Soft-tissue fat tumours: differentiating malignant from benign using proton density fat fraction quantification MRI.

AIM: To evaluate if quantifying proton density fat fraction (PDFF) would be useful in separating lipoma, atypical lipomatous tumour (ALT) and liposarcoma in the extremities and trunk. In addition, differentiating ALT versus non-classical lipomas using magnetic resonance imaging (MRI)-based fatty acid composition (FAC) and three-dimensional (3D) texture analysis was tested. MATERIAL AND METHODS: This prospective study (undertaken between 2014-2017; comprising 20 women, 21 men) was approved by the Regional Ethical Review Board and informed consent was obtained from all participants. For PDFF and FAC 3D spoiled gradient multi-echo images were acquired. PDFF was analysed in 16 lipomas (25-76 years), 14 ALTs (42-78 years) and 11 myxoid liposarcomas (31-68 years). The difference of mean PDFF was tested with one-way analysis of variance. A support vector machine algorithm was used to find the separating mean PDFF values. RESULTS: Mean PDFF for lipomas was 90% (range 76-98%), for ALT 83% (range 62-91%), and for liposarcoma 4% (range 0-21%). The difference of mean PDFF for liposarcomas versus ALT and lipoma was significant (p=0.0001, for both), and for ALT versus lipoma (p=0.021). The optimal threshold for separating liposarcoma from ALT and lipoma was 41.5%, and for ALT and lipoma 85%. Texture analysis could not separate ALT and non-classical lipomas, while the difference for FAC unsaturation degree was significant (p=0.013). CONCLUSION: Measuring PDFF is a promising complement to standard MRI, to separate liposarcomas from ALT and lipomas. Lipomas that are not solely composed of fat cannot confidently be separated from ALT using PDFF, FAC, or texture analysis.

Telomeric associations correlate with telomere length reduction and clonal chromosome aberrations in giant cell tumor of bone.

Giant cell tumor of bone (GCTB) is characterized cytogenetically by frequent telomeric associations (tas). To explore the mechanisms behind the formation of tas in GCTB and to investigate their karyotypic consequences, the frequencies of tas and clonal aberrations other than tas in 20 GCTBs were compared to telomere length and status, as assessed by quantitative PCR, fluorescence in situ hybridization (FISH), and expression levels of four genes involved in telomere maintenance. Based on the G-banding results, the tumors were divided into two groups, one with a high frequency of tas and one with a low frequency. Clonal aberrations were found to be restricted to the group with a high level of tas, and the same group showed a significantly larger reduction in telomere length in tumor cells compared to peripheral blood cells. Furthermore, 65 out of 66 tas analyzed by FISH were negative for telomeric sequences. The expression levels of TERT, TERF1, TERF2, and POT1 did not correlate with telomere length or the frequency of tas. Thus, the present findings provide strong support for the notion that decreased telomere length is a prerequisite for tas in GCTBs and that the clonal changes occurring in GCTBs are derived from tas.

Growth inhibition of human osteosarcomas in nude mice by human interferon-alpha: significance of dose and tumor differentiation.

The sensitivity of 11 human osteosarcoma xenografts in nude mice to human interferon-alpha (IFN-alpha) was studied. Growth inhibition could be demonstrated in all tumors but the necessary IFN-alpha dose ranged from 1 X 10(5)-1 X 10(6) IU/day. IFN-alpha had to be given daily to attain growth arrest and growth resumed after reduction of the IFN-alpha dose. The xenografts could be divided in two groups based on their sensitivity to IFN-alpha: one group of five xenografts that were growth arrested by IFN-alpha, 2 X 10(5) IU/day, and another group of six xenografts in which this dose was insufficient to arrest growth. The proportions of S-phase cells, determined by DNA flow cytometry of untreated control xenografts, were lower in the former group compared to the latter less IFN-alpha sensitive group. Histological examination revealed that in four of the five more IFN-alpha sensitive xenografts, tumor tissue was replaced by normal bone and marrow tissue. This was not seen in the respective control xenografts and not in any of the six less sensitive IFN-alpha treated xenografts. It appears that less proliferative osteosarcoma xenografts are more sensitive to growth inhibition by IFN-alpha. Interestingly the antitumor effect by IFN-alpha on these xenografts was expressed not only by growth arrest but also by tumor differentiation.

