Bone marrow transplantation in childhood leukaemia--experience and strategies in the Federal Republic of Germany

BMT has gained its place in the treatment of childhood leukaemia. Nevertheless, there are still many questions open. In acute lymphoblastic leukaemia children should normally be grafted in 2nd remission (CR). Some high risk cases, however, should probably be grafted in 1st CR. It is not clear whether children with late relapses benefit more from BMT than from renewed chemotherapy. Children with a relapse during maintenance therapy, however, have a better survival rate with BMT. In acute nonlymphoblastic leukaemia certain high risk patients should be grafted in 1st CR but it has still to be shown that BMT is superior to chemotherapy in such cases. It is not clear whether children with a relapse following intensive chemotherapy (such as the BFM-protocols) will benefit from BMT at all. In chronic myelocytic leukaemia, BMT in chronic phase should be performed. Thus, for the first time cure has become possible for this disease. Waiting for acceleration or even the occurrence of a blast crisis decreases the chance of survival after BMT dramatically. Since complications of BMT such as graft-versus-host reaction or severe infections are less frequent in children, relapses remain the main problem after BMT in childhood leukaemia.     

Fundamentals, trends and our experiences with total body irradiation (TBI) before bone marrow transplantation (BMT)

At the end of the sixties and to beginning of the seventies years the total body irradiation (TBI) was introduced in the concept of bone marrow transplantation (BMT). The aim is the destruction of leukaemic or normal stem cells surviving the chemotherapy or the overcoming of the immunological defense. From March 1980 to January 1987 we have treated 84 patients with single exposure of 8.5 to 10.5 Gy midline dose for body and lung in cases of leukaemia and of 6 to 7 Gy for patients with aplastic anaemia. We used a dose rate of about 5.5 cGy/min delivered by a linear accelerator. The results were comparable with other centres but a further indicator for the effectiveness of a irradiation technique is also the idiopathic interstitial pneumonitis (IIP). Our incidence of IIP was 10.7 per cent and the mortality was 2.4 per cent. Additional we have had 8.3 per cent interstitial pneumonitis (IP) caused by an infection. All patients with a combination of IP and GVHD had a fatal prognosis. In present time a tendency is to see to fractionation techniques in total body irradiation for decreasing of the pneumonitis rate, the reduction of severe acute and delayed side effects, for a better homogenisation of the dose in the whole body and for using of synchronizing effects on the stem cells.     

The induction and abolition of specific immunosuppression of heart allografts in rats by use of donor blood and cyclophosphamide.

Intravenous pretreatment of WAG/Rij rats with BN blood 14 days before transplantation leads to permanent survival of BN heart allografts. Pretreatment with donor blood in the reverse donor-host combination gives rise to accelerated rejection of WAG/Rij hearts. Addition of 100 mg/kg Cy to the pretreatment with donor blood in the WAG/Rij to BN model resulted in permanent graft acceptance, presumably due to the phenomenon of immunologic enhancement. This effect could be observed only if the interval between drug administration and antigen pretreatment was short. The use of Cy had a more profound and long-lasting effect on humoral than on cellular immunity. Addition of 100 mg/kg Cy to the active enhancement protocol in the "easy" BN to WAG/Rij combination produced permanent graft survival in an immunologic setting reminiscent of immunologic tolerance. The combined pretreatment with donor blood and 50 mg/kg Cy in this donor-host combination abolished the operation of immunologic enhancement, which could be induced if donor blood was given alone.     

Bone marrow transplantation in children with neuroblastoma

Neuroblastoma is the third most frequent malignant tumor in childhood. One-third of the patients over one year of age at the time of diagnosis suffer from the disseminated form (stage IV). Despite highly aggressive chemotherapy survival rates are poor. One hundred and eighty-seven patients with neuroblastoma stage IV have been treated according to the German protocol NB 85. The probability of disease free survival is only 15% after 70 months. Treatment strategy in our protocol includes autologous and allogeneic bone marrow transplantation (BMT) for patients with stage IV (and greater than 1 year old). Twenty-two patients were grafted (7 allogeneic and 15 autologous). The conditioning regimen consisted mainly of high-dose melphalan (180 mg/m2) and total body irradiation (TBI) (3.4 Gy). Survival rates are discussed in the context of the chemotherapy protocol. Our own experience with autologous BMT is poor, despite of different purging methods. For this reason we decided to focus on allogeneic BMT. We have grafted five patients within the last 3 years. Three of them are alive and well, on died from veno-occlusive disease 70 days after BMT, and the remaining patient, grafted from a syngeneic donor, died from relapsing tumor. The main problem in neuroblastoma stage IV is resistance to chemotherapy. Intensification of the conditioning regimen or double autografting leads to a rate of toxic deaths close to 20% (Zucker, EBMT 1987) which is not tolerable. New improvements in the conditioning regimen have to be found to increase the effect of BMT.     

