One out of nine Canadian males would suffer prostate cancer (PC) during his lifetime. Life expectancy of males with PC has increased with modern therapy and 90% live >10 years. However, 20% of PC-affected males would develop incurable metastatic diseases. Bone metastases (BM) are present in ~80% of metastatic PC patients, and are the most severe complication of PC, generating severe pain, fractures, spinal cord compression, and death. Interestingly, PC-BMs are mostly osteoblastic. However, the structure of this newly formed bone and how it relates to pain and fracture are unknown. Due to androgen antagonist treatment, different PC phenotypes develop with differential dependency on androgen receptor (AR) signaling: androgen-dependent (AR+), double negative (AR-) and neuroendocrine. How these phenotypes are related to changes in bone structure has not been studied. Here we show a state-of-the-art structural characterization of PCBM and how PC phenotypes are associated to abnormal bone formation in PCBM.

Cadaveric samples (n=14) obtained from metastases of PC in thoracic or lumbar vertebrae (mean age 74yo) were used to analyze bone structure. We used micro-computed tomography (mCT) to analyze the three-dimensional structure of the bone samples. After imaging, the samples were sectioned and one 3mm thick section was embedded in epoxy-resin, ground and polished. Scanning electron microscopy (SEM)/energy-dispersive X-ray spectroscopy (EDS) and quantitative backscattering electron (qBSE) imaging were used to determine mineral morphology and composition. Another section was used for histological analysis of the PC-affected bone. Collagen structure, fibril orientation and extracellular matrix composition were characterized using histochemistry. Additionally, we obtained biopsies of 3 PCBM patients undergoing emergency decompression surgery following vertebral fracture and used them for immunohistological characterization.

By using mCT, we observed three dysmorphic bone patterns: osteolytic pattern with thinned trabecula of otherwise well-organized structures, osteoblastic pattern defined as accumulation of disorganized matrix deposited on pre-existing trabecula, and osteoblastic pattern with minimum residual trabecula and bone space dominated by accumulation of disorganized mineralized matrix. Comparing mCT data with patho/clinical parameters revealed a trend for higher bone density in males with larger PSA increase. Through histological sections, we observed that PC-affected bone, lacks collagen alignment structure, have a higher number of lacunae and increased amount of proteoglycans as decorin.

Immunohistochemistry of biopsies revealed that PC-cells inside bone organize into two manners: i) glandular-like structures where cells maintain their polarization in the expression of prostate markers, ii) diffuse infiltrate that spreads along bone surfaces, with loss of cell polarity. These cells take direct contact with osteoblasts in the surface of trabecula. We define that PCBM are mostly composed by AR+ with some double negative cells. We did not observe neuroendocrine phenotype cells.

PCBMs generate predominantly osteoblastic lesions that are characterized by high lacunar density, lack of collagen organization and elevated proteoglycan content. These structural changes are associated with the infiltration of PC cells that are mostly androgen-dependent but have lost their polarization and contact directly with osteoblasts, perhaps altering their function. These changes could be associated with lower mechanical properties that led to fracture and weakness of the PCBM affected bone.

It is known that wide variability exists among patients in the susceptibility to and outcome from infection. Polymorphisms in genes coding for proteins involved in the response to bacterial pathogens as tumor necrosis factor-alpha(TNF-a), interleukin (IL)-1alpha, IL-1beta, IL-1 receptor agonist, IL-6, IL-10 can influence the amount or function of the protein produced in response to bacterial stimuli. These genetic polymorphisms may influence the susceptibility to and outcome from infection.

The aim of the study was to investigate whether genetic variation in genes coding for components of the innate immune response might be a critical determinant of the inflammatory response and the risk for and outcome from severe bacterial infection in individuals with musculoskeletal infections. The relationship between single nucleotide polymorphisms (SNPs) in the above mentioned genes and susceptibility to infection was evaluated.

Forty patients with musculoskeletal infections hospitalised at the Orthopaedic Clinic of University Hospital of Larissa, as well as 80 healthy controls were included in the study. Genomic DNA was isolated from peripheral blood from all cases and controls and was extracted according to standard procedures. The following genes with their polymorphic positions were studied: IL 1α (IL 1α promoter −889), IL 1β (IL 1β promoter −511, pos. +3962), IL 1R (IL 1R pos. pst1 1970), IL 1RA (IL 1RA pos. mspa1 11100), IL 4Rα (IL 4Rα pos. +1902), IL 12 (IL 12 promoter −1188), TGF-β (TGF-β exon 1 codon 10, codon 25), TNF-α (TNF-α promoter −308, −238), IL 2 (IL 2 promoter −330, pos. +166), IL 4 (IL 4 promoter −1098, −590, −33), IL 6 (IL 6 promoter −174, pos. +nt 565) and IL 10 (IL 10 promoter −1082, −819, −592). Genotype distribution and allele frequencies in patients and controls were evaluated.

There was a significant difference in genotype and allele frequency of IL-1a (T/C −889) p=0.000 (CC, TC) between patients and the control group. Moreover, 2 SNPs of interleukin 4 [IL-4 (T/G −1098) p=0.000 (GG, GT) p=0.009 (TT) and IL-4 (T/C-590) p=0.000 (CC, CT) p=0.006 (TT)] showed significant genotypic and allelic differences between the two groups. Finally, 2 SNPs of interleukin 6 [IL-6 (G/C-174) p=0.000 (CC) p=0.014 (GG), IL-6 G/A nt565) p=0.000 (AA,GA,GG)] and TNF-a [(G/A-308) p=0.034 (AG)] showed significant differences in genotype and allele frequencies between patients and the control group.

We observed, for the first time, significant differences in genotype and allele frequencies of TNF-a (G/A-308), IL-1a (T/C -889), IL-4 (T/G -1098), IL-4 (T/C-590), IL-6 (G/C-174) and IL-6 G/A (nt565) in patients with musculoskeletal infections, a fact which points towards the involvement of cytokine gene polymorphisms in the pathogenesis of infection.