SDF-1/CXCR4 inhibition prevents paradoxical generation of cisplatin-induced pro-metastatic niches

Platinum-based chemotherapy remains widely used in advanced non-small cell lung cancer (NSCLC) despite its ineffectiveness in long-term control of metastasis.Here, we uncover the interconnected pathways subtending cisplatin-induced metastasis promotion.We report that cisplatin treatment of tumor-free mice results in bone-marrow expansion of CCR2+CXCR4+Ly6Chigh inflammatory monocytes (IM) concomitantly with increased levels in the lungs of stromal SDF-1, the CXCR4 ligand. In experimental metastasis assays, cisplatin-induced IM favor tumor cells extravasation and expansion of CD133+CXCR4+ metastasis initiating cells (MICs), facilitating lung metastasis formation. At the primary tumor, cisplatin reduces tumor size but induces tumor release of SDF-1 triggering MICs expansion and recruitment of pro-invasive CXCR4+ macrophages. Co-recruitment of MICs and CCR2+CXCR4+ IM at SDF-1-enriched distant sites also promotes spontaneous metastasis. Combination treatment with a CXCR4 inhibitor prevents cisplatin-induced IM/MICs recruitment and interaction thus precluding metastasis overgrowth. Finally, we observe in NSCLC patients’ specimens that SDF-1 levels are higher in platinum-treated samples and correlate with worse outcome.Our findings suggest a possible novel combination therapy based on CXCR4 blockade to control metastatic disease, paradoxically promoted by cisplatin.

VITAMIN D, CALCIUM AND PHOSPHOR IN PATIENTS WITH β-THALASSEMIA MAJOR

There has been many reports that patients with β-thalassemia major have bone problems such as thinning of the bone, bone fragility and pathological fractures. For so many years it was believed that the bone problems is mainly caused by marrow expansion due to compenstation of the bone marrow to handle the chronic anaemia and hiypoxia in β-thalassemia major. Recently, there is evidence to suggest that in β-thalassemia major there is hypocalcemia and hypovitaminosis D. So, this study is to clarify if hypovitaminosis D is trully the cause of bone problem in thalassemia. Forty five subjects were recruited in this study, 35 were β-thalassemia major patients and 10 normal subjects as controls. Ten mL of venous blood were taken from median vein for investigations of total vitamin D [25(OH) vitamin D], total calcium and phosphor using the Enzyme-Linked Fluorescent Assay (ELFA), metallochromic dye (Arsenazo III) and chemical reaction of inorganic phosphate with amonium mollybdate respectily. Mean ± SD of vitamin D in β-thalassemia major is 21.28 ± 6.36 ng/ml and in control 34.85 ± 3.50 ng/ml (p<0.05); total calcium in β-thalassemia major is 8.58 ± 0.68 mg/dl and in control 9.22 ± 0.35 mg/dl (p<0.05); and phosphor in β-thalassemia major 3.98 ± 0.53 mg/dl and control 3.89 ± 0.49 mg/dl (p>0.1). There was no significant correlation (r = 0.17, p>0.05), when vitamin D was analysed against calcium for the correlation study,. This study demostrates that there was state of hypovitaminosis D and hypocalcemia in β-thalassemia major but hypovitaminosis D is not the only causative factor of the calcium levels. There should be another factor responsible for the calcium levels in β-thalassemia major and marrow expansion may remain the factors responsible for bone abnomarlities.

Haemoglobinopathies

Inherited abnormalities of the globin genes are the commonest single-gene disorders in the world and fall into two main groups: thalassaemias and sickle cell disease. Thalassaemias are due to quantitative defects in globin chain synthesis which cause variable anaemia and ineffective erythropoiesis. Thalassaemia was initially thought to be a disease of the bones due to uncontrolled bone marrow expansion causing bony distortion, although this is now unusual with appropriate blood transfusions. Osteopenia, often severe, is a feature of most patients with thalassaemia major and intermedia, caused by bone marrow expansion, iron overload, endocrinopathy, and iron chelation. Treatment with bisphosphonates is generally recommended. Other rheumatological manifestations include arthropathy associated with the use of the iron chelator deferiprone. Sickle cell disease involves a group of conditions caused by polymerization of the abnormal -globin chain, resulting in abnormal erythrocytes which cause vaso-occlusion, vasculopathy, and ischaemic tissue damage. The characteristic symptom is acute bone pain caused by vaso-occlusion; typical episodes require treatment with opiate analgesia and resolve spontaneously by 5 days with no lasting bone damage. The frequency of acute episodes varies widely between patients. The incidence of osteomyelitis is increased, particularly with salmonella, although it is much rarer than acute vaso-occlusion. Avascular necrosis can affect the hips, and less commonly the shoulders and knees. Coincidental rheumatological disease sometimes complicates the condition, particularly systemic lupus erythematosus (SLE) which is more prevalent in populations at increased risk of sickle cell disease.

