Accuracy of CT and MRI to assess resection margins in primary malignant bone tumours having histology as the reference standard.

AIM: To evaluate the accuracy of magnetic resonance imaging (MRI) and computed tomography (CT) in assessing the resection margins of primary malignant bone tumours. MATERIALS AND METHODS: Resected primary malignant bone tumour specimens removed from 46 patients (27 male; mean age: 48±22 years) were imaged using MRI (fat-saturated proton density-weighted and three-dimensional fat-suppressed T1-weighted gradient-recalled-echo) and CT immediately after surgery. A radiologist and an orthopaedist evaluated bone and soft-tissue margins of the specimens on both examinations. Histological evaluation was performed by a senior orthopaedic oncology pathologist. Margins were classified as R0 (safe margins), R1 (residuals between 0 and 1 mm), and R2 (macroscopic residuals). Cohen's k, chi-square, and McNemar's statistics were used. RESULTS: Having histology as the reference standard, reproducibility of the radiologist ranged from moderate (k=0.544) to substantial (k=0.741) for bone and soft-tissue margins on CT, respectively, while that of the orthopaedist ranged from fair (k=0.316) to moderate (k=0.548). When comparing R2 and R0+R1 scores, reproducibility of readers' evaluation of bone margins increased ranging from substantial (k=0.655) to perfect (k=1.000). Inter-reader agreement ranged from fair (k=0.308) to substantial (k=0.633). Accuracy of the radiologist and orthopaedist ranged from 76% to 83% and from 68% to 72%, respectively. When comparing R2 and R0+R1 scores, the accuracy of both readers ranged from 83% to 100%. There was no association between local recurrence and margin scores of histology, MRI, and CT (p≥0.058). CONCLUSIONS: MRI and CT may be useful for extemporaneous analysis of resection margins of primary malignant bone tumours, although wide accuracy variability between the different imaging methods was observed.

Wnt signaling pathway inhibitors as promising diagnostic serum markers of osteolytic bone metastasis.

Despite the clinical importance of bone metastases, we still know little about their onset and progression and current diagnostic tools lack the sensitivity and specificity required for clear early diagnosis. We therefore need to continue studying the pathogenesis of bone metastatic invasion in order to improve diagnosis. The Wnt pathway has been described as having an important role in bone carcinogenesis and metastatic progression. This study investigated the diagnostic potential of the two main Wnt inhibitors, sclerostin and DKK-1, to improve the detection of osteolytic bone metastases. We measured sclerostin and DKK-1, MMP-2 and MMP-9, the bone resorption marker TRAP5b and the metastatic marker survivin in a control group of healthy patients, in patients with primary tumors and in a group with metastasis. Sclerostin and DKK-1 were clearly high in primary tumor patients and even higher in metastatic patients, compared to controls. The close correlations with metastatic markers and bone resorption markers make sclerostin and DKK-1 promising as new biomarkers in the diagnosis of bone osteolytic metastases.

Circulating sRAGE in the diagnosis of osteolytic bone metastasis.

Despite the clinical importance of metastasis to the skeleton, the diagnostic tools for early detection and monitoring of bone metastasis lack sensitivity and specificity. We evaluated a promising new serum biomarker, the soluble form of the Receptor of Advanced Glycosylated End-products (sRAGE). sRAGE is involved in the Wnt-signaling pathway, and has been reported to reduce the risk of cancer. We investigated the diagnostic potential of sRAGE to improve the detection and monitoring of bone metastasis. We measured sRAGE in the serum of control healthy subjects, patients with primary tumors and patients with bone metastasis. sRAGE was also correlated with the Wnt inhibitors DKK-1 and sclerostin, the bone resorption markers MMP-2, MMP-9 and TRAP5, and the metastatic marker survivin. sRAGE was significantly lower in primary tumor and metastatic patients than in healthy subjects. sRAGE also showed a strong negative correlation with DKK-1, sclerostin, MMP-2, MMP-9, TRAP5b and survivin. These results indicated that sRAGE might play a protective role in bone metastasis progression, and it may diagnostic significance for detecting and monitoring osteolytic metastases.

