Complications and reoperations of the SB Charité lumbar disc prosthesis : experience in 75 patients Artificial disc prosthesis show fair to good short - and mid-term results . Long-term results are becoming apparent now , however , the incidence of late complications with this procedure remain poorly understood . In this report we will analyse late complications and discuss our experiences with salvage operations in patients with persistent pain after SB Charité disc prosthesis implantation . Seventy-five patients with persistent leg and back pain after insertion of an artificial disc prosthesis were enrolled in the study . In this negative selection frequently occurring late-complications were subsidence , wear , adjacent disc degeneration , facet joint degeneration and migration . In 15 patients we performed a posterior fusion without disc removal , and in 22 patients we removed 26 prostheses and performed a posterior and anterior fusion . The visual analogue scale -LRB- VAS -RRB- and Oswestry were examined before the salvage operation and after a follow-up period of at least 1 year , which is not yet available in all patients . The VAS and Oswestry decreased in the posterior group -LRB- n = 10 -RRB- respectively from 8.0 -LRB- SD 0.9 -RRB- to 6.3 -LRB- SD 2.1 -RRB- and from 57.0 -LRB- SD 17.0 -RRB- to 44.6 -LRB- SD 20.4 -RRB- ; and in the disc removal group -LRB- n = 14 -RRB- respectively from 8.0 -LRB- SD 0.9 -RRB- to 5.6 -LRB- SD 2.7 -RRB- and from 56.3 -LRB- SD 14.0 -RRB- to 43.0 -LRB- SD 20.7 -RRB- . Serious late complications may occur following total disc replacement . Removal of the SB Charité artificial disc is feasible but with inherent risks . Removal of the disc prosthesis gives slightly better results than posterior fusion alone after a follow-up of at least 1 year . Introduction Degenerative disc disease -LRB- DDD -RRB- is a major cause of pain and disability , with great social and financial impact , playing an increasing role in modern society -LSB- 21 -RSB- . Several surgical techniques have been developed to treat DDD . Spinal fusion is seen as the `` gold standard '' , but nowadays artificial disc prostheses are an alternative method -LSB- 13 -RSB- . The artificial disc should preserve motion , stability and normal function of a spinal segment . Also , less adjacent segment degeneration is expected -LSB- 6 , 7 , 13 , 15 -RSB- . In theory , this procedure has many advantages over spinal fusion . A spinal fusion does eliminate motion and can cause overloading and early degeneration of the adjacent levels , although usually appearing only after 10 -- 15 years -LSB- 1 , 11 , 12 , 17 -RSB- . Results after implantation of the SB Charité artificial disc prosthesis are diverse . short - and mid-term results are fair to good -LSB- 3 , 5 , 13 , 22 -RSB- . The FDA-IDE study , in which the SB Charité artificial disc was tested for approval in the US , showed a FDA-defined , overall clinical success rate of 57.1 % after 2-year follow-up -LSB- 2 , 15 -RSB- . For the IDE study , the procedure was only judged to be successful if four criteria were satisfied : -LRB- 1 -RRB- greater than 25 % improvement in Oswestry disability index ; -LRB- 2 -RRB- no device failure ; -LRB- 3 -RRB- no major complications ; and -LRB- 4 -RRB- no neurological deterioration compared to preoperative status . Consequently , the composite definition of success employed in the IDE study makes it difficult to compare with other studies that employed different success criteria . In a 10-year follow-up period , Lemaire et al. -LSB- 13 -RSB- found that 90 % of patients had good to excellent clinical outcome . On the other hand , in the only available long-term study of Putzier et al. -LSB- 16 -RSB- with an average follow-up of 17 years , the investigators found no evidence that long-term results of the disc prosthesis were superior to spinal fusion -LSB- 16 -RSB- . It remains unclear whether the early fair to good results obtained with an artificial disc will be consistently maintained with a longer follow-up period -LSB- 9 , 16 , 18 , 20 -RSB- . In the Netherlands , more than 1,000 patients have been implanted with a SB III Charité -LRB- Link , Germany -RRB- disc prosthesis starting in 1989 . As these patients received the implant during routine clinical practice , they were not the subjects of a randomized trial . However , 50 patients -LRB- 75 arthroplasties -RRB- were enrolled in a prospective observational trial , and at 2 years the clinical success rate was found to be 70 % -LSB- 22 -RSB- . Since 1989 , there have been many changes in the available implant sizes , surgical instrumentation , and patient indications . Although the basic `` SB III '' design has remained the same throughout the past 18 years , there have also been evolutionary changes in the polyethylene -LRB- PE -RRB- resin , sterilization , and endplate fixation technology . Starting in 2004 , the SB III design has been renamed the Charité artificial disc and is currently produced by a different manufacturer -LRB- DePuy Spine , Raynham , MA , USA -RRB- . Despite these aforementioned changes , it remains crucially important to fully understand the long-term clinical failure modes of early total disc replacements , as they form the foundation for judging the performance of contemporary implant designs and treatment paradigms . At present , we have treated 75 patients at our clinic with persistent leg and back pain after insertion of the SB Charité disc prosthesis . The incidence of complications following Charité artificial disc implantation at our institution has proven difficult to deduce because all operations were performed elsewhere . At 2 years of follow up , investigators from the hospital implanting the disc prosthesis reported that 17 out of 50 -LRB- 34 % -RRB- patients required secondary surgery , and there were three -LRB- 6 % -RRB- reported serious complications -LSB- 22 -RSB- . The purpose of this study is to analyse late-complications after insertion of a disc prosthesis , and to describe our experiences with salvage operations in this difficult patient group . Materials and methods Patients Approximately more than 1,000 Dutch patients , suffering from serious and constant back and leg pain , have been implanted with a SB III Charité -LRB- Link , Germany -RRB- artificial disc prosthesis in the lower lumbar spine . Over the last 10 years , 75 patients of this cohort , with persisting back and leg pain and being unsatisfied with their clinical situation , were seen at the orthopaedic outpatient clinic of the University Hospital of Maastricht -LRB- UHM -RRB- . They were seen by a different orthopaedic surgeon -LRB- AvO and LvR -RRB- than the surgeon who performed the prosthesis implantation . Twenty-seven of these cases have already been reported previously -LSB- 20 -RSB- . Forty-one patients were female with an average age at the time of disc implant of 42 years and 7 months -LRB- 30 -- 63 years -RRB- and 34 patients were male with an average age of 40 years and 9 months -LRB- 30 -- 51 years -RRB- at the time of disc implant -LRB- Table 1 -RRB- . The operated levels and the year the patients received their artificial disc implantation is shown in Table 1 . Table 1Patient characteristicsSexNAge at artificial disc insertionMales3440 years and 9 months -LRB- 30 -- 51 -RRB- Females4142 years and 7 months -LRB- 30 -- 63 -RRB- Operated level First level L2 -- L31 L3 -- L43 L4 -- L522 L5 -- S130 Second levels L3 -- L4 , L5 -- S11 L4 -- L5 , L5 -- S116Third levels L3 -- L4 , L4 -- L5 , L5 -- S11 L2 -- L3 , L4 -- L5 , L5 -- S11Time disc implantation 1989 -- 199425 1995 -- 199930 2000 -- 200520 Forty-six out of these 75 patients needed one or more salvage operations after their artificial disc implant . Indicators for reoperations were primary absence of pain relief , new pathology according to radiography , CT-scan or MRI in adjacent segment -LRB- s -RRB- , subsidence , facet joint arthrosis or migration of the prosthesis . In our hospital , we performed posterior fusion in 15 patients without disc removal , and in 22 patients we removed 26 prostheses and performed an anterior and posterior fusion . In addition , seven