Concentrations of ciclosporin in allogeneic bone marrow recipients. Comparison of assay methods

Thirteen patients in complete remission from acute nonlymphoblastic leukaemia or in chronic phase of chronic myelocytic leukaemia were treated with total body irradiation, cyclophosphamide and allogeneic bone marrow transplantation (BMT). Ciclosporin (CS) was administered for the prevention and the treatment of Graft versus Host Disease. Blood concentrations of CS were determined by Radioimmunoassay (RIA) and by High Performance Liquid Chromatography (HPLC). Trough levels of CS in peripheral blood as measured by RIA exceeded HPLC derived levels in nearly all (56/58) samples with a ratio of RIA:HPLC ranging from 2.43 +/- 1.42 at day 12 to 3.65 +/- 1.86 at day 26 after BMT (means +/- SD). A comparable ratio was found as regards the peak concentrations of CS in peripheral blood. Neither the dose of CS (0.5-3.0 mg/kg/day intravenously; 3.0-5.0 mg/kg/day per os) nor the duration of treatment (12, 19, 26 or 33 days after start of CS) were a significant factor as regards the ratio between HPLC and RIA. Concentrations of CS were also determined in bone marrow nucleated cells at 1 hour after the drug infusion had started. Here the ratio of RIA versus HPLC varied upon the duration of CS treatment with a highest ratio of 8.75 +/- 8.74 at day 12 after BMT. Bone marrow levels corresponded well with blood trough concentrations (p less than 0.01). It is concluded that the concentrations of CS in blood and bone marrow as determined by RIA and HPLC differ significantly, though consistently. At present, no advantage can be attributed to either method of analysis for routine clinical monitoring, as long as detailed information on the immunosuppressive and the toxic characteristics of CS metabolites in humans is lacking.     

Status of allogeneic bone marrow transplantation in childhood in the GDR

Of 25 HLA-identical, MLC negative transplants 10 patients had acute lymphoblastic leukaemia (ALL), 8 acute nonlymphoblastic leukaemia (ANLL), 3 severe aplastic anaemia, 2 malignant histiocytosis, 1 patients neuroblastoma and 1 Fanconi anaemia. 3 HLA nonidentical, MLC positive transplants were performed, two children had malignant infantile osteopetrosis and 1 child had a severe combined immunodeficiency disease. Patients with ALL and ANLL received cyclophosphamide and single dose total body irradiation. 3 patients received fractionated TBI. The results for the allogeneic group overall indicate that the actuarial disease free survival rate is 0.62. 16 of 25 patients are in continuous complete remission (CCR) periods of 3-78 months posttransplant. All three transplanted children with severe aplastic anaemia alive disease-free for periods of 21-81 months. 10 patients with ALL were transplanted (2 in first remission for high risk ALL, 8 in second remission). 7 of 10 patients are alive and disease-free (CCR rate 0.67). 8 patients underwent BMT for ANNL while in first remission in 7 patients and in third partial remission in 1 patient. 4 of 8 patients are alive and disease-free for periods of 25-56 months (CCR rate 0.50). 1 patient with neuroblastoma stage IV survives 24 months, 1 child with Fanconi anemia died on day +25 of GVHD and septicaemia. 1 of the 2 patients transplanted for malignant histiocytosis relapsed 3 months posttransplant, 1 patient is alive and disease-free 5 months posttransplant. In none of the HLA-nonidentical and MLC positive transplantations T-cell depleted marrow engrafted.     