Diffusion-weighted MR imaging in differentiation between osteoporotic and neoplastic vertebral fractures.

PURPOSE: To assess the usefulness of magnetic resonance imaging (MRI) with spin-echo echo-planar diffusion-weighted imaging (SE-EPI-DWI) in differentiation between vertebral osteoporotic fractures and pathological neoplastic fractures. MATERIALS AND METHODS: Thirty-three patients with both osteoporotic or neoplastic vertebral fractures diagnosed with X-ray or TC were studied with MRI exam, (1.5 T unit) with DWI sequences. DWI sequences were qualitatively analyzed. Apparent diffusion coefficient (ADC) values were also determined and compared to the definitive histologic diagnosis. RESULTS: DWI of neoplastic lesions showed hyperintensity signal in 22 out of 23 cases. Mean ADC value of neoplastic fractures was 1.241 ± 0.4 × 10(-3) mm(2)/s; mean ADC value of osteoporotic fractures was 0.646 ± 0.368 × 10(-3) mm(2)/s. Neoplastic fractures showed ADC values significantly higher than osteoporotic ones (p < 0.001). DWI imaging and histology showed a significant correlation. CONCLUSION: DWI provides reliable information to support MRI diagnosis of neoplastic versus osteoporotic fractures. ADC value appears as a useful adjunctive parameter.

Surgical management of recurrent thoracolumbar spinal sarcoma with 4-level total en bloc spondylectomy: description of technique and report of two cases.

INTRODUCTION: The descriptions of total spondylectomy and further development of the technique for the treatment of vertebral sarcomas offered for the first time the opportunity to achieve oncologically sufficient resection margins, thereby improving local tumor control and overall survival. Today, single level en bloc spondylectomies are routinely performed and discussed in the literature while only few data are available for multi-level resections. However, due to the topographic vicinity of the spinal cord and large vessels, the multisegmental resections are technically demanding, represent major surgery and only few case reports are available. Surgical options are even more limited in cases of revision surgery and local recurrences when en bloc spondylectomy was considered to be not feasible due to high risk of vital complications in expanding resection margins. Deranged anatomy, implants in situ and extensive intra-/paraspinal scar tissue formation resulting from previously performed approaches and/or radiation are considered the principal complicating factors that usually hold back spine surgeons to perform revision for resection leaving the patient to palliative treatment. METHODS: We present two patient cases with previously performed piecemeal vertebrectomy in the thoracic spine due to a solitary high-grade spinal sarcoma. After extensive re-staging, both patients underwent a multi (4)-level en bloc spondylectomy in our department (one patient with combined en bloc lung resection). Except a local wound disturbance, there was no severe intra- or postoperative complication. RESULTS: After multilevel en bloc spondylectomy both patients showed a good functional outcome without neurological deficits, except those resulting from oncologically scheduled resection of thoracic nerve roots. After a median follow-up of 13 months, there was no local recurrence or distant metastasis. The reconstruction using a posterior screw rod system that is interconnected to an anterior vertebral body replacement with a carbon composite cage showed no implant failure or loosening. In summary, the approach of a multilevel en bloc surgery for revision and oncologically sufficient resection in cases of spinal sarcoma recurrences seems possible. However, interdisciplinary decision making in a tumor board, realistic evaluation of surgical resectability to attain tumor free margins, advanced experiences in spinal reconstructions and involvement of vascular, visceral and thoracic surgical expertise are essential preconditions for acceptable oncological and functional outcome.

Induction of an antibody response in cultures of human peripheral blood lymphocytes.