Influence of human alpha-interferon on four human osteosarcoma xenografts in nude mice.

Growth-inhibiting effects of human alpha-interferon (HuIFN-alpha) were investigated in four human osteosarcoma xenografts in nude mice. In addition to effects on growth, the HuIFN-alpha treatment was evaluated by histological examination and DNA flow cytometric analysis. Daily doses of 2 X 10(5) IU HuIFN-alpha completely arrested the growth of two osteosarcoma xenografts and partially inhibited one, whereas 1 X 10(6) IU/day were necessary to arrest the growth of the fourth. Growth inhibition was reversible and tumor size independent. The histological appearance, including mitotic indices, and S-phase proportions were unchanged in three xenografts. The mechanism of the HuIFN-alpha-induced growth inhibition of these three xenografts was therefore not considered to be a direct antiproliferative effect, but rather due to increased cell loss and/or increased cell cycle time. The modal DNA value of one xenograft was changed from aneuploid to diploid during HuIFN-alpha treatment. Histologically, these xenografts were partly replaced by normal appearing bone and bone marrow. The S-phase proportion was also reduced in these xenografts, implying that HuIFN-alpha can also have a direct antiproliferative effect.

Intra-Articular Synovial Sarcomas: Incidence and Differentiating Features from Localized Pigmented Villonodular Synovitis.

Purpose. To determine the incidence of intra-articular synovial sarcomas and investigate if any radiological variables can differentiate them from localized (unifocal) pigmented villonodular synovitis (PVNS) and if multivariate data analysis could be used as a complementary clinical tool. Methods. Magnetic resonance images and radiographs of 7 cases of intra-articular synovial sarcomas and 14 cases of localized PVNS were blindedly reviewed. Variables analyzed were size, extra-articular growth, tumor border, blooming, calcification, contrast media enhancement, effusion, bowl of grapes sign, triple signal intensity sign, synovial low signal intensity, synovitis, age, and gender. Univariate and multivariate data analysis, the method of partial least squares-discriminant analysis (PLS-DA), were used. Register data on all synovial sarcomas were extracted for comparison. Results. The incidence of intra-articular synovial sarcomas was 3%. PLS-DA showed that age, effusion, size, and gender were the most important factors for discrimination between sarcomas and localized PVNS. No sarcomas were misclassified as PVNS with PLS-DA, while some PVNS were misclassified as sarcomas. Conclusions. The most important variables in differentiating intra-articular sarcomas from localized PVNS were age, effusion, size, and gender. Multivariate data analysis can be helpful as additive information to avoid a biopsy, if the tumor is classified as most likely being PVNS.

Interferon-inhibited human osteosarcoma xenografts induce host bone in nude mice.

The growth of human osteosarcoma xenografts in nude mice can be inhibited by human interferon-alpha (IFN-alpha). Histologic examination of growth-inhibited tumors has revealed mineralization and partial replacement of the tumor by normal bone tissue. We have investigated whether the normal bone tissue was formed by differentiated tumor cells or by induction of host stroma to differentiate into bone tissue. Employing antibodies to both murine and human type I collagen, it was found that the normal bone produced in IFN-inhibited osteosarcomas was host derived. These results suggest that IFN induced the osteosarcoma cells to produce a bone-inductive agent that interacts with the host cells, and leads to the formation of mature normal bone tissue in a heterotopic site.

Expression of deoxycytidine kinase and phosphorylation of 2-chlorodeoxyadenosine in human normal and tumour cells and tissues.