Acute and late effects in the course of and after total body irradiation with following bone marrow transplantation in patients with different forms of leukaemia

The acute reaction in the course of a total body irradiation (TBI) appears in an organ-specific damage of the stem cells. Moreover, there are unspecified central-nervous stress reactions. Clinical reactions are obtained by the study and symptomatic therapy is proposed. 90 patients with different forms of leukaemia were observed. We documented the course in a specific "protocol system". Reactions like an increase of body temperature, changes of pulse and blood pressure were registered. The occurrence of gastro-intestinal reactions is a typical symptom of the acute radiation syndrome e.g. vomiting and diarrhoea are demonstrated in dependence of the applicated dose of irradiation. Further symptoms of TBI appeared in the later period. Mucositis, parotitis, a decreased function of the salivary glands and diarrhoea as well as vomiting are characterized by different intensity and temporary termination. A difference between allogeneic and autologous transplantation is caused by a medicamental additional treatment. During the late period these symptoms will disappear completely. Moreover, after TBI and BMT late effects are a cataract and some changes in the hormonal system demanding a specific correction or substitution respectively.     

Analysis of the DNA content of urethane-induced liver tumors in mice

Administration of urethan (three times per 1 mg calculated per 1 g of the animal mass) after partial hepatectomy resulted in the development of liver tumours classified as adenomas in 62.8 per cent of mice 12 months after treatment. In the cells isolated from 85 adenomas and from the surrounding liver, DNA-fuchsin content was determined cytophotometrically. Three types of DNA distribution were distinguished, with the mode in the region of near-diploid (26%), near-tetraploid (71.8%), or near-octaploid (2.2%) DNA value. Most commonly DNA distributions were polymodal, but unimodal ones also occurred. The number of binucleated cells in tumours was significantly decreased. In the liver of one of the same animal, morphologically similar tumours of all the three types of DNA distribution could be found. The increase in the hepatocyte ploidy level in the initial parenchyma by preliminary repeated treatments with CCl4, had no effect either on the occurrence of tumours, or on their ploidy level. No correlation was found between the DNA content, the size and histological structure of tumours.     

Allogeneic bone marrow transplantation for leukaemia and aplastic anaemia. Results of the Leipzig BMT Group

In acute leukaemias there was a stable plateau in the survival curve at 45% after two years if grafted in first complete remission (n = 20) but only 13% of the patients are disease-free alive if grafted in a more advanced stage of the disease (n = 8). In 16 patients transplanted for chronic myeloid leukaemia the overall survival is 40%, in cases with graft-versus-host disease (GVHD) prevention by cyclosporine survival rate could be improved. Only 8 patients with severe aplastic anaemia, partially in low performance status were able to be transplanted; three died of infections, another by acute GVHD. The fatal complications in our study characterize the international well-known major problems in BMT: GVHD, interstitial pneumonitis, infections, graft failure in aplastic anaemia and recurrence of leukaemia, especially in more advanced leukaemia stage.     

The carcino-embryonic antigen of experimental tumors of the intestine]

The occurrence of a tumour-specific antigen in the intestinal tumours (induced in 51 albino rats by subcutaneous injection of 1,2-dimethylhydrazine), as well as in the blood serum was studied by double immunodiffusion in agar gel. Intestinal tumours proved to contain a water-soluble antigen absent in other tissues, including the colonic mucosa of control animals. This antigen was found in 70 per cent of the tumour-bearing rats. The antigen is perchloric acid-solubilized and is also present in the embryonic tissues in toto on the 7th and the 9th days of rat gestation. The features of this antigen were analogous with those of the carcino-embryonic antigen in human digestive tract tumours.     

Effects of oxidant stress on inflammation and survival of iNOS knockout mice after marrow transplantation

In a model of idiopathic pneumonia syndrome after bone marrow transplantation (BMT), injection of allogeneic T cells induces nitric oxide (.NO), and the addition of cyclophosphamide (Cy) generates superoxide (O.) and a tissue-damaging nitrating oxidant. We hypothesized that.NO and O. balance are major determinants of post-BMT survival and inflammation. Inducible nitric oxide synthase (iNOS) deletional mutant mice (-/-) given donor bone marrow and spleen T cells (BMS) exhibited improved survival compared with matched BMS controls. Bronchoalveolar lavage fluids obtained on day 7 post-BMT from iNOS(-/-) BMS mice contained less tumor necrosis factor-alpha and interferon-gamma, indicating that.NO stimulated the production of proinflammatory cytokines. However, despite suppressed inflammation and decreased nitrotyrosine staining, iNOS(-/-) mice given both donor T cells and Cy (BMS + Cy) died earlier than iNOS-sufficient BMS + Cy mice. Alveolar macrophages from iNOS(-/-) BMS + Cy mice did not produce.NO but persisted to generate strong oxidants as assessed by the oxidation of the intracellular fluorescent probe 2',7'-dichlorofluorescin. We concluded that.NO amplifies T cell-dependent inflammation and addition of Cy exacerbates.NO-dependent mortality. However, the lack of.NO during Cy-induced oxidant stress decreases survival of T cell-recipient mice, most likely by generation of.NO-independent toxic oxidants.     