A culture system is descirbed which provides adequate conditions for in vitro immunization of humand peripheral blood lymphocytes to heterologous erythrocytes. Making use of this method we could obtain, with a number of different donors, an antibody response which peaked at about day 8 of culture with 30-300 plaque-forming cells (PFC) per 10(6) input lymphocytes. However, in a number of experiments poor or negative results were obtained, even with donors that had previously given good response. This variability in the results was shown not to be due to a too low number of precursor cells present in the blood and could be overcome by treating the cells, before initiation of the culture, with a factor produced by mouse T cells educated to sheep erythrocytes (SRBC). Under these conditions a PFC responce was obtained which peaked at about day 8 and which in some experiments could be as high as 20,000 PFC per 10(6) input lymphocytes. Paralleling the increase in PFC was an increase in cell number. The cells recovered from the treated cultures were at all times more numerous than in the nontreated cultures. The height of both the proliferative and antibody-producing responses varied from experiment to experiment, a higher proliferative response, accompanying a higher PFC response. Although the mechanisms that are at the basis of the antibody response in vitro described in this paper still need to be clarified, this system may become a useful tool in studying the immune response in man.

Electrophoretically homogeneous antibody synthesized by spleen foci of irradiated repopulated mice.

10(6) splenocytes from primed donors were injected, together with sheep erythrocytes (SRBC), into X-irradiated syngeneic mice. 8 days later the spleens were excised and cut into small fragments, keeping track of their location in the organ. Each fragment was cultured individually for 24 hr in the presence of (14)C amino acids and the culture fluids were assayed for antibody activity. The antibody-producing fragments were found to be clustered in few restricted areas (foci) surrounded by negative tissue. The anti-SRBC antibody from single foci was purified by absorption on stroma followed by acid elution. Thereafter, it was subjected to electrophoresis and immunoelectrophoresis. The radioautography of the runs showed a considerable degree of homogeneity. Distinct and sharp spikes were localized in the beta and gamma region. The pattern of each focus is unique from the point of view of the number of spikes and their mobility. Eluates obtained from many pooled fragments gave a broad radioactive smear in beta-gamma region. Many foci synthesized antibody migrating as a single band. This homogeneous protein is probably the product of a clone of cells homogeneously differentiated. However, some foci producing two and probably more antibody bands were also encountered. Two interpretations of the finding can be given. Either more than one precursor may participate in the formation of a focus or a differentiation switch may occur during the clonal expansion.

Antibody response of rabbit blood lymphocytes in vitro. Kinetics, clone size, and clonotype analysis in response to streptococcal group polysaccharide antigens.

Peripheral blood lymphocytes (PBL) of rabbits previously hyperimmunized against streptococcal groups A and A-variant antigens were stimulated in vitro by the corresponding vaccines to produce group-specific antibody. This response was dependent on an optimal cell density (2 X 10(6) cells/ml), on the presence of antigen, it was specific and cross-reactive due to a shared rhamnose backbone of the two polysaccharide antigens, and it was highly selective, such that in a 42-55-day culture 1 out of 20 viable cells was a specific PFC. During the exponential increase of the antibody concentration at a constant number of PFC, antibodies were secreted at a rate of 2.4 X 10(4) molecules/s per cell until a plateau level of antibody (40 mug/culture) was reached. The microculture system was used to determine the minimal frequency of group polysaccharide-specific precursor cells in the blood. Independent of the time elapsed since the last immunization this frequency was 1-3 X 10(-5), i.e., in the range of 1-2.8 X 10(2) precursor cells per ml blood. This number was further used together with the clonotype analysis of the culture supernates to calculate the frequencies of precursors of major and minor clonotypes. A hierachy of persisting clonal memory precursor cells was found indicating that clonal dominance is determined by locked-in frequency patterns and therefore it is a phenomenon based on numbers of cells that respond to the antigen.

The sacral chordoma margin.

OBJECTIVE: Aim of the manuscript is to discuss how to improve margins in sacral chordoma. BACKGROUND: Chordoma is a rare neoplasm, arising in half cases from the sacrum, with reported local failure in >50% after surgery. METHODS: A multidisciplinary meeting of the "Chordoma Global Consensus Group" was held in Milan in 2017, focusing on challenges in defining and achieving optimal margins in chordoma with respect to surgery, definitive particle radiation therapy (RT) and medical therapies. This review aims to report on the outcome of the consensus meeting and to provide a summary of the most recent evidence in this field. Possible new ways forward, including on-going international clinical studies, are discussed. RESULTS: En-bloc tumor-sacrum resection is the cornerstone of treatment of primary sacral chordoma, aiming to achieve negative microscopic margins. Radical definitive particle therapy seems to offer a similar outcome compared to surgery, although confirmation in comparative trials is lacking; besides there is still a certain degree of technical variability across institutions, corresponding to different fields of treatment and different tumor coverage. To address some of these questions, a prospective, randomized international study comparing surgery versus definitive high-dose RT is ongoing. Available data do not support the routine use of any medical therapy as (neo)adjuvant/cytoreductive treatment. CONCLUSION: Given the significant influence of margins status on local control in patients with primary localized sacral chordoma, the clear definition of adequate margins and a standard local approach across institutions for both surgery and particle RT is vital for improving the management of these patients.