Deoxycytidine kinase (dCK) activates several clinically important drugs, including the recently developed antileukaemic compound 2-chlorodeoxyadenosine (CdA). The distribution of dCK in cells and tissues has previously been determined by activity measurements, which may be unreliable because of the presence of other enzymes with overlapping substrate specificities. Therefore we have measured dCK polypeptide levels in extracts of normal and malignant human peripheral blood mononuclear cells, gastrointestinal tissues and sarcomas, using a specific immunoblotting technique, as well as the phosphorylation of CdA in the same extracts. High levels of dCK were found in all major subpopulations of normal mononuclear leucocytes (120 +/- 19 ng dCK/mg protein) and in B-cell chronic lymphocytic leukaemia (81 +/- 30 ng/mg, n = 23). Hairy-cell leukaemia contained lower levels (28 +/- 23 ng/mg, n = 7), as did three samples of T-cell chronic lymphocytic leukaemia (18 +/- 14 ng/mg). Phytohaemagglutinin stimulation of normal lymphocytes did not lead to any substantial increase in either dCK activity or protein expression (less than 2.5-fold). The human CEM wt T-lymphoblastoid cell line contained 56 +/- 1 ng/dCK/mg protein, while in the CEM ddC50 and AraC8D mutants that lack dCK activity, no dCK polypeptide could be detected. In colon adenocarcinomas, the dCK content was significantly higher (20 +/- 9 ng/mg, n = 20) than in normal colon mucosa (8 +/- 3.5 ng/mg, n = 19, P < 0.05). A similar pattern of dCK expression was found in gastric adenocarcinomas (21 +/- 13 ng/mg, n = 5) and normal stomach mucosa (6 +/- 5 ng/mg, n = 5, P < 0.15). One leiomyosarcoma and one extra-skeletal osteosarcoma showed dCK levels comparable with those found in normal lymphocytes (84 +/- 6 and 109 +/- 4 ng/mg, respectively), while other sarcoma samples contained lower levels, comparable to the gastrointestinal adenocarcinomas (20 +/- 7 ng/mg, n = 12). Thus, dCK is expressed constitutively and predominantly in lymphoid cells, but it is also found in solid non-lymphoid tissues, with increased levels in malignant cells. The phosphorylation of CdA in crude extracts showed a close correlation to the dCK polypeptide level.

Scandinavian Sarcoma Group Osteosarcoma Study SSG VIII: prognostic factors for outcome and the role of replacement salvage chemotherapy for poor histological responders.

From 1990 to 1997, 113 eligible patients with classical osteosarcoma received neo-adjuvant chemotherapy consisting of high-dose methotrexate, cisplatin and doxorubicin. Good histological responders continued to receive the same therapy postoperatively, while poor responders received salvage therapy with an etoposide/ifosfamide combination. With a median follow-up of 83 months, the projected metastasis-free and overall survival rates at 5 years are 63 and 74%, respectively. Independent favourable prognostic factors for outcome were tumour volume < 190 ml, 24-h serum methotrexate > 4.5 microM and female gender. The etoposide/ifosfamide replacement combination did not improve outcome in the poor histological responders. In conclusion, this intensive multi-agent chemotherapy results in > 70% of patients with classical osteosarcoma surviving for 5 years. The data obtained from this non-randomised study do not support discontinuation and exchange of all drugs used preoperatively in histological poor responders. As observed in previous Scandinavian osteosarcoma studies, female gender appears to be a strong predictor of a favourable outcome.

Comparison of technetium-99m-MIBI and technetium-99m-tetrofosmin uptake by musculoskeletal sarcomas.

UNLABELLED: Technetium-99m-MIBI was initially developed for heart studies but it can also be used to depict tumors, predict multidrug resistance and evaluate chemotherapy. Recently, 99mTc-tetrofosmin, which exhibits similar physical properties, has been launched for heart studies. Tumor uptake and prediction of multidrug resistance have also been reported regarding the latter tracer. A comparison of these two tracers regarding the detectability of musculoskeletal sarcoma has been made. METHODS: Twenty patients with musculoskeletal sarcoma of the extremities or pelvis underwent planar examination after the administration of 99mTc-MIBI and 99mTc-tetrofosmin with an interval of 2-7 days. The tumor activity was compared with one ipsilateral and one contralateral background region. RESULTS: There was a small, but not significant, difference in favor of 99mTc-MIBI with regard to both background regions. CONCLUSION: Technetium-99m-MIBI and 99mTc-tetrofosmin can both be used to visualize musculoskeletal sarcomas. The choice may depend on which agent is used routinely for myocardial studies in the laboratory.

Nonrandom secondary chromosome-aberrations in liposarcomas with t(12, 16).