Haemoglobinopathies

Inherited abnormalities of the globin genes are the commonest single-gene disorders in the world and fall into two main groups: thalassaemias and sickle cell disease. Thalassaemias are due to quantitative defects in globin chain synthesis which cause variable anaemia and ineffective erythropoiesis. Thalassaemia was initially thought to be a disease of the bones due to uncontrolled bone marrow expansion causing bony distortion, although this is now unusual with appropriate blood transfusions. Osteopenia, often severe, is a feature of most patients with thalassaemia major and intermedia, caused by bone marrow expansion, iron overload, endocrinopathy, and iron chelation. Treatment with bisphosphonates is generally recommended. Other rheumatological manifestations include arthropathy associated with the use of the iron chelator deferiprone. Sickle cell disease involves a group of conditions caused by polymerization of the abnormal -globin chain, resulting in abnormal erythrocytes which cause vaso-occlusion, vasculopathy, and ischaemic tissue damage. The characteristic symptom is acute bone pain caused by vaso-occlusion; typical episodes require treatment with opiate analgesia and resolve spontaneously by 5 days with no lasting bone damage. The frequency of acute episodes varies widely between patients. The incidence of osteomyelitis is increased, particularly with salmonella, although it is much rarer than acute vaso-occlusion. Avascular necrosis can affect the hips, and less commonly the shoulders and knees. Coincidental rheumatological disease sometimes complicates the condition, particularly systemic lupus erythematosus (SLE) which is more prevalent in populations at increased risk of sickle cell disease.

Haemoglobinopathies

Inherited abnormalities of the globin genes are the commonest single-gene disorders in the world and fall into two main groups: thalassaemias and sickle cell disease. Thalassaemias are due to quantitative defects in globin chain synthesis which cause variable anaemia and ineffective erythropoiesis. Thalassaemia was initially thought to be a disease of the bones due to uncontrolled bone marrow expansion causing bony distortion, although this is now unusual with appropriate blood transfusions. Osteopenia, often severe, is a feature of most patients with thalassaemia major and intermedia, caused by bone marrow expansion, iron overload, endocrinopathy, and iron chelation. Treatment with bisphosphonates is generally recommended. Other rheumatological manifestations include arthropathy associated with the use of the iron chelator deferiprone. Sickle cell disease involves a group of conditions caused by polymerization of the abnormal -globin chain, resulting in abnormal erythrocytes which cause vaso-occlusion, vasculopathy, and ischaemic tissue damage. The characteristic symptom is acute bone pain caused by vaso-occlusion; typical episodes require treatment with opiate analgesia and resolve spontaneously by 5 days with no lasting bone damage. The frequency of acute episodes varies widely between patients. The incidence of osteomyelitis is increased, particularly with salmonella, although it is much rarer than acute vaso-occlusion. Avascular necrosis can affect the hips, and less commonly the shoulders and knees. Coincidental rheumatological disease sometimes complicates the condition, particularly systemic lupus erythematosus (SLE) which is more prevalent in populations at increased risk of sickle cell disease.

Haemoglobinopathies

♦ Haemoglobinopathies are commonly inherited disorders of haemoglobin synthesis♦ Thalassaemia is commonest around the Mediterranean countries and has skeletal manifestations due to massive marrow expansion with thinning of the cortex♦ Sickle cell crises occur in homozygotes and are a result of venous occlusion causing avascular necrosis. Infection and exposure to cold can sometimes precipitate these painful events♦ If surgery is needed blood cross-match must be carefully performed in advance, the theatre should be kept warm, and the hydration and acid/base balance of the patient monitored carefully.

Bone Marrow Iron Concentration as a Marker of Iron Accumulation and Marrow Expansion in Patients with Beta Thalassemia Major.