Long term retention of 237Np in rats

This interim report summarizes the results of observations during the first year after a single injection of 237Np nitrate (0.2 or 1.0 mg/kg body weight) into adult female rats and further preliminary data obtained with young animals. The retention of 237Np was followed by whole body counting and serial sacrifice of groups of animals. The retention data could be fitted to three-component exponential equations which show no major differences between the two 237Np dose levels. The half-times and extrapolated initial fractions calculated from the first two exponential terms indicate that one fraction, representing about 40 per cent of the injected 237Np was excreted within the first 5 days and an additional 15 per cent within the first 5 months, while the rest was excreted with a half-time of about 3.5 years. This final long term component is assumed to indicate the rate of loss of 237Np from the skeletal compartment. In young animals both whole-body and skeletal retention of 237Np during the first 5 months of observation was about 50 per cent higher than in the adults. Several soft tissue tumours, mostly mammary tumours, have appeared to approximately the same extent in both control and 237Np treated adult rats but no osteosarcomas were detected up to 15 months after injection of 237Np.     

The antioxidant status of patients subjected to total body irradiation.

BACKGROUND AND PURPOSE: Total body irradiation (TBI) is a routine preconditioning procedure for the treatment of leukemia and aplastic anemia, prior to bone marrow transplantation (BMT). Ionizing radiation generates reactive oxygen derived species (ROS) that can be removed by antioxidants. Our purpose is to determine the antioxidant status of patients undergoing TBI by evaluating the oxidant stress and their antioxidant capacity. MATERIAL AND METHODS: We evaluated by cyclic voltammetry (CV) the total antioxidant capacity (TAC) in plasma of 14 patients undergoing TBI prior to BMT. The levels of the antioxidants, ascorbic acid (AA) and uric acid (UA) were determined by HPLC-ECD. The oxidant stress level was calculated by the ratio [dehydro ascorbic acid]/total ascorbic acid]. RESULTS: TAC was reduced by 36% (p < 0.02) but after 4 months recovered to a level 22% higher than before the treatment (p < 0.05). Both, AA and UA, decreased following irradiation by 84% (p < 0.02) and 24% (p < 0.05) respectively, but returned to a level of 21% and 320% after 4 months compared to baseline values. The changes in [UA] were affected by Allopurinol (xanthine oxidase inhibitor), given as a routine pretransplant therapy until day -1. The [dehydroascorcbic acid]/[total ascorbic acid] (%) was 45% (range of normal controls = 13.2 +/- 1.5%) and increased by 69% following TBI. In order to obtain a decrease in the TAC of plasma in vitro, comparable to that in vivo, a 1000 fold higher dose of irradiation was required. CONCLUSIONS: TBI caused a pronounced decrease in antioxidant capacity and an excessive increase in oxidant stress. We assume that TBI alters antioxidant homeostasis greatly enhancing the stress damage. CV measurements may lead to a better understanding of the balance between oxidant stress and antioxidant utilization, and to a reconsideration of the routine use of Allopurinol as pretreatment for TBI, and antioxidant support before and/or after TBI.     

Second malignancies in Hodgkin's disease: a complication of certain forms of treatment.

A total of 764 patients with Hodgkin''s disease treated with radiotherapy (RT) or chemotherapy or both were reviewed 3-186 months (median 43 months) after initial treatment to assess the incidence of second malignancies. Incidence of solid tumours and acute non-lymphoblastic leukaemia (ANLL) were calculated by a life-table method and percentages of patients affected derived from life-table plots. Within 10 years after initial treatment the overall incidence of second solid tumours was 7.3%, and over a comparable period 2.4% of patients developed ANLL. Solid tumours occurred only in patients given RT with or without adjuvant chemotherapy, and ANLL occurred only after treatment with MOPP (mustine, vincristine, procarbazine, and prednisolone) or modified MOPP regimens. Neither solid tumours nor ANLL occurred in patients given ABVD (adriamycin, bleomycin, vinblastine, and dacarbazine). The highest incidence of leukaemia (5.4%) occurred after treatment with extensive RT plus (5.4%) occurred after treatment with extensive RT plus MOPP; hence the benefits of this approach in Hodgkin''s disease must be weighed against its carcinogenic potential.     