Bone sarcoma of the spine.

Primary malignant bone tumors of the vertebral column, i.e., bone sarcomas of the spine, are inherently rare entities. Vertebral osteosarcomas and chordomas represent the largest groups, followed by the incidence of chondro-, fibro-, and Ewing's sarcomas. Detailed clinical and neurological examination, complete radiographic imaging [radiographs, computed tomography (CT), magnetic resonance imaging (MRI)], and biopsy are the decisive diagnostic steps. Oncosurgical staging for spinal tumors can serve as a decision-guidance system for an individual's oncological and surgical treatment. Subsequent treatment decisions are part of an integrated, multimodal oncological concept. Surgical options comprise minimally invasive surgery, palliative stabilization procedures, and curative, wide excisions with complex reconstructions to attain wide or at least marginal resections. The most aggressive mode of surgical resection for primary vertebral column tumors is the total en bloc vertebrectomy, i.e., single- or multilevel en bloc spondylectomy. En bloc spondylectomy involves a posterior or combined anterior/posterior approach, followed by en bloc laminectomy, circumferential (360 degrees) vertebral dissection, and blunt ventral release of the large vessels, intervertebral discectomy and rotation/ en bloc removal of the vertebra along its longitudinal axis. Due to the complex interdisciplinary approach and the challenging surgical resection techniques involved, management of vertebral bone sarcomas is recommended to be performed in specific musculoskeletal tumor centers.

En bloc spondylectomy reconstructions in a biomechanical in-vitro study.

Wide surgical margins make en bloc spondylectomy and stabilization a referred treatment for certain tumoral lesions. With a total resection of a vertebra, the removal of the segment's stabilizing structures is complete and the instrumentation guidelines derived from a thoracolumbar corpectomy may not apply. The influence of one or two adjacent segment instrumentation, adjunct anterior plate stabilization and vertebral body replacement (VBR) designs on post-implantational stability was investigated in an in-vitro en bloc spondylectomy model. Biomechanical in-vitro testing was performed in a six degrees of freedom spine simulator using six human thoracolumbar spinal specimens with an age at death of 64 (+/- 20) years. Following en bloc spondylectomy eight stabilization techniques were performed using long and short posterior instrumentation, two VBR systems [(1) an expandable titanium cage; (2) a connected long carbon fiber reinforced composite VBR pedicle screw system)] and an adjunct anterior plate. Test-sequences were loaded with pure moments (+/- 7.5 Nm) in the three planes of motion. Intersegmental motion was measured between Th12 and L2, using an ultrasound based analysis system. In flexion/extension, long posterior fixations showed significantly less range of motion (ROM) than the short posterior fixations. In axial rotation and extension, the ROM of short posterior fixation was equivalent or higher when compared to the intact state. There were only small, nonsignificant ROM differences between the long carbon fiber VBR and the expandable system. Antero-lateral plating stabilized short posterior fixations, but did not markedly effect long construct stability. Following thoracolumbar en bloc spondylectomy, it is the posterior fixation of more than one adjacent segment that determines stability. In contrast, short posterior fixation does not sufficiently restore stability, even with an antero-lateral plate. Expandable verses nonexpandable VBR system design does not markedly affect stability.

Three-dimensional stiffness in a thoracolumbar en-bloc spondylectomy model: a biomechanical in vitro study.