Liver iron concentration (LIC) is a known and accurate marker of iron accumulation and is widely utilized to monitor iron chelation therapy in multiply transfused patients with Beta thalassemia major. Iron concentration in the bone marrow has not been studied and reported before. We utilized atomic absorption spectrophotometry to measure the iron content of marrow biopsy (BIC) in 102 thalassemia patients in various phase of treatment, 74 of them during the course of the disease and 28 patients after successful allogeneic marrow transplant. We observed BIC values below 0.5 mg/g dry weight in 7 healthy donors used as controls. Mean and median BIC were 4.67 and 2.70 (range 0.05 – 59.9) mg/g dw, in 101 valuable patients. Bone Marrow iron concentration (BIC) was calculated at the same time of LIC for each patient and a ratio LIC/BIC was generated. LIC/BIC ratio below 3, ratio between 3 and 10, and above 10 identified three categories of patients each with significant linear correlation between LIC and BIC (0.74; 0.76; 0.81 respectively). Twenty eight patients were in the first category, 48 in the second, and 25 in the third. Mean BIC was 9.47, 3.7, 1.2 mg/g dw and mean LIC was 12.7, 19.6, 22.3 mg/g dw respectively for CAT1, CAT2 and CAT3. Patients were also classified in class of risk for transplant, based on hepatomegaly, presence of liver fibrosis and history of regular or irregular iron chelation. BIC was higher in class 3 patients and in the irregularly chelated patients. Patients in class 1 were prevalently in CAT1 or 2, patients in class 3 were prevalently in CAT2 or 3. BIC value in each of the 3 categories correlated significantly with the whole body iron (WBI) amount, and WBI was significantly different in the three categories being lower in CAT1, that have higher BIC ( 3927 mg; 5894 mg, 7030 mg in CAT1, 2 and 3; p 0.01). Iron chelation quality (regular vs irregular) correlated with BIC value and with BIC category, majority of regularly chelated patients were in the Category 1 vs Category 2 or 3 (p 0.01). Patients before transplant were prevalently in CAT1 and 2, while those post transplant were mostly in CAT3, twelve patients that were studied both before and after transplant, changed from cat 1 to 2 or from cat 2 to 3. Their BIC changed significantly decreasing after BMT (median BIC was 7.8 before transplant and 2.25 after, p=0.002) To investigate the role of genetic hemochromatosis mutations, H63D region and Hamp region in 63 patients were also studied. Mean BIC was 3.08 in 16 patients with H63D mutation compared to 5.59 in those without mutation, and 11/16 had BIC below the median, p=0.03. Significantly more patients with H63D mutation were in CAT3 (p 0.02). Apparently H63D mutation favours accumulation of iron in the body, that was higher in CAT3, but participate also to lowering utilization of introduced iron. In conclusion, patients in category 1 had lower LIC and higher BIC, comprehended 50% of the patients in class 1 of risk, and 45% of the regularly chelated patients. On the contrary, majority of patients post transplant independently of having or not performed an iron removal program were in CAT3. We can draw conclusion that BIC related categories, as described here, give information on the balance between accumulation and utilization of iron. Patients belonging to BIC-Cat 1 correspond to patients that have been treated adequately and with lower iron body burden, better chelation history. They have higher BIC value and lower LIC value. They also are in pre-transplant phase, with active beta-thalassemia, when ineffective erythropoiesis and marrow expansion are more prominent. Further studies will be necessary to confirm the association of marrow iron content with the marrow functionality and expansion.

Zoledronic Acid Increases Bone Mineral Density in Patients with Thalassemia Intermedia-Induced Osteoporosis Regardless of the Incessant Bone Marrow Expansion.

Osteoporosis represents an important cause of morbidity in adult patients with thalassemia. Its pathogenesis is multifactorial, and includes mainly bone marrow expansion, endocrine dysfunction and iron overload. Patients with thalassaemia intermedia (TI) seem to have a more expanded bone marrow with pressure on cortical bone, which causes pain and bone loss in several cases. The measurement of soluble transferrin receptor (sTfR) and erythropoietin (Epo) in the serum is considered as accurate marker of erythropoietic activity in thalassemia. Bisphosphonates are potent inhibitors of osteoclast activity and have been used for the management of thalassemia-induced osteoporosis. The aim of this study was to evaluate the effect of zoledronic acid, the most potent aminobisphosphonate, on bone mineral density (BMD) of patients with TI and explore possible correlations with bone marrow expansion and erythropoietic activity. Thirty-five patients with TI and osteopenia or osteoporosis (13M/22F, median age 45 years) were evaluated. Twenty-three were randomized to receive zoledronic acid, 4 mg, IV, every 3 months (n=12) or every 6 months (n=11) for one year, while 12 patients received placebo every 3 months. There was no difference in terms of the presence of gonadal dysfunction between the three studied groups. BMD of the lumbar spine (L), femoral neck and forearm was determined in all patients, using DEXA, before and 12 months after treatment. Bone marrow expansion was assessed by the measurement of sTfR and Epo serum levels, using an ELISA methodology (R&D Systems, Minneapolis, MN, USA), before and 12 months post zoledronic acid or placebo administration. In all patients markers of bone remodelling, such as C-telopeptide of collagen type-I (CTX) and bone specific alkaline phosphatase (bALP) were also measured by ELISA (Nordic Bioscience Diagnostics, Herlev, Denmark, and Quidel, San Diego, CA, USA, respectively). Patients were asked to quantify their degree of bone pain on Huskisson’s visual analogue scale and the McGill-Melzack scoring system before and 12 months post-therapy. All patients had increased values of sTfR, Epo, CTX, and bALP compared with 40 controls of similar age and gender (p&lt;0.001). Patients who received zoledronic acid showed a significant increase in their L-BMD (p=0.01), which was accompanied by a dramatic reduction in CTX and bALP values ((p&lt;0.001). There was no difference in terms of L-BMD changes between zoledronic acid groups. Placebo group showed an aggravation of L-BMD (p=0.041) and markers of bone remodelling at 12 months. No other changes were observed in the BMD of other sites. Zoledronic acid reduced bone pain, which remained stable in placebo group during the study period. There was only weak correlation between baseline sTfR levels and L-BMD, while there was no correlation between Epo or hemolytic parameters (indirect bilirubin, reticulocytes counts, and LDH) with BMD of all studied sites. Serum sTfR and Epo values showed a significant elevation after 12 months of therapy in all studied groups (p&lt;0.01, p&lt;0.02, and p&lt;0.01, respectively); this elevation was irrespective of the L-BMD changes. This study suggests that the increase of BMD produced by zoledronic acid in TI is irrespective of the continuous increase of bone marrow expansion, which is considered a major cause of bone loss in this hemoglobinopathy.

