IGF-1R pathway activation as putative biomarker for linsitinib therapy to revert tamoxifen resistance in ER-positive breast cancer

Preclinical studies indicate that activated IGF-1R can drive endocrine resistance in ER-positive (ER+) breast cancer, but its clinical relevance is unknown. We studied the effect of IGF-1R signaling on tamoxifen benefit in patients and we searched for approaches to overcome IGF-1R-mediated tamoxifen failure in cell lines. Primary tumor blocks from postmenopausal ER+ breast cancer patients randomized between adjuvant tamoxifen versus nil were recollected. Immunohistochemistry for IGF-1R, p-IGF-1R/InsR, p-ERα(Ser118), p-ERα(Ser167) and PI3K/MAPK pathway proteins was performed. Multivariate Cox models were employed to assess tamoxifen efficacy. The association between p-IGF-1R/InsR and PI3K/MAPK pathway activation in MCF-7 and T47D cells was analyzed with Western blots. Cell proliferation experiments were performed under various growth-stimulating and -inhibiting conditions. Patients with ER+, IGF-1R-positive breast cancer without p-IGF-1R/InsR staining (n = 242) had tamoxifen benefit (HR 0.41, p = 0.0038), while the results for p-IGF-1R/InsR-positive patients (n = 125) were not significant (HR 0.95, p = 0.3). High p-ERα(Ser118) or p-ERα(Ser167) expression was associated with less tamoxifen benefit. In MCF-7 cells, IGF-1R stimulation increased phosphorylation of PI3K/MAPK proteins and ERα(Ser167) regardless of IGF-1R overexpression. This could be abrogated by the dual IGF-1R/InsR inhibitor linsitinib, but not by the IGF-IR-selective antibody 1H7. In MCF-7 and T47D cells, stimulation of the IGF-1R/InsR pathway resulted in cell proliferation regardless of tamoxifen. Abrogation of cell growth was regained by addition of linsitinib. In conclusion, p-IGF-1R/InsR positivity in ER+ breast cancer is associated with reduced benefit from adjuvant tamoxifen in postmenopausal patients. In cell lines, stimulation rather than overexpression of IGF-1R is driving tamoxifen resistance to be abrogated by linsitinib.     

Maximising the efficiency of clinical screening programmes: balancing predictive genetic testing with a right not to know

We explored the dilemma between patients'' right not to know their genetic status and the efficient use of health-care resources in the form of clinical cancer screening programmes. Currently, in the Netherlands, 50% risk carriers of heritable cancer syndromes who choose not to know their genetic status have access to the same screening programmes as proven mutation carriers. This implies an inefficient use of health-care resources, because half of this group will not carry the familial mutation. At the moment, only a small number of patients are involved; however, the expanding possibilities for genetic risk profiling means this issue must be addressed because of potentially adverse societal and financial impact. The trade-off between patients'' right not to know their genetic status and efficient use of health-care resources was discussed in six focus groups with health-care professionals and patients from three Dutch university hospitals. Professionals prefer patients to undergo a predictive DNA test as a prerequisite for entering cancer screening programmes. Professionals prioritise treating sick patients or proven mutation carriers over screening untested individuals. Participation in cancer screening programmes without prior DNA testing is, however, supported by most professionals, as testing is usually delayed and relatively few patients are involved at present. Reducing the number of 50% risk carriers undergoing screening is expected to be achieved by: offering more psychosocial support, explaining the iatrogenic risks of cancer screening, increasing out-of-pocket costs, and offering a less stringent screening programme for 50% risk carriers.     