patients received posterior fusion elsewhere , and in two patients the disc prosthesis was removed elsewhere -LRB- Fig. 1 -RRB- . Fig. 1Overview of reoperations after disc prosthesis implantation -LRB- UHM : University Hospital Maastricht -RRB- Surgical method of disc prosthesis removal The disc prosthesis was removed by a team consisting of a vascular surgeon and an orthopaedic surgeon . The patient was placed under general anaesthesia and positioned in supine position -LRB- for level L5 -- S1 -RRB- or in a semilateral position -LRB- for level L4 -- 5 or higher -RRB- . A disc prosthesis at level L4 -- 5 was removed via anterolateral lumbotomy , without mobilisation of the great vessels . A disc prosthesis at level L5 -- S1 was removed through the original left retroperitoneal approach or alternatively via right retroperitoneal , between the great vessels . To remove the disc prosthesis one of the endplates was released by undercutting and removal of some bone after cleaning of fibrous tissue . The metal endplate was gripped and removed with twisting and pulling manoeuvres . Then the core and second endplate was easily released and removed . The gap between the vertebral bodies after retrieval of the disc prosthesis was mostly 2.5 -- 3 cm high . This was filled with an autologous strut graft in 2 patients , artificial bone in 3 patients and allograft strut grafts and allograft bone chips from femoral heads in 17 patients . We now favour the insertion of allograft strut grafts from the bone bank . Clinical performance and complications Before the orthopaedic surgeons in our outpatients clinic performed reoperation , they evaluated the images of X-ray , CT-scans , MRI and if necessary discography on the presence of late complications . Clinical examination included the ten-point visual analogue scale -LRB- VAS -RRB- , ranging from 0 points for `` no pain '' and 10 points for `` severe pain '' , and the Oswestry low back pain disability questionnaire . The VAS and Oswestry were examined before the salvage operation and after a follow-up period of at least 1 year after this operation . The 1 year follow-up VAS and Oswestry are not yet available in all patients . Statistical analysis Analyses were performed using SPSS , release 12.0.1 . Non-parametric tests , i.e. Mann -- Whitney and Wilcoxon , were used to test mean . Significance was indicated by P values less than 0.05 . Results Late complications An overview of late complications after disc implantation is shown in Table 2 . They included the following : 39 patients showed subsidence of the disc prosthesis , in 24 of these patients the disc prosthesis was considered too small . Thirty-six patients showed signs of adjacent disc degeneration , narrowing of the disc and osteophytes on conventional X-rays . In 17 patients this was not obvious before artificial disc insertion on plain X-rays and discography . Eleven patients with multi-level adjacent disc degeneration had developed degenerative lumbar scoliosis . In 25 patients facet joint degeneration was seen on CT-scans . In six patients the disc prosthesis showed anterior migration and in two patients posterior migration of the disc prosthesis occurred . In ten patients we discovered breakage of the metal wire around the core -LRB- Fig. 2 -RRB- . Table 2Overview of late complications after receiving a disc prosthesis -LRB- patients can have more than one complication -RRB- Late complicationsNumber of patientsSubsidenceDisc prosthesis too small3924Adjacent disc degeneration36Degenerative scoliosis11Facet joint degeneration on CT scan25Anterior migration6Posterior migration2Breakage metal wire10Wear5Severe osteolysis1Subluxation PE core1Fig . 2Example of removed polyethylene core L4 -- 5 -LRB- left -RRB- and L5 -- S1 -LRB- right -RRB- -LRB- 6.5 years after insertion -RRB- with a fracture of the metal wire and damage of disc prosthesis Less often the surgeons noticed the subsequent late complications . In one case severe osteolysis was seen in the sacrum in a ruptured and severely worn L5 -- S1 case . In another case a subluxation of the PE core and an adjacent osteoporotic compression fracture was noticed . In five patients radiological wear of the disc prosthesis was obvious due to loss of height of the core , or sclerosis and cysts around the prosthesis on CT-scan . Study population The group of 15 patients receiving posterior fusions in our hospital without removing the prosthesis , consisted of 8 men and 7 women . Mean age at their revision surgery was 49 years and 9 months -LRB- 34 -- 76 years -RRB- and mean time-interval between their disc implant and revision surgery was 7 years and 11 months -LRB- 2 -- 15 years -RRB- . Facet joint degeneration was noticed during all operations . Afterwards , we removed the disc prosthesis in four patients of this group because of persisting pain . Nowadays , we advise disc prosthesis removal in conjunction with fusion surgery , assuming that the disc prosthesis can remain a pain source even after solid posterior fusion . So far , we retrieved 26 prostheses in 22 patients -LRB- 17 females , 5 males -RRB- . The additional posterior fusion took place in nine patients 2 weeks before or 2 weeks after the removal of the disc prosthesis . In the other 13 patients , posterior fusion was done as a second operation on the same day as the removal . Nowadays this is the standard procedure in our hospital . The mean interval between insertion and retrieval of the disc prosthesis was 8 years and 11 months -LRB- 3 -- 16 years -RRB- . The mean age at retrieval of the disc prosthesis was 50 years -LRB- 40 -- 72 years -RRB- . A pre - and post-operative radiograph of one case , in which we removed the disc prosthesis L4 -- 5 and L5 -- S1 and performed a posterior and anterior fusion , is shown in Figs. 3 and 4 . Fig. 3a Anteroposterior and b lateral radiograph of the lumbar spine 3 years and 9 months after disc replacement at L4 -- 5 and L5 -- S1Fig . 4a Anteroposterior and b lateral radiograph of the lumbar spine 9 months after revison surgery Intraoperatively we twice encountered a lesion of the left common iliac vein -LRB- in L5 -- S1 cases -RRB- , once a lesion of the left common iliac artery -LRB- in a L4 -- L5 case -RRB- and once a small incomplete colon lesion . These complications could all be controlled by the vascular surgeon with relatively little blood loss . Mean blood loss for the anterior procedure was 753 cc -LRB- 60 -- 5,100 cc -RRB- . In one patient profound bleeding was encountered from the vertebral body bone and possibly the epidural plexus underneath the distal endplate of a L5 -- S1 disc prosthesis . This was controlled by packing with bone bank chips and gel foam , however the total blood loss in this two-level case was 5,100 cc . In another patient we planned to remove the disc prosthesis , however , due to a rupture of the small intestine during the access phase , we decided only to perform a posterior fusion . Preliminary clinical results General clinical information for both revision strategies , with a follow-up period of at least 1 year , is shown in Table 3 . Table 3Clinical information for both revision strategies -LRB- with a follow-up period of at least 1 year -RRB- NrSexYear index surgeryAge index surgeryLevelsYear re-operationRemoval/fusionExtra procedures % improvement VAS % improvement OswestryClinically improved Oswestry -LRB- > 25 % -RRB- 1F199344L2 -- L32003FusionNone41 .1827.50 Yes2M199541L4 -- L51997FusionNone − 33.33 − 44.44 No3F199263L5 -- S12005FusionNone − 14.29 − 24.34 No4M199730L5 -- S12001FusionNone27 .7816.67 No5F200042L4 -- L52003FusionNone12 .5014.71 No6F199642L4 -- L5 , L5 -- S12003FusionNone27 .786.45 No7F200238L4 -- L5 , L5 -- S12003FusionNone29 .4116.13 No8M199748L5 -- S12004FusionNone86 .6782.76 Yes9M199537L5 -- S12005FusionNone18 .750.00 No10F199734L5 -- S12002Fusion None0 .0074.07 Yes2005RemovalNone29 .41 MissingMissing11M199237L4 -- L52005RemovalNone − 12.5025.00 Yes12F199533L4 -- L52006Removal None − 12.505.41 No13M198944L4 -- L52005RemovalNone12 .5030.00 Yes14F199139L4 -- L52002RemovalNone87 .5056.25 Yes15F199539L5 -- S12004RemovalNone76 .4773.08 Yes16F199946L4 -- L5 , L5 -- S12005RemovalNone62 .5062.50 Yes17F199233L3 -- L4 , L5 -- S12005RemovalNone11 .1112.50 No18M199846L4 -- L52004RemovalNone9 .0938.10 Yes19F199232L2 -- L3 , L4 -- L5 , L5 -- - S12004RemovalNone55 .5616.80 No20M200147L4 -- L52005RemovalNone − 6.670.00 No21M199539L4 -- L52005RemovalDynesys62 .503.33 No22F200237L4 -- L5 , L5 -- S12005RemovalNone11 .1110.00 No23F199055L4 -- L52006RemovalNone14 .2913.97 No Ten patients receiving posterior fusion without removal of the prosthesis , have at this moment a follow-up period of more than 1 year since their posterior fusion . The mean VAS before posterior fusion was 8.0 -LRB- SD 0.9 -RRB- and after posterior fusion 6.3 -LRB- SD 2.1 -RRB- -LRB- Fig. 5 -RRB- . Fig. 5VAS scores before and after salvage operation Furthermore , 14 patients had a follow-up period of more than 1 year since their disc prosthesis removal . Two disc removal patients had insertion of the Dynesys fixation system -LRB- one patient had a follow-up of at least 1 year -RRB- , in addition to the fused disc prosthesis level , for multiple adjacent levels degeneration . This multilevel Dynesys instrumentation was recently removed in both patients due to screw loosening . The VAS score in this group decreased significantly from 8.0 -LRB- SD 0.9 -RRB- before disc prosthesis removal to 5.6 -LRB- SD 2.7 -RRB- after removal -LRB- P < 0.05 -RRB- -LRB- Fig. 5 -RRB- . The percentage of improvement after revision surgery in both groups is shown in Fig. 6 . Fig. 6Percentage change in VAS scores in both revision strategies The mean Oswestry decreased in the posterior fusion group -LRB- n = 10 -RRB- from 57.0 -LRB- SD 17.0 -RRB- to 44.6 -LRB- SD 20.4 -RRB- , and in the disc removal group -LRB- n = 13 -RRB- from 56.3 -LRB- SD 14.0 -RRB- to 43.0 -LRB- SD 20.7 -RRB- -LRB- Fig. 7 -RRB- . This questionnaire is missing in one patient from the disc removal group . According to the abovementioned IDE-criteria , in which an improvement of ≥ 25 % was considered to be clinically improved , 3 out of 10 patients in the fusion group and 6 out of 13 patients in the disc removal group were clinically improved -LRB- Table 3 ; Fig. 8 -RRB- . Fig. 7Oswestry scores before and after salvage operationFig . 8Percentage change in Oswestry scores in both revision strategies Postoperative complications Two patients from the posterior fusion group developed pseudo-arthrosis postoperatively . We encountered five postoperative complications in the disc removal group . One patient developed deep venous thrombosis -LRB- DVT -RRB- of the left leg after suturing a left common iliac vein lesion . In two patients , decreased sensitivity in the left groin and upper leg was noticed , which was partially reversible . Two patients have severe pain and decreased diffuse strength in the left leg postoperatively . In one of these patients these complaints are diminishing at the moment . Presumably , excessive retraction of the lumbosacral nerves in the psoas muscle played a role in these left leg complications . Discussion Seventy-five patients with persistent leg and back pain after insertion of SB Charité disc prosthesis were studied . The causes of persisting pain were thought to be related to the following late-complications : subsidence , migration , wear of the disc prosthesis , facet joint degeneration or adjacent degeneration in various combinations . Good placement and good sizing of the disc prosthesis appeared problematic for many of the patients in our series . In 39 patients we saw subsidence of the disc prosthesis , and in our judgement X-rays showed that in 24 of these patients the disc prosthesis was too small . Whether suboptimal sizing and placement resulted from initial surgical technique , or from historical limitations in instrumentation and/or sizing availability remains unclear . Fixation of the disc prosthesis can be inadequate , giving subsidence and migration of the disc prosthesis . Migration can probably be prevented by coating of the metal plate , but subsidence probably not . Previous studies suggested that adjacent degeneration is prevented after disc replacing surgery -LSB- 4 , 8 , 14 -RSB- . However , in our study 36 patients showed adjacent disc degeneration . This could be the result of the DDD itself , spreading to multiple levels of the spine , and/or be the consequence of stresses on adjacent levels , generated from the unphysiological motion and functioning of the disc prosthesis . Concerning the 25 patients in which facet joint degeneration was visible on CT-scans , we hypothesized that an instability is created after removal of the anterior longitudinal ligament and the annulus fibrosis . Replacement with an unconstrained prosthesis will accentuate this instability , especially in extension and axial rotation . The facet joints will be the only restrictor of axial rotation and will degenerate with time . Because of persisting back and leg pain we performed a posterior fusion without disc removal in 15 patients . Initially , the results were disappointing in most patients , and we therefore started to remove the disc prosthesis . In all patients with removal of the disc prosthesis , PE wear was observed in minor or major degree with surrounding inflammatory fibrous tissue containing PE wear debris . The association between periprosthetic tissue inflammatory reactions and clinical manifestations of pain is not clear at the present time and will be studied further . It is speculated that with removal of the disc prosthesis and the inflammatory periprosthetic tissue , the patient may obtain better pain relief than after only posterior fusion -LSB- 19 -RSB- . This hypothesis obviously needs further support with a larger number of more carefully selected patients . Clinical results after disc prosthesis revision performed in our hospital were diverse . Patients with short segment fusions seemed to fare better than patients with long segment fusions or long flexible fixations with the Dynesys system . At present , the small number of patients and the large number of potential factors influencing the outcomes -LRB- Table 3 -RRB- , precluded assessment of a significant association between revision strategy and outcome in the current study . The mean VAS for pain after disc prosthesis removal was reduced significantly with 2.4 points , from 8.0 to 5.6 , whereas the VAS in patients with posterior fusion showed a smaller decrease from 8.0 to 6.3 . However , the analyses are based on a small number of cases and a greater number of patients with longer follow up are necessary for a more definitive conclusion . In a collaborative study by Kurtz et al. -LSB- 10 -RSB- it was concluded that artificial discs exhibited wear mechanisms similar to artificial hips and knees . Since the operated patients are mostly between 30 and 50 years at the time of their disc implantation and these young patients will be likely very active , wear may become a clinically significant issue at long-term follow-up . The clinical relevance of wear in total disc replacements is the subject of intense , ongoing research at our institutions . A major point to consider is that , in contrast with a posterior fusion , retrieval of an artificial disc prosthesis can be dangerous because of nearby vascular structures and scar tissue from the original surgery . However , in our hands , disc removal surgery proved feasible in all but one case in this patient group -LRB- 26/27 disc retrievals , 96 % -RRB- . Due to the availability of a vascular surgeon during the retrieval surgery average blood loss could be restricted . In conclusion , serious complications may arise following total disc replacement surgery , however , as yet relatively few data are available to provide the basis for treatment of patients with clinically failed artificial discs . Removal of the disc prosthesis with anterior and posterior fusion gives slightly better results than posterior fusion alone after a follow-up of at least 1 year . Removal of the SB Charité artificial disc was feasible but with inherent risks . As more data become available , perhaps revision artificial disc surgery may become a more common operation for spine surgeons in the near future .