Ten liposarcomas were analyzed cytogenetically after short-term culturing. Eight tumors had a t(12;16) (q13;p11) and two tumors had complex translocations involving chromosomes 7, 12, and 16 and 2, 9, 12, 16 and 20, respectively. Among the secondary aberrations seen in five tumors, +8 was found in two tumors and i(7)(q10) in four tumors. Trisomy 8 has previously been described as a nonrandom secondary aberration in myxoid liposarcoma, but i(7q) has only been reported in a single case before. All recurrent chromosome aberrations reported in liposarcomas with recombination between 12q13 and 16p11 (42 cases) were surveyed and compared with their frequencies in liposarcomas without this recombination (33 cases). Trisomy 5 and 8 were found in both tumor groups, whereas +19, t(3;15)(p23;q15), del(6)(q21), i(7q), and rearrangements of 1p11 and 2q35 were found exclusively in tumors with 12q13 and 16p11 aberrations.

A new cytogenetic subgroup in lipomas: loss of chromosome 16 material in spindle cell and pleomorphic lipomas.

Six spindle cell lipomas and two pleomorphic lipomas were analyzed cytogenetically. One spindle cell lipoma had a supernumerary ring chromosome as the sole anomaly. The other five spindle cell lipomas and both pleomorphic lipomas had hypodiploid stemlines with monosomy 16 or unbalanced aberrations leading to loss of 16q13-qter, a feature distinguishing these lipoma subtypes from other benign and borderline adipose tissue tumor histotypes. unbalanced aberrations of chromosomes 13 and 10 were found in five and three cases respectively; 13q12 was lost in all of these cases, whereas there was no common deleted segment in chromosome 10. No aberrations involving 12q13-15, which are frequent in typical lipomas, were found. Both pleomorphic lipomas, but none of the spindle cell lipomas, had hypotetraploid sidelines, multiple nonclonal aberrations, and telomeric associations. The present findings reveal a new cytogenetic/histopathological association in adipose tissue tumors.

Chromosome aberrations and cytogenetic intratumor heterogeneity in chondrosarcomas.

Clonal chromosome aberrations identified after short-term culture are presented for 13 chondrosarcomas; in 5 cases both the primary tumors and local recurrences were studied. The stemline chromosome number was hypodiploid or hyperhaploid in 9 tumors. The most frequent numerical anomalies were, in falling order of frequency, loss of chromosomes Y, 10, 13, and 6, and gain of chromosomes 7 and 20. No recurrent structural rearrangement was found, but chromosome bands 5q13, 1q21, 7p11, and 20q11 were each involved in three different rearrangements. Karyotypic heterogeneity was assessed in two different ways: as the presence of more than one clone in one sample and as the presence of different clones in different samples from the same surgical specimen. Clonal karyotypic evolution was demonstrated in 6 of the 7 cases in which two or more samples could be investigated. All 6 showed intersample heterogeneity. Intrasample heterogeneity was found in only 5 of the 28 samples with aberrations. By comparing the incidences of the nonrandomly occurring aberrations in stemlines and sidelines in the heterogeneous tumors, it was possible to conclude that loss of chromosome 13 and rearrangement of band 5q13 were early events in the clonal evolution.

Supernumerary ring chromosomes in five bone and soft tissue tumors of low or borderline malignancy.

Five tumors (two myxoid malignant fibrous histiocytoma, two dermatofibrosarcoma protuberans, and one parosteal osteosarcoma) with ring chromosomes as the sole cytogenetic anomaly or as the only structural rearrangement were observed in a series of 60 karyotypically abnormal, nonlipogenic bone and soft tissue tumors (BST). All five tumors were of borderline or low malignancy. These findings support the suggestion that supernumerary ring chromosomes as the sole structural chromosomal aberration are not associated with any particular histopathologic diagnosis but may characterize a group of BST of borderline or low malignancy.

Cytogenetic analysis of four angiosarcomas from deep and superficial soft tissue.

Angiosarcomas are rare malignant vascular tumors, most commonly found in the skin or superficial soft tissue. We found clonal chromosome aberrations in four short-term cultured angiosarcomas. Two cases were diagnosed as epithelioid angiosarcomas, and one as postmastectomy angiosarcoma. Two of the tumors were deep-seated and two were superficial. Angiosarcomas from deep, soft tissue are extremely rare and have never been cytogenetically investigated before. The chromosome number ranged from hypodiploid to hypertriploid. When the results from the present study were combined with data on the four previously reported cytogenetically aberrant angiosarcomas, the most frequently rearranged chromosomes were 5, 7, 8, 13, 15, 20, 22, and the Y chromosome. Recurrent changes, each found in three of these eight angiosarcomas, were gains of 5pter-p11, 8p12-qter, and 20pter-q12, losses of 7pter-p15 and 22q13-qter, and -Y in two of three men.