The Pisotriquetral Joint: Osteoarthritis and Enthesopathy

Pisotriquetral (PT) osteoarthritis (OA) and enthesopathy of the flexor carpi ulnaris (FCU) are pathologies of the hypothenar eminence which both often remain undiagnosed, but can cause ulnar wrist pain. This study determined the prevalence of these pathologies in an older donor population. Twenty wrists were obtained from 10 cadavers with an age ranging from 65 to 94 years. Radiographs were taken of all wrists with the hand in pisotriquetral view and were assessed for osteoarthritic changes of the PT joint and signs of enthesopathy of the FCU. Ten wrists were grossly dissected and the other ten wrists were sagitally sectioned at a thickness of 10 μm. The wrists were analyzed for type and grade of osteoarthritis and signs of enthesopathy. On radiology, 2 out of 20 wrists showed no signs of osteoarthritis, 5 wrists showed severe changes. One wrist showed signs of enthesopathy. On macroscopy, 9 out of 10 wrists showed osteoartritic changes; 5 of these were severely osteoarthritic. On microscopy, all wrists showed some degree of osteoarthritis of which five showed severe changes. Signs of enthesopathy were seen in seven wrists. Pisotriquetral osteoarthritis has a high prevalence in the older donor population and may therefore be a cause of ulnar sided wrist pain. It should therefore always be considered in the differential diagnosis of ulnar sided wrist pain. By performing clinical examination with these pathologies in mind, diagnosis could be a lot faster. Furthermore, based on our results, radiographs seem to be not accurate in diagnosing osteoarthritis of the PT joint and enthesopathy of the FCU.     

Selective peripheral denervation: comparison with pallidal stimulation and literature review

Patients with cervical dystonia who are non-responders to Botulinum toxin qualify for surgery. Selective peripheral denervation (Bertrand’s procedure, SPD) and deep brain stimulation of the globus pallidus (GPi-DBS) are available surgical options. Although peripheral denervation has potential advantages over DBS, the latter is nowadays more commonly performed. We describe the long-term outcome of selective peripheral denervation as compared with GPi-DBS, along with the findings of literature review. Twenty patients with selective peripheral denervation and 15 with GPi-DBS were included. Tsui scale, a visual analogue scale, and the global outcome score of the Toronto Western Spasmodic Torticollis Rating Scale were used to define a “combined global surgical outcome”. The “combined global surgical outcome” for patients with selective peripheral denervation or pallidal stimulation was respectively “bad” for 65 and 13.3 %, “fair-to-good” for 30 and 26.7 %, and “marked” improvement for 5 and 60 % (p < 0.001). Improvement on visual analogue scale (p < 0.002), global outcome score (p < 0.002), and Tsui score (p < 0.000) was larger for the pallidal stimulation group. Seventy-five percent of patients with selective peripheral denervation and 60 % of patients with pallidal stimulation reported side effects. Seven patients with selective peripheral denervation successively underwent GPi-DBS, with a further significant improvement in the Tsui score (?48.6 ± 17.4 %). GPi-DBS is to be preferred to selective peripheral denervation for the treatment of cervical dystonia because it produces larger benefit, even if it can have more potentially severe complications. GPi-DBS is also a valid alternative in case of failure of SPD.     

Factors Influencing the Use of Sentinel Lymph Node Biopsy in the Netherlands

Background

In the US, whether a sentinel lymph node biopsy (SLNB) is performed depends on tumor and patient factors, including socioeconomic status (SES) and type of health care insurance. We analyzed which patient and tumor characteristics influenced the use of SLNB in a country where every patient has equal access to healthcare.

Methods

Patients diagnosed with a cutaneous invasive melanoma of ≥1 mm between 2004 and 2011 and living in the northeastern part of the Netherlands were selected from the Netherlands Cancer Registry. Regression analysis was performed to assess the association of patient and tumor characteristics and SLNB use.

Results

SLNB was performed in 42 % of the 2,413 included patients. The frequency of performing SLNB increased between 2004 and 2011 from 24 to 55 % (p < 0.001). Patients were less likely to undergo SLNB if they had a melanoma located in the head and neck area (p < 0.001), when they were over 55 years (p = 0.001), and if they had a low SES (p = 0.03). SLNB use was more likely when the diagnosis of melanoma was made in the university hospital (p = 0.045) or when the Breslow thickness was 2.01–4.0 mm (p = 0.03).

Conclusions

The use of SLNB has increased significantly between 2004 and 2011. However, in 2011 it was still performed in only 55 % of the Dutch patients with a melanoma ≥1 mm. In patients with head and neck melanoma, older patients, and patients with low SES, SLNB was less frequently performed. Patients with T3 melanomas and a diagnosis made in the university hospital more often had an SLNB performed.     