B O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B 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O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O

artificial disc
retrieval surgery
complications


Hynic-rh-Annexin

V

scintigraphy

for

in

vivo

imaging

of

apoptosis

in

patients

with

head

and

neck

cancer

treated

with

chemoradiotherapy

Purpose

The

purpose

of

this

study

was

to

determine

the

value

of

Hynic-rh-Annexin-V-Scintigraphy

-LRB-

TAVS

-RRB-

a

non-invasive

in

vivo

technique

to

demonstrate

apoptosis

in

patients

with

head

and

neck

squamous

cell

carcinoma

Introduction

Apoptosis

is

an

important

mechanism

of

cell

death

in

response

to

treatment

with

radiation

and

many

chemotherapeutic

agents

-LSB-

-RSB-

To

what

extent

apoptosis

contributes

to

the

overall

cytotoxic

effect

of

an

anti-cancer

treatment

modality

has

been

the

topic

of

intense

research

during

the

last

decade

-LSB-

-RSB-

The

relative

contribution

of

apoptosis

to

the

occurrence

of

cell

death

varies

greatly

both

between

different

tumour

types

and

normal

tissues

-LSB-

-RSB-

Recently

in

vivo

imaging

of

apoptosis

has

proven

to

be

feasible

by

using

radiolabelled

Annexin

V

-LSB-

-RSB-

This

endogenous

human

protein

has

a

high

affinity

for

membrane-bound

lipid

phosphatidylserine

-LRB-

PS

-RRB-

which

becomes

exposed

at

the

outer

leaflet

of

the

cell

membrane

bilayer

at

an

early

stage

of

the

apoptotic

process

-LSB-

-RSB-

PS

then

serves

as

a

recognition

site

for

macrophages

that

digest

and

remove

apoptotic

cells

The

reproducibility

of

the

scintigraphy

has

been

demonstrated

in

head

and

neck

squamous

cell

carcinoma

-LRB-

HNSCC

-RRB-

patients

by

serial

imaging

in

untreated

patients

-LSB-

-RSB-

in

which

the

mean

difference

in

uptake

was

We

have

recently

demonstrated

that

V

scintigraphy

-LRB-

TAVS

-RRB-

correlates

with

radiation-induced

cytologically

confirmed

apoptosis

in

non-Hodgkin

lymphoma

-LSB-

-RSB-

and

can

be

used

to

identify

patients

that

have

a

favourable

prognosis

-LSB-

-RSB-

The

current

standard

of

practice

for

patients

with

advanced

stage

HNSCC

is

treatment

with

concurrent

cisplatin-based

chemoradiation

-LSB-

-RSB-

Although

this

is

an

effective

treatment

it

also

is

accompanied

by

more

severe

toxicity

than

radiation

alone

We

reasoned

that

TAVS

early

during

treatment

might

be

used

to

monitor

apoptosis

induction

in

tumour

and

normal

tissue

and

to

give

an

indication

of

the

radiosensitivity

of

these

structures

In

future

this

would

also

offer

the

possibility

to

adapt

the

treatment

strategy

at

an

early

stage

on

a

patient-by-patient

basis

In

the

present

study

we

therefore

aimed

at

assessing

the

feasibility

of

TAVS

as

a

non-invasive

technique

to

demonstrate

treatment-induced

apoptosis

in

vivo

in

patients

with

HNSCC

early

during

treatment

with

concurrent

chemoradiation

The

purpose

was

to

determine

the

degree

of

uptake

on

TAVS

in

normal

tissue

primary

tumour

and

lymph

node

metastases

and

to

evaluate

the

treatment-induced

Annexin

uptake

in

relation

to

radiation

dose

Furthermore

we

questioned

whether

the

differences

in

uptake

would

correlate

with

treatment

response

Materials

and

methods

Patients

included

in

this

study

were

recruited

between

January

and

March

from

a

randomised

phase

III

trial

in

advanced

stage

HNSCC

investigating

the

optimal

route

of

cisplatin

delivery

during

cisplatin-based

chemoradiation

-LRB-

RADPLAT

-RRB-

Randomisation

was

between

two

treatment

arms

Arm

standard

intravenous

-LRB-

IV

-RRB-

administration

of

cisplatin

-LRB-

h

before

radiotherapy

at

days

and

-RRB-

or

arm

high-dose

selective

intra-arterial

-LRB-

IA

-RRB-

delivery

of

cisplatin

at

a

dose

of

-LRB-

on

days

and

within

h

after

radiotherapy

delivery

-RRB-

Main

eligibility

criteria

included

inoperable

squamous

cell

carcinoma

of

the

oral

cavity

oropharynx

or

hypopharynx

TNM

stage

status

of

primary

oral

cavity

or

oropharyngeal

tumours

and

for

hypopharyngeal

tumours

with

any

N-status

-LRB-

functionally

-RRB-

inoperable

disease

no

distant

metastases

age

at

least

years

ability

to

give

informed

consent

and

no

prior

cerebro-vascular

accident

Both

the

randomised

trial

and

the

TAVS

study

were

approved

by

the

medical

ethics

committee

of

the

hospital

Patients

were

informed

about

the

nature

of

the

study

protocol

and

signed

informed

consent

before

enrolment

separately

for

both

the

randomised

trial

and

the

TAVS

protocol

Radiotherapy

was

given

with

MV

photon

linear

accelerators

Target

volume

included

the

primary

tumour

and

the

bilateral

neck

for

a

dose

of

Gy

in

fractions

A

boost

was

given

to

the

known

macroscopic

tumour

extensions

at

the

primary

tumour

site

and

lymph

node

metastases

to

a

dose

of

Gy

in

fractions

The

total

dose

delivered

was

Gy

in

fractions

five

fractions

per

week

with

an

overall

treatment

time

of