Postnatal development and neoplastic disease pattern in NMRI mice after combined treatment with ethylnitrosourea and X-irradiation on different days of the fetal period

Mice were X-irradiated on day 14, 15 or 16 of gestation with 1.0 Gy. This did not result in an increased tumour frequency in the offspring until 12 months. Mice treated with ethylnitrosourea (ENU) (45 mg/kg) on these gestation days developed a significantly increased tumour frequency in the lungs and liver, and in the ovaries after treatment on day 15 of gestation. Additionally this experiment was the first to show that ENU treatment on gestation day 14, 15 or 16 results in haemangiosarcomas of the subcutis at a low incidence (2.0, 2.4, 2.6 per cent). After combined treatment with these two agents in the sequence X+ENU and an interval of 4 h, a significantly increased incidence rate of animals with tumours was observed in the offspring treated on gestation day 14 or 16. Moreover, the treatment on gestation day 16 exhibited the highest tumour frequency per examined animal (5.7) of all treatment groups. This result is due to a relatively uniform increase of all tumor types. Within this pattern, the frequency of liver tumours was most marked. The diagnosed liver tumours were significantly augmented after X+ENU treatment on day 16. In the reverse sequence (ENU+X), the total tumour outcome was not significantly altered compared with the effects of ENU alone. However, detailed analysis also showed a significant augmentation of the liver tumour frequency with treatment on day 15.     

Gluconeogenesis in the liver of arthritic rats

The gluconeogenic response in the liver from rats with chronic arthritis to various substrates and the effects of glucagon were investigated. The experimental technique used was the isolated liver perfusion. Hepatic gluconeogenesis in arthritic rats was generally lower than in normal rats. The difference between normal and arthritic rats depended on the gluconeogenic substrate. In the absence of glucagon the following sequence of decreasing differences was found: alanine (-71.8 per cent) reverse similarglutamine (-71.7 per cent)>pyruvate (-60 per cent)>lactate+pyruvate (-44.9 per cent)>xylitol (n.s.=non-significant) reverse similarglycerol (n.s.). For most substrates glucagon increased hepatic gluconeogenesis in both normal and arthritic rats. The difference between normal and arthritic rats, however, tended to diminish, as revealed by the data of the following sequence: alanine (-48.9 per cent) reverse similarpyruvate (-47.6 per cent)>glutamine (-33.8 per cent)>glycerol (n.s.) reverse similarlactate+pyruvate (n.s.) reverse similarxylitol (n.s.). The causes for the reduced hepatic gluconeogenesis in arthritic rats are probably related to: (a) lower activities of key enzymes catalyzing most probably steps preceding phosphoenolpyruvate (e.g. phosphoenolpyruvate carboxykinase, pyruvate carboxylase, etc. ); (b) a reduced availability of reducing equivalents in the cytosol; (c) specific differences in the situations induced by hormones or by the individual substrates. Since glycaemia is almost normal in chronically arthritic rats, it seems that lower gluconeogenesis is actually adapted to the specific needs of these animals.     

Allogeneic bone marrow transplantation for hepatocellular carcinoma: hepatocyte growth factor suppresses graft-vs.-host disease

