a treatment option for the correction of recurrent dislocation of a total hip replacement. In this study, we reviewed our experience with this technique in order to define patient selection criteria and to report the outcome of treatment. METHODS: Of 2935 hips treated with primary porous-coated total hip arthroplasty, fourteen (in fourteen patients) that met certain preoperative and intraoperative criteria were treated with modular component exchange because of recurrent hip instability. The primary arthroplasties in these fourteen patients had been performed through a posterior approach. At the revisions, we removed any sources of osseous or soft-tissue impingement that contributed to dislocation. Acceptable stability at the completion of component exchange was defined as stability in maximum flexion, in full extension with external rotation, and in at least 45 degrees of internal rotation with the hip in 90 degrees of flexion and maximum adduction. RESULTS: One patient was lost to follow-up. At a mean of 5.8 years (range, 2.8 to 11.8 years) after the revision, ten of the remaining thirteen patients had not had a dislocation. Of the three patients in whom the hip dislocated after the modular component exchange, only one had recurrent dislocation; thus, recurrent dislocation was eliminated in twelve of thirteen patients. CONCLUSIONS: In selected cases, modular component exchange for the treatment of recurrent hip dislocation has a success rate comparable with that of more extensive operations. This method should be considered because it avoids the morbidity associated with revision of well-fixed components. However, to ensure the appropriateness of this surgical option, each patient must be thoroughly evaluated to identify all factors that contribute to instability and adequate intraoperative stability must be achieved.

Frequent expression of the tumor necrosis factor receptor-associated factor 1 in latent membrane protein 1-positive posttransplant lymphoproliferative disease and HIV-associated lymphomas.

The tumor necrosis factor receptor-associated factor 1 (TRAF1) participates in the signal transduction of various members of the tumor necrosis factor receptor (TNFR) family, including TNFR2, CD40, CD30, and the Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1). In vitro, TRAF1 is induced by LMP1, and previous studies have suggested that expression of TRAF1 is higher in EBV-associated tumors than in their EBV-negative counterparts. To determine whether this was the case in posttransplant lymphoproliferative disease (PTLD) and related disorders, we used immunohistochemistry to analyze expression of TRAF1 in a total of 42 such lesions arising in a variety of immunosuppressive states. The specimens consisted of 22 PTLD lesions, 18 acquired immunodeficiency syndrome-associated lymphomas, including 6 primary central nervous system lymphomas, and 2 cases of Hodgkin disease. The presence of latent EBV infection was determined by EBER in situ hybridization, and expression of EBV-LMP1 was detected by immunohistochemistry. Latent EBV infection, as determined by a positive EBER signal, was detected in 36 of 42 tumors. Of the EBER-positive specimens, 30 of 36 also expressed LMP1. Twenty-four of 30 LMP1-positive tumors, including both Hodgkin disease specimens, expressed TRAF1, compared with only 3 of 12 LMP1-negative tumors. This difference was statistically significant (P <.005). These results show frequent expression of TRAF1 at the protein level in LMP1-positive PTLD and related disorders and suggest an important role for LMP1-mediated TRAF1 signaling in the pathogenesis of EBV-positive tumors arising in immunosuppressive states.

Expression of the tumour necrosis factor receptor-associated factors 1 and 2 in Hodgkin's disease.

The tumour necrosis factor receptor-associated factors (TRAFs) 1 and 2 participate in the signal transduction of various members of the tumour necrosis factor receptor (TNFR) family, including TNFR1, TNFR2, CD40, CD30, and the Epstein-Barr virus (EBV)-encoded latent membrane protein-1 (LMP1). Previous in situ hybridization studies have demonstrated TRAF1 transcripts in the malignant cells of the majority of Hodgkin's disease (HD) tumours, where the expression of TRAF1 was higher in EBV-associated tumours than in their EBV-negative counterparts. In order to determine whether TRAF1 and also TRAF2 were expressed at the protein level in HD and whether there was any relationship to EBV status, immunohistochemistry has been used to detect these proteins in a series of HD specimens. TRAF1 protein was detected more frequently in Hodgkin/Reed-Sternberg (HRS) cells from EBV-positive tumours than in their EBV-negative counterparts. This difference was statistically significant (p=0.01). In contrast, TRAF2 expression by HRS cells appeared to be independent of EBV status. Using a sequential labelling approach, co-localization of LMP1 with either TRAF1 or TRAF2 was also demonstrated in HRS cells from EBV-positive tumours.