weeks

The

radiation

technique

was

either

a

conventional

three-field

beam

setup

-LRB-

using

conventional

simulation

or

virtual

simulation

with

CT

scan

-RRB-

or

an

intensity-modulated

radiotherapy

-LRB-

IMRT

-RRB-

plan

depending

on

resources

and

tumour

extent

The

IA

cisplatin

delivery

was

accomplished

by

a

selective

catheterisation

procedure

using

the

femoral

artery

according

to

the

earlier

described

RADPLAT

protocol

-LSB-

-RSB-

Concurrently

with

IA

cisplatin

sodium-thiosulphate

was

administered

to

neutralise

systemic

cisplatin

In

both

arms

prehydration

and

posthydration

were

given

V

scintigraphy

diagnostic

imaging

radiation

treatment

planning

and

image

fusion

Baseline

TAVS

was

performed

within

weeks

before

the

start

of

concurrent

chemoradiation

-LRB-

average

interval

days

range

-RRB-

Post-treatment

TAVS

was

done

within

h

after

the

start

of

cisplatin

chemotherapy

At

the

time

of

post-treatment

TAVS

patients

had

received

Gy

of

radiation

in

case

of

IV

cisplatin

Gy

in

case

of

IA

cisplatin

Each

patient

received

an

average

of

MBq

-LRB-

range

MBq

-RRB-

of

V

-LRB-

Theseus

Imaging

Corporation

Boston

USA

-RRB-

by

slow

intravenous

injection

h

before

the

planar

imaging

and

single-photon

emission

tomography

-LRB-

SPECT

-RRB-

imaging

Planar

images

were

used

to

assess

the

biodistribution

SPECT

of

the

head

and

neck

region

was

acquired

by

the

step-and-shoot

mode

one

step

per

s

per

frame

matrix

size

using

a

dual-head

gamma

camera

-LRB-

Genesis

Philips

Best

The

Netherlands

-RRB-

equipped

with

low-energy

high-resolution

collimators

For

SPECT

reconstructions

an

iterative

algorithm

was

used

and

the

images

were

postfiltered

using

a

Butterworth

filter

-LRB-

cutoff

frequency

order

-RRB-

The

use

of

absolute

quantitative

analysis

of

SPECT

data

was

validated

by

comparison

of

iterative

and

FBP

reconstruction

methods

Transaxial

coronal

and

sagittal

slices

were

visually

examined

to

evaluate

tracer

uptake

at

the

tumour

sites

and

in

normal

tissues

The

intensity

of

the

obtained

images

was

corrected

by

normalisation

for

the

injected

radioactive

dose

and

body

weight

Baseline

diagnostic

imaging

with

MRI

-LRB-

system

Somatom

Siemens

Medical

Systems

Erlangen

Germany

-RRB-

or

spiral

CT

-LRB-

Tomoscan

Philips

Best

The

Netherlands

or

HiSpeed

CT

GE

Medical

Systems

USA

-RRB-

was

performed

within

weeks

before

the

start

of

treatment

and

repeated

weeks

after

the

end

of

the

treatment

for

the

evaluation

of

treatment

response

Radiation

treatment

planning

was

done

with

our

clinical

treatment

planning

system

-LRB-

TPS

U-MPlan

University

of

Michigan

Ann

Arbor

USA

-RRB-

Six

patients

were

treated

by

IMRT

and

the

treatment

plans

were

recalculated

for

the

dose

at

the

time

of

first

post-treatment

TAVS

The

regions

of

interest

-LRB-

ROI

-RRB-

were

delineated

manually

for

each

patient

on

the

CT

scan

These

included

the

gross

tumor

volume

-LRB-

GTV

-RRB-

of

the

primary

tumour

and/or

lymph

nodes

the

parotid

glands

and

the

submandibular

glands

In

the

other

seven

patients

a

standard

three-field

technique

was

used

by

virtual

simulation

with

a

CT

scan

In

these

latter

patients

the

CT

scan

was

imported

into

the

TPS

in

which

the

delineations

were

done

The

treatment

fields

were

reconstructed

and

the

clinically

applied

dose

distribution

at

the

time

of

TAVS

was

recalculated

in

the

TPS

The

primary

tumour

and

lymph

node

volumes

were

calculated

by

reconstruction

of

the

delineated

GTV

SPECT

MRI

and

CT

were

performed

separately

For

the

SPECT

scan

we

obtained

reproducibility

of

the

positioning

of

the

head

and

neck

as

during

RT

by

the

use

of

immobilisation

mask

fixed

to

the

table

by

adhesive

tape

All

images

and

the

radiation

dose

data

were

transferred

for

image

fusion

to

our

in-house

developed

workstation

for

co-registration

-LRB-

Worldmatch

Workstation

-RRB-

in

DICOM

format

-LSB-

-RSB-

Keeping

the

limitations

of

the

mask

fixation

in

mind

it

was

decided

that

for

more

accuracy

matching

was

performed

on

different

anatomical

regions

for

different

ROIs

e.g.

for

the

analysis

of

parotid

glands

or

oropharyngeal

tumours

the

co-registration

was

done

in

such

a

way

that

it

ensured

adequate

matching

for

that

area

only

For

co-registration

of

ROIs

in

the

neck

-LRB-

e.g.

lymph

nodes

or

hypopharyngeal

tumours

-RRB-

only

the

neck

and

vertebral

column

was

used

in

the

matching

procedure

Accurate

matching

of

body

contours

and

bone

structures

was

visually

verified

SPECT

composite

SPECT/SPECT

CT

and

SPECT/CT

images

obtained

using

a

colourwash

technique

were

simultaneously

examined

using

linked

cursor

to

evaluate

Annexin

V

uptake

in

tumour

and

normal

tissues

The

tumour

and

normal

tissue

uptake

at

baseline

and

post-treatment

were

calculated

as

follows

ROIs

were

delineated

in

the

planning

CT.