Cytogenetic and quantitative DNA analysis of primary and xenografted human osteosarcomas.

We have analyzed, cytogenetically and by flow DNA cytometry, three human osteosarcomas. From one case, both the primary tumor (T9) and three of its serial passages in nude mice were analyzed. From the two other tumors (T1, T4), nude mouse xenografts were examined. Complex karyotypic rearrangements were invariably found in the short-term culture preparations. The major clones were hypodiploid in T1 but near-triploid in all other tumor samples. No rearrangement common to all tumors could be identified. The DNA indexes were 1.0/2.0 (T1), 1.4 (T4), 0.9/1.8 (T9 primary), and 1.7 (all T9 xenografts). Thus, there was good correlation between the DNA indexes and the chromosome numbers. Chromosomal evolution could be studied in one case, in which both the primary tumor (T9) and its xenograft passages (1, 4, and 7), obtained 1, 7, and 11 months after the first transplantation of T9 cells to nude mice, were analyzed. All but one of the clonal marker chromosomes found in the primary tumor were retained in all passages. On the other hand, several new clonal markers developed.

A highly aggressive primitive mesenchymal tumor with a translocation (1;19)(q12;q13.2).

Soft tissue sarcomas constitute a heterogeneous group of malignant tumors of mesenchymal origin, the classification of which may present a diagnostic challenge. We present here the cytological, histopathological, immunohistochemical, and cytogenetic findings of an unusual case of a highly aggressive sarcoma. Based on the morphology and the immunohistochemical profile, this primitive tumor and its metastases could not be conclusively classified as any of the defined subtypes of sarcomas, although the findings were suggestive of a variant of rhabdomyosarcoma. Cytogenetic characterization using G-banding, SKY, FISH, and CGH revealed almost identical chromosomal compositions of the primary tumor and the metastasis. The hypertetraploid karyotype was characterized by numerical imbalances as well as by an unbalanced translocation t(1;19)(q12;q13.2), which has not been previously reported.

Radiation-associated sarcomas are characterized by complex karyotypes with frequent rearrangements of chromosome arm 3p.

Ionizing radiation is a well-known risk factor for sarcoma development. To investigate whether radiation-associated sarcomas are characterized by chromosome aberrations that distinguish them from de novo sarcomas, we identified those patients in our series of more than 500 cytogenetically abnormal sarcomas that fulfilled the following criteria: (1) each patient should have been irradiated for another malignancy at least 3 years prior to the sarcoma diagnosis, and (2) the sarcoma should have developed within the field of radiation. Ten patients fulfilling these criteria could be retrieved (median age at sarcoma diagnosis was 55 years, range 17-79; median latency period between primary tumor and radiation-associated sarcoma was 9 years, range 4-30). The diagnoses were typical for radiation-associated sarcomas: 2 each of malignant fibrous histiocytoma, leiomyosarcoma, and pleomorphic sarcoma, and 1 each of osteosarcoma, fibrosarcoma, myxofibrosarcoma, and spindle cell sarcoma. All 10 cases had relatively complex karyotypes with multiple, mostly unbalanced, structural rearrangements, similar to what has been reported in de novo sarcomas of the corresponding histologic subtypes. The only cytogenetic features that were unusually frequent among the radiation-associated sarcomas were the finding of unrelated clones in 3 cases, and loss of material from chromosome arm 3p, in particular 3p21-3pter, in 8 cases. Loss of the same chromosome segment has been described in 4 of the 8 previously published cases of radiation-associated sarcomas that have been analyzed after short-term culturing, which makes this imbalance significantly (P < 0.001) more frequent among radiation-associated sarcomas (12 of 18 cases) than among unselected cases of the corresponding histologic subtypes (74 of 282 cases). In contrast to the cytogenetic results, no 3p deletions were detected among the 6 cases of the present series that could be analyzed by comparative genomic hybridization (CGH). The most frequent imbalance detected by CGH was gain of 15cen-q15 (3 cases), followed by loss of chromosome 13 and gain of 5p, and 7cen-q22, each detected in 2 cases.

Properties and levels of deoxynucleoside kinases in normal and tumor cells; implications for chemotherapy.