Working parents: what factors are involved in their ability to take time off from work when their children are sick?

BACKGROUND: A series of studies has demonstrated that sick children fare better when their parents are present. OBJECTIVE: To examine working conditions that determine whether parents can spend time with and become involved in the care of their children when they are sick. DESIGN: Survey with a multivariate analysis of factors influencing parental care of sick children. PARTICIPANTS: Mixed-income urban working parents aged 26 to 29 years participating in the Baltimore Parenthood Study. RESULTS: Only 42% of working parents in our sample cared for their young children when they became sick. A multivariate logistic regression analysis was conducted to predict which parents stayed at home when their children were sick. Those parents who had either paid sick or vacation leave were 5.2 times as likely to care for their children themselves when they were sick. Of parents with less than a high school education, 17% received paid leave, compared with 57% of parents with a general equivalency diploma, 76% of parents with a high school diploma, and 92% of parents with more than a high school education (P<.001). CONCLUSIONS: The finding that many parents were unable to care for their sick children themselves is important for pediatric care. While low-income children are more likely to face marked health problems and to be in need of parental care, they are more likely to live in households in which parents lack paid leave and cannot afford to take unpaid leave.

Ambient solar UVR, personal exposure and protection.

Ambient solar ultraviolet radiation (UVR) has been monitored around Australia by the Australian Radiation Laboratory (ARL) and its successor ARPANSA since the mid 1980's using a network of radiometric detectors and a spectroradiometer (SRM) for spectral measurements, based in Melbourne. In a continent the size of Australia, the levels vary markedly, basically following a latitude gradient increasing towards the equator but with local geographical and weather effects also evident. ARL also conducts personal exposure studies of various population groups in collaboration with other research centres to gather information on what fraction of the ambient UVR people receive. ARL also undertakes studies on the UVR protection provided by sunscreens, clothing, hats, sunglasses and other materials in an attempt to improve UVR protection used by the public.

Hip arthroplasty in chronic dialysis patients.

We retrospectively reviewed 15 patients (24 hips) on chronic renal dialysis who underwent hip arthroplasty between 1970 and 1990. The average age at surgery was 39 years; the average follow-up was 8 years (range, 1-19 years). All follow-up of less than 5 years relates to those patients who died. Of these 24 hips, 14 (58%) failed or were failing due to loosening; the average time to revision was 7 years (range, 1.5-14 years). A complicated course was experienced in 16 hips (66%), primarily related to medical difficulties. There was one perioperative death. The following orthopedic complications afflicted 5 hips (21%): one femur fracture during revision; one femur fracture at 2 months after revision; one dislocation during seizure; one displacement of acetabular cup requiring recementing; and late generalized septic death of one patient (with both hips involved). Within an average of 3 years (range, 15 months to 7 years) after their index surgery, 6 of the 15 patients (40%) died. The patients who lived were chronically ill, and all but three remained on long-term dialysis. The functional level of all those remaining on dialysis steadily declined, and none reached a quality of life comparable to an osteoarthritic patient. This study confirms a previously reported high mortality and morbidity rate in this population. Despite their difficulties, 22/24 primary hips were relieved of pain and increased in function; six patients returned to work. We see no better alternative for pain relief in total hip arthroplasty, particularly in view of contemporary surgical techniques and improved medical management.

Solar UVR exposures of primary school children at three locations in Queensland.

The ultraviolet radiation (UVR) exposures of primary school children in Brisbane, Toowoomba and Mackay (latitudes 27 degrees 30', 27 degrees 33' and 21 degrees 15' south, respectively) were assessed over a period of 2 weeks at each location using UVR-sensitive polysulfone (PS) film badges attached at the shoulder. The students filled in questionnaires on their time spent outdoors for each day of the study. These data in conjunction with the ambient UVR measured by a detector/datalogger unit at each site were used to correlate the calculated exposures with those measured using the PS badges. Overall, the questionnaires indicated that the males spent more time outdoors and had higher measured UVR exposures than females. For both boys and girls at each location, there was a strong correlation between the mean measured UVR exposure and the ambient solar UVR at that location.