Each

ROI

was

projected

using

the

registration

transformation

onto

the

baseline

and

post-treatment

SPECT

scans

The

area

was

next

sampled

by

random

points

and

for

each

point

the

Annexin

uptake

was

determined

in

the

SPECT

scans

by

trilinear

interpolation

Of

these

samples

the

maximum

and

mean

values

were

computed

In

this

way

the

uptake

in

the

ROI

was

accurately

sampled

although

the

pixel

size

in

the

SPECT

scans

is

relatively

large

-LRB-

about

mm

-RRB-

For

the

quantification

of

Annexin

uptake

no

attenuation

correction

was

performed

because

the

images

were

obtained

on

a

conventional

gamma

camera

without

a

hybrid

system

Subsequently

the

difference

-LRB-

ΔU

-RRB-

between

the

post-treatment

and

baseline

uptake

was

determined

by

subtraction

of

the

baseline

scan

from

the

post-treatment

scan

The

subtraction

was

performed

on

a

point

by

point

basis

for

all

points

Then

the

mean

or

maximum

value

of

the

difference

of

both

scans

-LRB-

subtraction

scan

-RRB-

was

computed

and

expressed

as

ΔU

No

correction

for

background

activity

was

made

as

this

was

automatically

eliminated

by

subtraction

of

the

baseline

activity

from

the

post-treatment

activity

assuming

that

the

background

activity

is

equal

in

the

baseline

and

post-treatment

scan

Radiation

dose

parameters

-LRB-

mean

and

maximum

dose

in

cGy

-RRB-

within

each

ROI

were

calculated

and

correlated

with

corresponding

ΔU

parameters

It

was

observed

that

the

maximum

ΔU

and

the

mean

ΔU

were

closely

related

a

linear

relationship

between

both

two

parameters

was

found

for

each

ROI

-LRB-

tumour

r

p

parotid

gland

r

p

submandibular

gland

r

p

lymph

node

r

p

-RRB-

indicating

that

both

values

would

have

been

representative

for

the

whole

ROI

Evaluation

of

treatment

Six

to

eight

weeks

after

the

end

of

treatment

the

results

of

therapy

were

evaluated

by

means

of

radiological

investigations

-LRB-

by

MRI

or

CT

scan

and/or

ultrasound

-RRB-

and

examination

under

general

anaesthesia

with

biopsies

taken

in

case

of

suspicious

findings

For

residual

disease

in

the

neck

at

the

time

of

evaluation

salvage

neck

dissection

was

performed

if

the

patient

was

judged

operable

Follow-up

visits

were

planned

every

months

in

the

first

year

after

therapy

every

months

in

the

second

year

and

less

frequent

thereafter

A

follow-up

chest

X-ray

was

performed

annually

Statistical

analysis

For

quantitative

comparison

of

continuous

data

Student

's

t

test

was

applied

Chi-squared

and

Fisher

's

exact

test

were

used

for

analysis

of

categorical

data

The

Pearson

and

Spearman

rank

correlation

coefficient

r

were

used

to

calculate

correlations

between

Annexin

uptake

and

treatment

parameters

Locoregional

control

and

survival

data

were

calculated

from

the

start

of

treatment

using

the

Kaplan

Meier

method

and

log

rank

testing

Two-sided

P

values

of

were

considered

statistically

significant

Results

Sixteen

patients

gave

their

consent

for

participating

in

the

TAVS

study

However

one

patient

refused

post-treatment

TAVS

scans

in

one

patient

no

CT

scan

in

radiation

treatment

position

was

available

and

in

one

patient

co-registration

with

SPECT

was

unsuccessful

due

to

misalignment

because

no

immobilisation

mask

and

no

tongue

depressor

could

be

used

during

SPECT

due

to

pain

-LRB-

Fig.

-RRB-

The

number

of

patients

included

in

this

analysis

on

V

scintigraphy

in

advanced

stage

HNSCC

is

therefore

The

mean

age

of

patients

was

years

-LRB-

range

years

-RRB-

See

Table

for

the

patient

tumour

and

treatment

characteristics

Fig.

projection

of

TAVS

imaging

co-registered

with

planning

CT

scan

The

solid

line

represents

the

contour

of

the

patient

during

RT-planning

CT

scan

including

tongue

depressor

The

projected

image

is

the

TAVS

a

Baseline

TAVS

b

post-treatment

TAVS

Note

the

mismatch

of

contours

of

the

head

and

neck

mostly

due

to

different

position

of

the

chin

in

b

The

misalignment

of

the

lower

neck

in

a

is

due

to

the

flexion

of

the

neck

For

the

analysis

the

matching

procedure

was

focused

on

the

mandibular

region

This

patient

was

excluded

from

analysis

because

of

misalignment

in

bTable

tumour

and

treatment

characteristicsPatient

numberGenderAge

-LRB-

years

-RRB-

Tumor

siteTNM

stageTumor

volume

-LRB-

-RRB-

Max

ΔU

in

primary

tumourInterval

cisplatin

infusion

and

Annexin

scan

-LRB-

h

-RRB-

RT

techniqueMode

of

cisplatin

Conventional

Conventional

Conventional

Conventional

Conventional

Conventional

Conventional

fieldIntravenouslyM

Male

F

female

IMRT

intensity

modulated

radiotherapy

Annexin

uptake

in

normal

tissue

and

tumour

At

baseline

scintigraphy

reproducible

physiologic

Annexin

uptake

was

detected

on

the

planar

images

in

liver

kidneys

spleen

gall

bladder

bone

marrow

colon

and

urinary

bladder

as

described

earlier

-LSB-

-RSB-

In

the

parotid

glands

weak

baseline

uptake

was

present

in

all

cases

The

post-treatment

TAVS

showed

moderate

to

strong

increase

in

uptake

in

of

parotid

glands

-LRB-

Fig.

-RRB-

The

average

of

the

mean

number

of

counts

increased

from

to

-LRB-

p

-RRB-

Visual

analysis

of

the

increase

in

uptake

showed

that

these

changes

were

related

to

the

radiation

portals

and

the

dose

given

to

the

parotid

glands

-LRB-

Fig.

-RRB-

The

difference

in

Annexin

uptake

between

the

post-treatment

and

baseline

TAVS

-LRB-

ΔU

-RRB-

in

each

parotid

gland

was

correlated

with

the

radiation

dose

at

the

time

of

post-treatment

scintigraphy

-LRB-

n

patients

n

parotids

-RRB-

The

mean

ΔU

showed

a

positive

correlation

with

the

mean

radiation

dose

-LRB-

Pearson

coefficient

r

p

-RRB-

parotid

glands

that

received

a

higher

dose

of

radiation

showed

a

higher

Annexin

uptake

-LRB-

Fig.

-RRB-

The

increase

in

Annexin

uptake

in

parotid

glands

that

were

treated

with

parotid-sparing

IMRT

was

less

than

in

parotid

glands

that

were

treated

with

a

conventional

three-field

technique

-LRB-

IMRT

counts

three-field

technique

counts

p

-RRB-

The

given

radiation

dose

at

the

time

of

post-treatment

TAVS

-LRB-

or

Gy

-RRB-

and

the

mode

of

cisplatin

administration

-LRB-

IA

or

IV

-RRB-

did

not

affect

the

uptake

in

parotid

glands

As

xerostomia

scoring

was

not

documented

sufficiently

detailed

in

the

files

we

interviewed

patients

alive

at

last

follow-up

for

xerostomia

grading

for

the

purpose

of

this

study

In

nine

patients

alive

at

last

follow-up

the

treatment-induced

Annexin

uptake

in

parotid

glands

was

related

to

the

subjective

xerostomia

rating

using

the

EORTC

QLQ

questionnaire

-LSB-

-RSB-

No

relation

could

be

established

in

this

small

set

of

patients

-LRB-

data

not

shown

-RRB-

Fig.