BACKGROUND: In selected cases, en-bloc spondylectomy is the only option to reach wide resection margins for patients with malignant tumours of the thoracolumbar spine. These patients must be also provided a secure initial stabilization of the spine and this is the role of vertebral body replacements employed with posterior fixation systems. The aim of this study was to determine the postimplantation stiffness of a connected vertebral body replacement pedicle screw system in different implantation scenarios following an en-bloc spondylectomy. Reconstruction was varied by posterior fixation lengths and axial compression forces during implantation. METHODS: Three-dimensional stiffness was assessed in 6 fresh frozen human spinal specimens (Th11-L3) using a six degree of freedom spine simulator. Following en-bloc spondylectomy reconstruction was performed using a carbon composite fibre vertebral body replacement connected to a posterior fixation system by two artificial pedicles. The spines were loaded with pure moments (7.5Nm) in the three main motion planes. The intersegmental rotations were measured between Th12 and L2. FINDINGS: Reconstructions using long posterior fixation modes demonstrated significant (P<0.05) higher stiffness compared to short posterior fixations in all motion planes. In axial rotation short posterior fixation modes failed to reach the values of the intact state. Neither high nor low axial compression force during implantation showed a significant impact on postfusional stiffness. INTERPRETATION: In this biomechanical model, the employed system should be implanted with a posterior fixation of two adjacent segments to the lesion in order to achieve a secure stabilization of the treated segment.

Primary antibody response of rabbit blood lymphocytes in vitro.

This paper describes a method for in vitro induction of a primary response of rabbit peripheral blood lymphocytes to sheep red blood cells. The response is measured by visualizing and enumerating the plaque-forming cells (PFC). Removal of an adhering suppressor cell and use of a low cell concentration in culture are among the crucial requirements. Maximum response was usually reached after 10--15 days of culture. The number of PFC then decreased or stayed at roughly plateau level at least up to the fourth week of culture, when most of the experiments were terminated. In several instances the response had a cyclical character with repeating peaks of PFC. Only plaques of the direct type were found.

A two-stage culture system for the induction of antibody-forming cell clones in cultures of normal human blood lymphocytes.

A two-stage culture method is described for the induction of a specific antibody response to sheep red cells (SRC) in microcultures at limiting dilutions of human peripheral blood lymphocytes (PBL). PBL from normal donors were cultured for 4 days with antigen and EBV using well defined conditions. The cells were then distributed in 10 microliter microcultures at different cell densities in order to estimate the frequency of responding units. The culture wells were tested for the presence of anti-SRC antibody by the spot test. The results show that the expression of antibody-forming cell clones in the second stage microcultures is strictly dependent on the presence of both antigen and EBV during the first stage cultures. The efficiency of the system was improved by the addition of 4% polyethylene glycol (PEG, MW 6000) in the first stage and its removal in the second stage and by the use of human serum (instead of fetal calf) in both stages. This approach permits the separation of different cellular events, occurring when human B cells are stimulated by antigen and represents a useful approach for studying the mechanisms of the specific immune response in man.

Induction of antibody forming cells with specificity for Candida albicans mannoproteins in cultures of human peripheral blood lymphocytes.

A method is described for the induction of a specific antibody response to Candida albicans in cultures of normal human peripheral blood lymphocytes (PBL). PBL were cultured in the presence of whole C. albicans cells and the antibody response was evaluated by the ELISPOT technique, on plastic wells coated with a purified candidal cell well mannoprotein (MP). Under the conditions described here, a specific antibody response was obtained in all of the eight donors tested. The response was antigen-dependent and antigen-specific, peaked around day 10-12 of culture and the antibodies belonged to both the IgM and the IgG isotypes. By testing the cultured cells on MP from different Candida species, the method permitted the detection of antibodies directed against MP epitopes shared by C. albicans and C. parapsilosis.

The antigen-specific induction of normal human lymphocytes in vitro is down-regulated by a conserved HIV p24 epitope.

