The Neuroradiology Journal 21: 683-692, 2008
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Localized Amyloidosis and Alzheimer’s Disease: the Rationale for Weekly Long-Term Low Dose Amyloid-Based Fractionated Radiotherapy F. BISTOLFI Radiotherapy Department, Galliera Hospital; Genoa, Italy
Key words: amyloidosis, Alzheimer, Creutzfeldt-Jacob, fractionated radiotherapy, Diapulse, pulsed short waves
SUMMARY – Amyloidosis, a plasma cell dyschrasia, is characterized by accumulation in the intercellular spaces of fibrillar proteins with a typical beta-sheet pattern. Vascular-cerebral amyloidosis is the hallmark of Alzheimer’s disease and spongiform encephalopathy (Creutzfeldt-Jacob and the like). Current treatment of primary systemic amyloidosis is neither free from complications nor - in some presentations - a mortality rate. Localized tracheo-bronchial amyloidosis (TBA) has been successfully treated with high energy beams of radiation (20 Gy in 10×200 cGy in two weeks). The CT response to radiation takes several months after completion of treatment. As 20 Gy in two weeks are followed by inflammatory reactions, this dosage cannot be suggested in the treatment of amyloidotic radiosensitive organs (e.g. kidneys, liver), or in the hypothetical treatment of Alzheimer’s disease. On the basis of the following points: 1) plasma cells in amyloid deposits are not numerous; 2) plasma cells are radioresistant, both in vitro and in vivo (radiotherapy of solitary plasmocytoma); 3) the effects of radiotherapy (20 Gy/2 w) on TBA localizations cannot be exclusively due to plasma cell killing, this study postulates a biophysical mechanism of radiation-induced H-bond breaks in the beta-sheet structure of amyloid, together with depolymerization of glucosaminoglycans, very radiosensitive molecules invariably associated with amyloid fibrils. As both biophysical effects are DNAindependent, the adoption of a definite time/dose ratio (e.g. 20 Gy/2 w) loses much of its importance. Therefore an innovative alternative might be a weekly long-term low-dose fractionated radiotherapy, matching the very slow response of amyloid to radiation. Before being applied to Alzheimer’s disease, the proposed radiotherapy (RT) schedule should be tried in TBA patients to compare the new results of long-term fractionated RT with the old results of 20 Gy/2 w. Should long-term fractionated RT prove equally (or almost equally) effective, but certainly much less toxic than 20 Gy/2 w, its application to Alzheimer patients might become an effective and safe treatment, provided clinical and objective control by means of current imaging techniques (MRI, PET) can be assured.
Amyloidosis Amyloidosis 1,2,3,4,5,6,6b is accumulation in the tissues of various insoluble fibrillar proteins in an amount sufficient to impair normal functions. The classical clinical classification 6b recognizes five forms: primary amyloidosis, myeloma-associated amyloidosis, secondary amyloidosis, localized and pseudotumoral amyloidosis and familial-hereditary amyloidosis.
Primary systemic amyloidosis (AL, immunoglobulin light chain amyloid) results from the extracellular deposition of insoluble immunoglobulin light (AL) or heavy chains (AH), which disrupt organ function and ultimately lead to the death of the patient. The heart, lung, skin, tongue, thyroid gland and intestinal tract may be involved. Localized amyloid ‘tumors’ may be found in the tracheo-bronchial system or other sites. Hepatomegaly, nephrotic 683
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proteinuria and congestive cardiomyopathy are common, as well as painful peripheral neuropathy. Secondary amyloidosis (AA) is associated with chronic diseases (tuberculosis, bronchiectasis, osteomyelitis, leprosy, rheumatoid arthritis, granulomatous ileitis) and shows a predilection for the spleen, liver, kidney, adrenals and lymphnodes. Amyloid associated with multiple myeloma has the same distribution as primary systemic amyloidosis. Familial forms often exhibit distinctive types of neuropathy, nephropathy and cardiopathy. Other forms of amyloidosis are the hemodialysis-related amyloidosis and two brain diseases: Alzheimer’s disease and spongiform encephalopathies. The current classification of amyloidosis is based on the composition of the subunit fibril protein and recognizes the following associations 1,6 b,13: Primary systemic amyloidosis and Myeloma
monoclonal immunoglobulin light chains (AL) or heavy chains (AH)
Secondary amyloidosis
AA
serum amyloid A protein
Hemodialysis related amyloidosis
A β2 M
β2 -microglobulin
Familial and senile amyloidosis
ATTR
transthyretin
Alzheimer
A β PP
β-amyloid precursor protein
Spongiform encephalopathies
prion protein.