-LRB-

diamonds

-RRB-

and

post-treatment

-LRB-

squares

-RRB-

Annexin

activity

in

parotid

glands

-LRB-

a

-RRB-

submandibular

glands

-LRB-

b

-RRB-

and

tumour

-LRB-

c

-RRB-

for

all

patients

The

data

for

parotid

and

submandibular

glands

represent

the

right

and

left

gland

within

each

patientFig

imaging

co-registered

with

planning

CT

scan

in

frontal

plane

and

axial

plane

a

and

c

at

baseline

b

and

d

after

treatment

from

patient

number

The

Annexin

uptake

is

represented

by

colourwash

In

b

and

d

the

isodose

lines

show

the

dose

distribution

in

relation

to

the

parotid

gland

and

primary

tumour

-LRB-

isodose

lines

shown

and

from

outer

to

inner

side

of

patient

-RRB-

Note

the

increased

treatment-induced

Annexin

uptake

in

the

right

parotid

gland

in

correspondence

to

the

higher

radiation

dose

distribution

when

compared

to

the

left

parotid

gland

Also

note

the

weak

increase

in

primary

tumour

Annexin

uptake

in

the

right

oropharynx

after

treatment

and

in

the

anterior

floor

of

mouth

-LRB-

d

-RRB-

Fig.

plot

of

mean

radiation

dose

-LRB-

cGy

-RRB-

and

the

post-treatment

increase

in

Annexin

uptake

mean

ΔU

in

parotid

glands

-LRB-

a

-RRB-

normalised

for

tracer

dose

and

body

weight

Circles

represent

parotid

glands

treated

with

IMRT

triangles

parotids

treated

with

conventional

technique

In

b

the

correlation

between

mean

radiation

dose

-LRB-

cGy

-RRB-

and

the

post-treatment

increase

in

Annexin

uptake

mean

ΔU

in

submandibular

glands

is

given

For

the

submandibular

glands

a

similar

pattern

as

with

the

parotids

was

observed

absent/weak

baseline

uptake

and

moderate

to

strong

increase

after

the

start

of

chemoradiation

-LRB-

Fig.

-RRB-

The

average

of

the

mean

number

of

counts

increased

from

to

-LRB-

p

-RRB-

No

correlation

between

the

ΔU

and

radiation

dose

was

noted

-LRB-

Fig.

-RRB-

probably

as

all

submandibular

glands

were

located

within

the

high-dose

region

The

baseline

TAVS

showed

moderate

to

strong

Annexin

uptake

in

the

primary

tumour

in

all

patients

indicative

of

spontaneous

apoptosis

or

necrosis

On

the

post-treatment

TAVS

the

uptake

in

tumour

clearly

increased

in

nine

cases

in

the

other

four

little

or

no

changes

occurred

-LRB-

Fig.

-RRB-

The

average

of

the

maximum

number

of

counts

increased

from

to

-LRB-

p

-RRB-

The

difference

between

post-treatment

and

baseline

TAVS

-LRB-

ΔU

-RRB-

in

primary

tumour

was

not

correlated

with

primary

tumour

volume

-LRB-

Pearson

coefficient

r

p

-RRB-

nor

with

the

mode

of

chemotherapy

administration

-LRB-

p

-RRB-

In

nine

patients

lymph

node

metastases

were

present

in

the

neck

-LRB-

mean

lymph

node

volume

range

-RRB-

The

treatment-induced

Annexin

uptake

-LRB-

maximum

ΔU

-RRB-

in

primary

tumour

was

positively

correlated

with

the

uptake

in

corresponding

lymph

node

metastases

-LRB-

r

p

-RRB-

as

shown

in

Fig.

To

verify

that

the

observed

effects

of

Annexin

uptake

in

different

structures

could

not

be

attributed

solely

to

the

administered

radioactive

dose

per

patient

but

was

tissue-specific

we

calculated

the

correlation

between

the

ΔU

in

tumour

and

parotid

glands

No

evidence

of

such

an

effect

was

found

-LRB-

r

p

-RRB-

Fig.

plot

of

the

treatment-induced

increase

in

Annexin

uptake

maximum

ΔU

in

primary

tumour

and

lymph

node

metastases

Each

colour

represents

an

individual

patient

Some

patients

had

multiple

lymph

node

metastases

originating

from

the

same

primary

tumour

In

these

cases

multiple

values

of

treatment-induced

Annexin

uptake

in

lymph

nodes

-LRB-

on

the

y-axis

-RRB-

correspond

to

a

single

value

of

the

primary

tumor

-LRB-

on

the

x-axis

-RRB-

Response

to

treatment

locoregional

control

and

survival

The

median

follow-up

of

all

patients

alive

at

last

follow-up

was

months

-LRB-

range

-RRB-

The

complete

response

-LRB-

CR

-RRB-

rate

at

the

primary

tumour

site

was

CR

rate

in

the

neck

in

the

case

of

nodal

metastases

was

The

overall

CR

rate

was

The

estimated

locoregional

-LRB-

LR

-RRB-

tumour

control

rates

were

at

and

years

The

rates

for

disease-free

survival

-LRB-

DFS

-RRB-

were

at

and

years

Overall

survival

-LRB-

OS

-RRB-

rates

at

and

years

were

and

respectively

In

this

small

subset

of

patients

from

a

larger

randomised

phase

III

trial

there

were

no

differences

in

response

rates

LR

control

DFS

or

OS

between

the

two

treatment

arms

-LRB-

IV

vs

IA

-RRB-

No

correlation

could

be

established

between

baseline

or

treatment-induced

Annexin

uptake

-LRB-

ΔU

-RRB-

and

any

outcome

parameter

-LRB-

response

rate

recurrences

and/or

survival

-RRB-

Discussion

This

study

represents

our

first

clinical

experience

on

in

vivo

imaging

of

apoptosis

with

TAVS

in

HNSCC

patients

in

which

we

applied

co-registration

of

multiple

imaging

modalities

By

this

we

were

able

to

analyse

treatment-induced

changes

in

normal

and

tumour

tissue

early

during

therapy

after

one

cycle

of

cisplatin

chemotherapy

and

Gy

of

radiotherapy

Treatment-induced

Annexin

uptake

in

the

parotid

glands

could

be

visualised

indicative

of

early

treatment

related

apoptosis

at

mean

radiation

doses

as

low

as

Gy

-LRB-

see

Fig.