Osteopenia Is Commonly Present in Prepubertal Children with Severe Hb E/βthalassemia Despite Adequate Transfusion and Iron Chelation Therapy.

Objectives: Previous studies showed that low bone mineral density (BMD) is highly prevalent in Hb E/βthalassemia, who received occasional transfusion. Concerning the clinical heterogeneity of this syndrome, we determine whether adequate transfusion and chelation therapy in severe cases with Hb E/β0 thalassemia could ameliorate this complication. Methods: 50 pre-pubertal patients, age from 8–13, were recruited after informed consent. 26 were classified as severe group using Thalassemia International Federation (TIF) criteria, while 24 were mild. In severe patients, each received transfusion every 3 weeks (12–15 ml/kg) to keep their pre-transfusion Hb at 10 g/dL and they received iron chelation either by deferioxamine (20–40 mg/kg/d) or deferaxirox (25–30 mg/kg/d). Demographic data and history of chelation therapy were recorded. Serum free T4, TSH, PTH, cortisol, 25-OH Vit D, osteocalcin, alkaline phosphatase, serum ferritin (SF), Ca and P were determined. We measured the BMD of lumbar spines (L2-L4) and total body using DEXA (Lunar, Prodigy) and adjusted for height-age (HA) and bone age (BA). WHO criteria for BMD was Z-score −1 to −2.5 = osteopenia and &lt; −2.5 = osteoporosis. Results: They were compound heterozygotes of Hb E with either codon 41/41 (50.6%), codon 17 (19%), IVS2#654 (14%), IVS1#1 (4%), IVS1#5 (3%) and codon 71/72 (2%) mutations. In severe group, 15 cases had poor compliance for chelation therapy (group A) (average SF; 6027 ± 2564 ng/ml) while 11 cases had good compliance (group B) (average SF; 2374.3 ± 189 ng/ml) and the ferritin level in mild group (group C) were 197 ± 89.4 ng/ml. There were no statistical significances among these three groups regarding baseline Hb, age and sex-distribution, weight, height and their corrected Z-scores for the standard of Thai children. Only the onset of anemia was significant lower in group C (5.4 ±1.8 yrs) compared to 2.12 ± 1.35 and 2.24 ± 2 yrs in group A and B respectively. All had normal free T4, TSH, PTH, Ca and P. As expected, 7 cases (78%) of group A with marked iron overload had osteopenia while one (7%) had osteoporosis. A strong invert correlation between BMD and serum ferritin in this group was observed. To our surprise, 6 out of 11 (54%) in group B and 14 out of 24 (58%) in group C also had osteopenia. However, there was no correlation between BMD and baseline Hb, ferritin and other clinical parameters in the group B and C. Conclusion: Low BMD in thalassemia might be resulted from ineffective erythropoiesis, bone marrow expansion and iron deposition in the bone marrow. In severe Hb E/βthalassemia, despite adequate transfusion, osteopenia and osteoporosis remain highly prevalent especially when iron overload is a co-factor. However, detecting this complication in a “so-call’ milder group was surprising since this group of patients was less anemia and rarely required blood transfusion. These suggest that several factors beside anemia and bone marrow expansion might play roles on developing this complication. Moreover, the current standard transfusion and chelation protocol might not be preventable our patients from bone complications and they were at a greater risk of fracture in adulthood. It will be of interest to investigate whether calcium and Vit D supplement with or without osteoclast inhibitors could prevent osteopenia in this highly common hereditary anemia.