Deoxynucleoside kinases are key enzymes in deoxyribonucleoside salvage, activating several clinically important chemotherapeutic drugs. The four known kinases, cytosolic thymidine kinase (TK1) and deoxycytidine kinase (dCK) and the mitochondrial thymidine kinase (TK2) and deoxyguanosine kinase (dGK), have been purified and characterized as to the subunit structure as well as specificity with a large number of analogs. These results are summarized and used to establish selective assays for the four enzymes in crude extracts of normal and malignant human peripheral blood mononuclear cells, gastrointestinal tissues and sarcomas. TK2 and dGK activities were found at low levels in all tissues, possibly correlated to the content of mitochondria. TK1 activity was detected only in samples containing a significant number of S phase cells. We have measured dCK activity as well as dCK polypeptide level by immuno blotting in these extracts. High levels of dCK were found in normal mononuclear leukocytes (91-145 ng dCK/mg protein) and in B-cell chronic lymphocytic leukemia (80 +/- 30 ng/mg, n = 23). Hairy cell leukemia contained lower levels (28 +/- 23 ng/mg, n = 7), as did unexpectedly three samples of T-cell chronic lymphocytic leukemia (18 +/- 14 ng/mg). Phytohemaglutinine stimulation of normal lymphocytes did not lead to any substantial increase in either dCK activity or expression (less than 2.5-fold). In colon adenocarcinomas, the dCK content was significantly higher (21 +/- 9.3 ng/mg, n = 20) than in normal colon mucosa (8.2 +/- 3.7 ng/mg, n = 19, p < 0.05). A similar pattern of dCK expression was found in gastric adenocarcinomas (21 +/- 13 ng/mg, n = 5) and normal ventricular mucosa (6.2 +/- 5.4 ng/mg, n = 5, p < 0.15). One leiomyosarcoma and one extra-skeletal osteosarcoma showed a dCK levels comparable to those found in normal lymphocytes (84 +/- 6 and 109 +/- 4 ng/mg), while other sarcoma samples contained levels comparable to the gastrointestinal adenocarcinomas (20 +/- 7 ng/mg, n = 12). We confirm that dCK is expressed constitutively and predominantly in lymphoid cells, but conclude that a significant expression may be found in non-lymphoid tissues as well, with increased levels in the corresponding tumor tissue. 2-Chlorodeoxyadenosine (CdA), an antileukemic agent used in treatment of hairy cell leukemia and chronic lymphocytic leukemias (B-CLL), is phosphorylated by dCK which was used as the selective substrate for this enzyme. A study was performed to investigate if there was a correlation between the dCK levels and the response to CdA treatment.(ABSTRACT TRUNCATED AT 400 WORDS)

Bone resorption in orthotopic and heterotopic bone of dichloromethylene bisphosphonate-treated rats.

The effect of the bisphosphonate dichloromethylene bisphosphonate (Cl2MBP) on orthotopic and heterotopic bone, induced by implants of demineralized bone matrix (DBM) in rats, was analyzed, with special reference to bone resorption. The heterotopic bone was formed by induction for 3 weeks; at this time, the rats were given daily subcutaneous injections of 3 mg/kg of body weight of Cl2MBP or saline, until sacrifice. Prior to the start of treatment, the animals were given 45Ca and [3H]proline to label the inorganic and organic components of bone, respectively. Groups of rats were sacrificed at intervals from 1 to 31 days after isotope injection, and the net formation of bone and the elimination rates of the two isotopes were studied in the heterotopic bone, in diaphyseal and metaphyseal bone, and in teeth. The treatment with Cl2MBP caused a doubling of the daily net increase in mineral of the induced heterotopic bone, and a less pronounced increase in the ash content of tibiae. The treatment decreased the elimination rates of both isotopes in the orthotopic and heterotopic bone, showing that decreased bone resorption is the cause of the increased net bone formation.

Adjuvant interferon treatment in human osteosarcoma.

An update of the adjuvant trial on osteosarcoma in Sweden comparing patients receiving natural interferon (IFN) alpha with a high-dose chemotherapy group and a nonadjuvant group is presented. The overall survival for the IFN group is 49%, for the chemotherapy group 54%, and for the nonadjuvant group 35%. Trial evaluation was complicated by group differences with respect to various clinicopathologic features of prognostic significance. The role of IFN in the treatment of osteosarcoma can still not be established.