-RRB-

and

one

course

of

cisplatin

The

Annexin

uptake

in

the

parotids

showed

a

radiation

dose-response

relationship

Glands

that

had

received

higher

doses

of

radiation

demonstrated

increased

Annexin

uptake

Loss

of

parotid

gland

function

leading

to

xerostomia

is

an

important

long-term

side

effect

of

radiotherapy

affecting

quality

of

life

of

patients

It

has

well

been

shown

by

scintigraphy

studies

-LSB-

-RSB-

and

by

salivary

flow

rate

studies

-LSB-

-RSB-

that

reduction

of

radiation

dose

to

the

parotid

glands

can

maintain

parotid

function

decrease

xerostomia

and

improve

quality

of

life

Most

studies

on

salivary

gland

function

are

performed

after

a

full

course

of

RT

-LRB-

typically

Gy

in

weeks

-RRB-

-LSB-

-RSB-

Our

study

design

with

early

in

vivo

imaging

indicated

that

already

after

low

dose

of

RT

-LRB-

Gy

-RRB-

parotid

glands

may

be

affected

and

that

the

physiologic

process

leading

to

loss

of

parotid

gland

function

already

starts

early

This

is

in

agreement

with

observations

in

experimental

rodent

studies

in

which

apoptosis

was

induced

early

after

doses

of

up

to

Gy

-LSB-

-RSB-

Similar

results

were

obtained

in

a

monkey

model

with

early

radiation

induced

apoptosis

of

serous

acinar

cells

of

salivary

glands

-LSB-

-RSB-

It

was

suggested

that

these

early

effects

are

mainly

responsible

for

the

acute

sialoadenitis

which

at

higher

RT

doses

might

progress

in

chronic

xerostomia

by

damage

to

and

depletion

of

ductal

stem

cells

within

the

gland

In

the

nine

patients

assessed

for

xerostomia

no

relation

could

be

established

with

subjective

salivary

gland

function

after

radiotherapy

and

parotid

gland

Annexin

uptake

However

the

significance

of

this

analysis

is

limited

as

we

assessed

both

parotid

glands

separately

by

TAVS

and

xerostomia

questionnaires

give

an

overall

impression

of

function

We

did

not

include

follow-up

salivary

flow

studies

In

short

our

results

indicate

indirect

evidence

of

early

radiation-induced

salivary

gland

damage

and

in

future

studies

this

needs

to

be

correlated

with

functional

outcome

parameters

Due

to

the

large

inter-patient

variability

in

the

baseline

TAVS

-LRB-

Fig.

-RRB-

the

subtraction

method

for

assessing

treatment-induced

changes

was

chosen

over

a

relative

method

-LRB-

e.g.

percentage

change

-RRB-

In

this

figure

it

is

important

to

notice

that

the

maximum

uptake

value

of

the

baseline

and

post-treatment

scan

might

be

located

in

different

points

within

the

tumour

ROI

Therefore

these

data

can

not

be

simply

subtracted

but

for

the

calculation

of

the

maximum

ΔU

in

primary

tumour

and

lymph

nodes

the

baseline

scan

was

first

subtracted

from

the

post-treatment

scan

and

subsequently

these

parameters

were

calculated

from

the

subtraction

scan

The

treatment-induced

Annexin

uptake

in

primary

tumour

and

pathological

lymph

nodes

showed

a

positive

correlation

Patients

with

primary

tumours

with

a

high

Annexin

uptake

also

appeared

to

have

lymph

node

metastases

with

high

Annexin

uptakes

The

ΔU

in

primary

tumour

and

lymph

node

metastases

showed

large

inter-patient

differences

-LRB-

Fig.

-RRB-

This

variation

could

not

be

attributed

to

differences

in

the

route

of

cisplatin

administration

-LRB-

intravenous

or

intra-arterial

chemoradiation

-RRB-

nor

to

the

radiation

doses

given

-LRB-

or

Gy

-RRB-

nor

to

the

primary

tumour

volume

This

positive

correlation

for

ΔU

in

primary

tumour

and

lymph

nodes

as

well

as

the

large

inter-patient

differences

might

represent

a

tumour-specific

apoptotic

response

In

our

series

repositioning

of

the

patient

during

SPECT

scan

in

RT

position

was

standard

procedure

but

due

to

the

limitations

in

fixation

not

optimal

The

repositioning

facilitated

the

co-registration

of

SPECT

and

CT

scan

In

one

patient

misalignment

occurred

because

the

positioning

was

not

accurate

due

to

the

fact

that

the

RT

position

could

not

be

reproduced

-LRB-

Fig.

-RRB-

So

for

co-registration

of

-LRB-

functional

-RRB-

imaging

to

RT-planning

CT

scan

data

in

HNSCC

we

recommend

improvements

from

our

protocol

to

obtain

a

reproducible

positioning

like

the

use

of

an

immobilisation

mask

fixed

to

the

SPECT

table

the

use

of

the

same

mattress

under

the

patient

and

a

laser

alignment

system

In

ongoing

studies

we

have

now

incorporated

these

In

the

current

series

no

attenuation

correction

was

applied

because

the

images

were

obtained

on

a

conventional

gamma

camera

without

a

hybrid

system

For

the

purpose

of

this

study

the

quantification

of

Annexin

uptake

remains

valid

as

we

used

the

relative

increase

from

baseline

to

post-treatment

uptake

and

the

degree

of

attenuation

will

be

equal

on

both

scans

In

this

small

series

of

HNSCC

the

treatment-induced

Annexin

uptake

-LRB-

ΔU

-RRB-

in

primary

tumour

did

not

predict

outcome

No

correlation

between

Annexin

uptake

and

early

response

was

observed

probably

partly

due

to

the

high

initial

response

rates

-LRB-

-RRB-

In

addition

TAVS

also

did

not

predict

the

locoregional

control

rates

within

the

first

years

of

follow-up

In

our

previous

experience

on

TAVS

in

other

tumour

sites

-LRB-

mainly

lymphoma

-RRB-

a

strong

correlation

has

been

found

between

early

response

-LRB-

within

months

-RRB-

and

Annexin

uptake

-LSB-

-RSB-

One

explanation

for

the

lack

of

correlation

might

be

that

in

the

current

series

these

advanced

stages

HNSCC

with

large

tumours

harbour

more

necrosis

which

is

also

detected

by

TAVS

because

of

accessibility

of

PS

at

the

necrotic

cell

membrane

-LSB-

-RSB-

Van

de

Wiele

et

al.

-LSB-

-RSB-

showed

that

in

HNSCC

Annexin

scintigraphy

correlated

with

histopathological

apoptosis

-LRB-

using

TUNEL

assay

-RRB-

only

in

the

absence

of

necrosis

Indeed

in

this

series

baseline

Annexin

uptake

in

the

HNSCC

patients

was

clearly

demonstrated

whereas

it

was

not

present

in

our

previous

study

in

lymphoma

patients

-LSB-

-RSB-

This

may

suggest

the

presence

of

necrosis

in

large

tumours

from

solid

origin

Other

factors

that

may

affect

Annexin

uptake

are

the

intra

and

peri-tumoural

lymphocyte

infiltration

-LSB-

-RSB-

For

the

future

we

plan

to

increase

the

number

of

HNSCC

patients

to

undergo

the

TAVS

imaging

to

prove

whether

or

not

it

can

be

used

for

outcome

prediction

as

it

was

shown

in

patients

with

malignant

lymphoma

and

non-small

cell

lung

cancer

-LSB-

-RSB-

In

conclusion

co-registration

of

Annexin

V

scintigraphy

with

radiotherapy-planning

CT

scan

showed

a

radiation-dose-dependent

uptake

in

parotid

glands

indicative

of

early

apoptosis

during

treatment

The

inter-individual

spread

in

Annexin

uptake

in

primary

tumours

could

not

be

related

to

differences

in

treatment

schedule

or

tumour

volume

but

the

Annexin

uptake

in

tumour

and

lymph

nodes

were

closely

correlated

This

effect

might

represent

a

tumour-specific

apoptotic

response

O O O B O O O O O B O O O B I I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B I O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B I O O O O O O O O O O O O O O O B I O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B B O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O

apoptosis
annexin
scintigraphy
head and neck cancer
parotid gland