Under light microscopy, amyloid is a homogeneous highly refractile metachromatic substance with an affinity for Congo red. Under electron microscopy, amyloid consists of linear, nonbranching fibrils of about 5-15 nm×800 nm. Under X-ray diffraction, the heterogeneous fibril protein chains show a typical beta-sheet pattern, on which both the optical and tintorial properties of amyloid depend. Blake et Al 5, using high resolution electron microscopy and X-ray diffraction, concluded that all beta-sheets composing the protofilaments have a helical arrangement, and that the beta-sheet helix may be the generic core structure of amyloid. Therefore, all proteins in amyloid deposits share fibrillar structure and beta-sheet conformation: hence the comprehensive term of beta-fibrillosis to indicate all clinical forms of amyloidosis 6. Treatment of primary systemic amyloidosis is directed first to the blood marrow plasma 684
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cell dyschrasia. Current treatment 2,3,4,7,8,9,10,11,12 includes melphalan/prednisone or melphalan/ prednisone/colchicine 2,7, sometimes with the addition of other alkylating agents. However, therapy with multiple alkylating agents did not result in a higher response rate or longer survival time 4. Since 1995 autologous stem cell transplantation has been incorporated in the high-dose treatment of primary systemic amyloidosis. The response rates are substantially better than those for patients treated with lowdose traditional melphalan and prednisone 3,8,10, but the related mortality is very high, reaching 44% in some series. As a consequence, patients eligible to receive stem cell transplantation must represent a highly selected population 3. In cases of severe single organ involvement, organ transplantation such as heart, kidney or liver transplantation may have a successful outcome 2. An innovative treatment, aimed at directly reducing the amyloid deposits has been proposed by Merlini et Al 11,12 using I-DOX (4’-iodo4’-deoxydoxorubicin). This will be discussed below in view of its conceptual links with the proposed amyloid-based radiation treatment. Independently on the clinical response in terms of survival time and quality of life 2,3,4,7,8,9,10, what deserves attention is the fact that amyloid deposition is not an irreversible process inasmuch as amyloid deposits can be substantially reduced by therapy. Therefore, any new method of treating localized amyloid involvement of single organs must be welcome. Alzheimer’s disease and CreutzfeldtJacob’s encephalopathy as brain localized amyloidosis Amyloid-related diseases now include Alzheimer’s disease and transmissible spongiform encephalopathies 13. Alzheimer’s disease, now considered the most common form of amyloidosis, is due to vascular-cerebral amyloidosis and shows a series of pathological components: neuritic plaques, neurofibrillar interlacing, cell degeneration and congophilic angiopathy 6. Formation of senile plaques composed of amyloid β protein is a pathological hallmark of Alzheimer’s disease in human brain and precedes the onset of symptoms by many years. According to Higuchi et Al 14, noninvasive detection of such plaques could afford a presymptomatic diagnosis by MRI with the intravenous administration of a 19F containing amyloido-
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philic compound specifically labeling amyloid beta plaques in the brain. When injected in the vascular system, another β-amyloidophilic compound, the anylin-derived phenyl-hydrobenzothiazole (PHB), crosses the brain barrier and stains the amyloid senile plaques. By labeling PHB with 14C, the plaques may be demonstrated in the brains of living Alzheimer patients using positron emission tomography (PET) 15. A joint clinical study involving Swedish and USA institutions showed very impressive PET images in the brains of nine Alzheimer patients in comparison with five normal subjects. 