Synthetic peptides containing amino acid sequence 218-238 of the core protein p24 of human immunodeficiency virus type 1 (HIV-1) and progressively shorter sequences at its C-terminus, were tested for their effect on antigen dependent in vitro responses of peripheral blood lymphocytes (PBL) from normal human donors. A peptide as short as 7 amino acids, corresponding to a highly conserved sequence, was able to inhibit in a dose-dependent manner the induction of a specific primary antibody response to the sheep red cell (SRC) antigen, as well as the proliferative response to recall microbial antigens. The results of this study constitute additional evidence of the immunoinhibitory effects of HIV components and may help to unravel some of the pathogenic mechanisms of AIDS. Moreover, they are of potential relevance for the development of immunoprophylactic and therapeutic strategies.

Serum reactivity against an immunoregulatory sequence of HIV p24 in HIV-1-infected subjects.

The aim of this study was to assess the antibody reactivity in HIV-infected subjects against an HIV-1 p24 sequence, p226 (aa226-237), including a seven amino acid epitope showing immunosuppressive activity in vitro and to evaluate the relationship between anti-peptide antibody levels and disease progression. Sera of HIV-infected subjects, at different stages of disease, were compared to control sera in a retrospective evaluation. Recombinant HIV-1 p24 and p24- and control-peptides were used in an enzyme immunoassay as targets for antibodies present in the sera. Antibodies directed against the whole p24 protein and its peptides were found in all the sera studied but at different levels. The anti-p226 reactivity was not significantly different at different clinical stages. Nevertheless, it was inversely correlated to the reactivity directed against the whole protein, that was lower in subjects characterized by low CD4 cell numbers.

Induction of a specific antibody response to Bordetella pertussis antigens in cultures of human peripheral blood mononuclear cells.

The role of specific antibodies in protective immunity to Bordetella pertussis has not yet been clearly defined. In the present work, the induction of a specific antibody response to B. pertussis in cultures of human peripheral blood mononuclear cells (PBMC) was investigated, on the assumption that the capacity of circulating lymphocytes to mount a specific response in vitro may provide a useful parameter for the evaluation of protective immunity. When PBMC from normal adult donors were cultured with a heat-inactivated B. pertussis whole-cell suspension, cells secreting antibodies to pertussis toxin, pertactin and filamentous haemagglutinin were generated consistently. The antibody response peaked between days 7 and 11 of culture and the antibodies produced were exclusively of the IgM class.

A mannoprotein constituent of Candida albicans cooperates with antigen in the induction of a specific primary antibody response in cultures of human lymphocytes.

The effects of Candida albicans mannoproteins on the induction of a primary antibody response to a T-dependent antigen, sheep erythrocytes (SRBC), in cultures of human blood lymphocytes, were investigated. Two experimental systems (bulk and limiting dilution cultures) allowing the detection of both enhancing and inhibitory effects, were used. In bulk cultures, antigen alone elicited a small number of specific antibody forming cells, unless IL-2 was supplied. Addition of the fungal mannoprotein extract or of a purified constituent of it increased 5 to more than 10 times the specific response. When limiting dilution analysis was performed, we observed that: a) a similar number of specific precursor cells was induced by antigen and either IL-2 or mannoprotein; b) the plot of the number of seeded cells versus the log of the fraction of negative cultures was linear in antigen and IL-2 triggered cultures but constantly deviated from linearity when the candidal stimulant was added. Thus, more than one type of precursor cell was limiting in these cultures, and the immunoenhancing effect of mannoprotein may involve multiple cellular interactions.

Immunoregulatory effect of a synthetic peptide corresponding to a region of protein p24 of HIV.

The effect of a synthetic peptide, corresponding to a sequence of HIV-1 p24 protein (amino acids 218-237), on in vitro immune responses was studied. The peptide inhibited in a dose-dependent manner the induction of an anti-SRC antibody response and of a PPD-specific proliferative response of human PBL. On the other hand, PHA-induced proliferation of human PBL and PPD-induced proliferation of a PPD-specific human T-cell line were not modified by comparable amounts of the peptide. These results suggest that structures from a protein (p24), present in the serum throughout the course of HIV infection, are able to interfere with the inductive stages of specific immune responses. These findings may help to unravel some of the pathogenic mechanisms of AIDS and may contribute to the development of vaccine strategies.