Allogeneic bone-marrow transplantation (BMT) can induce a powerful graft-vs.-tumor (GVT) effect not only on hematological malignancies but also on solid tumors. However, graft-vs.-host disease (GVHD) is a major complication of allogeneic BMT. We assessed GVT effect on hepatocellular carcinoma (HCC) and the effects of hepatocyte growth factor (HGF) gene transduction on GVHD in HCC transplanted mice. (C57BL/6 x C3H/HeJ)F(1)(B6C3F1, H-2(bxk)) mice were used as recipients and C3H/HeJ(H-2(k)) mice were used as donors. Hepa1-a (a C57L mouse-derived hepatoma cell, H-2(b)) was subcutaneously injected into the recipient mice. Tumor bearing mice were treated in the following ways: group 1, no treatment; group 2, total body irradiation (TBI); group 3, TBI and BMT; group 4, TBI and BMT with empty vector; group 5, TBI and BMT with HGF gene transduction; group 6, TBI and BMT with administration of FK506, a representative immunosuppressive agent. Acute GVHD was assessed by histological examination of the liver, small intestines, and large intestines. Tumor growth was markedly suppressed in mice that received an allogeneic BMT. Donor-derived CD8(+) T cells had infiltrated into the tumor, and cytotoxic CD8(+) T cells against HCC were present. However, among the four groups that received a BMT, this suppressive effect was weaker in group 6 compared with the other three groups (groups 3, 4, and 5). HGF gene transduction improved GVHD while preserving the GVT effects. Allogeneic BMT markedly suppresses the growth of HCC. Simultaneous HGF gene transfer can suppress GVHD while preserving the GVT effect.     

A THERAPEUTIC REGIMEN FOR TUBERCULOUS MENINGITIS

The results of treatment of tuberculous meningitis in Highland Alameda County Hospital, since the advent of streptomycin, in 34 cases were: dead, 21 patients, or 61 per cent; alive, 10 patients, or 29 per cent. Three cases were excluded from the ultimate report due to inadequate follow-up. The mean survival time of the ten survivors at the time of report was 35 months, the longest being 74 months.The composite results of treatment elsewhere, as reported in the literature, have been: dead, 892, or 59 per cent; alive, 629, or 41 per cent.The pathogenesis of the disease and the effects of chemotherapy are discussed.A therapeutic regimen for tuberculous meningitis is presented, which has been used at Highland Hospital since INH became established as the most effective component in the combined chemotherapeutic attack on the disease.     

Tumour bed effect: hypoxic fraction of tumours growing in preirradiated beds

The reduction in tumour growth rate seen when tumours are implanted into preirradiated sites, the tumour bed effect (TBE), is believed to be due to radiation damage to vascular stroma, leading to defective angiogenesis in the tumour. The present work examined whether or not the functional inadequacy of irradiated stroma was accompanied by an increased hypoxic fraction in tumours growing in irradiated beds. Mouse flank skin was given 0 or 20 Gy X-rays and RIF-1 fibrosarcoma cells were implanted i.d. into the centre of the treatment field one week later. Tumours of 200 mm3 were irradiated under clamped or unclamped conditions and the hypoxic fraction measured from the displacement of the corresponding survival curves, assayed in vitro. Results indicated a small increase in the hypoxic fraction. Averaging values from three independent experiments, the percentage of hypoxic cells increased from 2.5 per cent for cells in tumours growing in unirradiated beds to 4.6 per cent for those from tumours in beds given 20 Gy. Thus an irradiated vascular bed is still to some extent able to maintain the proportion of oxic: hypoxic tumour cells found in tumours growing in unirradiated beds, despite manifest changes in tumour necrosis and growth rate.     

Imaging pancreatic cancer with a peptide-nanoparticle conjugate targeted to normal pancreas

Designing molecules that bind to targets that become upregulated or overexpressed as normal cells become cancerous is an important strategy for both therapeutic and diagnostic drug design. We hypothesized that pancreatic ductal adenocarcinoma (PDAC) might be imaged with the inverse strategy, that is by the design of a nanoparticle-conjugate targeted to bombesin (BN) receptors present on normal acinar cells of the pancreas. Using the fluorescein hapten visualization method to assess the presence of bombesin (BN) receptors, we first demonstrated BN receptors in the normal mouse and human pancreas, but then the lack of BN binding receptors in 13 out of 13 specimens of PDAC. The BN peptide-nanoparticle conjugate, BN-CLIO(Cy5.5), was synthesized and accumulated in the mouse pancreas in receptor dependent fashion, but not in a receptor dependent fashion in other tissues, based on tissue fluorescence measurements. The BN-CLIO(Cy5.5) nanoparticle decreased the T2 of normal pancreas and enhanced the ability to visualize tumor in a model of pancreatic cancer by MRI. The use of BN-CLIO(Cy5.5) nanoparticle as a normal tissue-targeted, T2-reducing contrast agent offers a promising approach to imaging PDAC.