Experimental optimization of the detection limit of one-step solid-phase radioimmunoassay

The minimal detection limit and the conditions of maximal sensitivity of a one-step solid-phase inhibition radioimmunoassay for human immunoglobulin A have been determined by application of statistical methods of experimental optimization. The choice of the optimal combination of qualitative variables, such as the origin of the antibody and the nature of the solid phase, was made by the study of a covariable under non-optimal conditions of the quantitative variables, such as the amount of antibody. The covariable was the avidity of the antibody, which is expected to have a large influence on the sensitivity. Only the difference in avidity between two immunosorbents with cellulose or Sepharose as solid-phase material proved to be statistically significant, and further study was done with cellulose. The experimental optimization of the sensitivity as a function of five quantitative variables yielded a reduction of the detection limit by a factor 5.6 (from 23.5 to 4.2 ng IgA). The variables determining the amount of insolubilized antibody in the assay had the largest influence on the value of the detection limit. The conditions of optimal sensitivity did agree with the predictions by a physical model of radioimmunoassay. The results are discussed in relation to the assay parameters such as the amount and the avidity of the insolubilized antibody and the initial percentage of binding, and in relation with theoretical optimization of the sensitivity.     

Pathogen elimination and prevention within a regulated,Designated Pathogen Free,closed pig herd for long‐term breeding and production of xenotransplantation materials

Background

We established a Source Animal (barrier) Facility (SAF ) for generating designated pathogen‐free (DPF ) pigs to serve as donors of viable organs, tissues, or cells for xenotransplantation into clinical patients. This facility was populated with caesarian derived, colostrum deprived (CDCD ) piglets, from sows of conventional‐specific (or specified) pathogen‐free (SPF ) health status in six cohorts over a 10‐month period. In all cases, CDCD piglets fulfilled DPF status including negativity for porcine circovirus (PCV ), a particularly environmentally robust and difficult to inactivate virus which at the time of SAF population was epidemic in the US commercial swine production industry. Two outbreaks of PCV infection were subsequently detected during sentinel testing. The first occurred several weeks after PCV ‐negative animals were moved under quarantine from the nursery into an animal holding room. The apparent origin of PCV was newly installed stainless steel penning, which was not sufficiently degreased thereby protecting viral particles from disinfection. The second outbreak was apparently transmitted via employee activities in the Caesarian‐section suite adjacent to the barrier facility. In both cases, PCV was contained in the animal holding room where it was diagnosed making a complete facility depopulation‐repopulation unnecessary.

Method

Infectious PCV was eliminated during both outbreaks by the following: euthanizing infected animals, disposing of all removable items from the affected animal holding room, extensive cleaning with detergents and degreasing agents, sterilization of equipment and rooms with chlorine dioxide, vaporized hydrogen peroxide, and potassium peroxymonosulfate, and for the second outbreak also glutaraldehyde/quaternary ammonium. Impact on other barrier animals throughout the process was monitored by frequent PCV diagnostic testing.

Result

After close monitoring for 6 months indicating PCV absence from all rooms and animals, herd animals were removed from quarantine status.

Conclusion

Ten years after PCV clearance following the second outbreak, due to strict adherence to biosecurity protocols and based on ongoing sentinel diagnostic monitoring (currently monthly), the herd remains DPF including PCV negative.

     

Characteristics of differentiated CD8+ and CD4+ T cells present in the human brain

Immune surveillance of the central nervous system (CNS) by T cells is important to keep CNS-trophic viruses in a latent state, yet our knowledge of the characteristics of CNS-populating T cells is incomplete. We performed a comprehensive, multi-color flow-cytometric analysis of isolated T cells from paired corpus callosum (CC) and peripheral blood (PB) samples of 20 brain donors. Compared to PB, CC T cells, which were mostly located in the perivascular space and sporadically in the parenchyma, were enriched for cells expressing CD8. Both CD4⁺ and CD8⁺ T cells in the CC had a late-differentiated phenotype, as indicated by lack of expression of CD27 and CD28. The CC contained high numbers of T cells expressing chemokine receptor CX3CR1 and CXCR3 that allow for homing to inflamed endothelium and tissue, but hardly cells expressing the lymph node-homing receptor CCR7. Despite the late-differentiated phenotype, CC T cells had high expression of the IL-7 receptor α-chain CD127 and did not contain the neurotoxic cytolytic enzymes perforin, granzyme A, and granzyme B. We postulate that CNS T cells make up a population of tissue-adapted differentiated cells, which use CX3CR1 and CXCR3 to home into the perivascular space, use IL-7 for maintenance, and lack immediate cytolytic activity, thereby preventing immunopathology in response to low or non-specific stimuli. The presence of these cells in this tightly regulated environment likely enables a fast response to local threats. Our results will enable future detailed exploration of T-cell subsets in the brain involved in neurological diseases.