14C PHB-PET will certainly become a useful method to control the objective effects of therapy in Alzheimer patients 15. It has also been claimed that some infective agents named prions (protein infective particles) can give rise to some forms of amyloidosis associated with Creutzfeldt-Jacob’s disease, a severe spongiform encephalopathy characterized by the presence of amyloid plaques in the brain and cerebellum. Prions are neither viruses nor bacteria but protein molecules able to polymerize in rod-like filaments, whose crystallographic presentation at the X-ray diffraction is that typical of beta-sheets forming amyloid fibrils. While Creutzfeldt-Jacob’s encephalopathy affects 50-70-year-old subjects, the atypical new variant (bovine spongiform encephalitis, BSE) commonly known as “mad cow disease” affects younger patients with atypical signs and is thought to be caused by the consumption of prion-infected beef 6,16. Localized amyloidosis in the tracheobronchial system: and external beam radiation therapy Tracheo-bronchial amyloidosis (TBA) is characterized by deposition of amyloid in the trachea and bronchi as submucosal plaques and/or pseudotumoral masses 17. In both cases various degrees of lumen narrowing ensue. The condition is not common and the severity of the clinical course depends on its respiratory and infective complications. Over the years, multimodal therapeutic approaches have been adopted, including bronchoscopic methods (laser resection, balloon dilatation, temporary placement of a silicon stent). Since the publication by Kurrus et Al in 1998 17 on an effective high energy radiation treatment in one 67-year-old patient, a few other papers appeared in the following
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ten years, confirming the usefulness of external beam radiation therapy (EBRT) in nine adult patients with TBA 18,19,20,21. CT scans are essential to show the extension of lesions prior to treatment planning and during follow-up. Amyloid deposits may be displayed as superficial plaques, submucosal infiltration at the level of the trachea, carina, mainstem bronchi, or bulky masses narrowing the bronchial lumen. Amyloid-induced collapse of one single lobe may also occur. The irradiation technique relies upon 4 MV, 6 MV, 10 MV x-ray beams through two parallel AP-PA opposing fields, sometimes 18 with an added right and left lateral field arrangement. Customized field techniques aim at limiting the dose to lung parenchyma. The given dose is a standard time/dose ratio of 20 Gy in ten daily 200 cGy fractions in two weeks (20 Gy/2 w.). In one case report 19 the dose was 24 Gy in 12 daily 200 cGy fractions and in another case report 17 the same schedule (20 Gy/2 w) was applied six months later because of a distal extension of the primary lesion treated with the same dose. Good symptomatic and objective results are described: the median time to a subjective response was four months (range 1-7 months) in Neben-Wittich’s series of seven patients 18. However, the objective results at bronchoscopy and CT scans are much slower, taking several months after completion of treatment (range 10-16 months) 17,20. One case report 19 found a resolution of objective signs starting one month after treatment and continuously progressing over the subsequent 16 months. Side effects are mainly grade 1 and 2 oesophagitis, but grade 2 pneumonitis was also observed 18. The median follow-up in Neben-Wittich’s series was 40 months (range 10-69 months). Follow-up in other case-reports reached 18 months 17,19 and 21 months 20. As EBRT can provide symptomatic as well as objective improvement, it becomes a treatment option for localized tracheo-bronchial amyloidosis causing airway obstruction. The mechanism of treatment: does EBRT affect only plasma cells?
The mechanism by which EBRT can reduce TBA deposits is unclear 18. What has been established is that TBA, like AL amyloidosis, arises from immunoglobulins 21. Therefore, it has been postulated that amyloid deposits are the result of light chain deposition by local plasma cells 18. According to O’Reagan et Al 21. “as plasma cells are radiosensitive” (yet, see further) low-dose radiation may offer an alternative approach to 685
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the pharmacological treatment of TBA. Monroe et Al 19 also based their treatment plan and dose (24 Gy) on the association of monoclonal plasma cell Ig light chain deposits in both multiple myeloma and amyloidosis, presuming that the successful low-dose radiation treatment in both cases depends on the inactivation of monoclonal plasma cells. However, available pathological data do not show large numbers or large clusters of plasma cells in areas of amyloidosis 17,18,20 , which somewhat weakens the hypothesis of TBA improvement exclusively through plasma cell injury leading to decreased amyloidogenic protein production and deposition 20. In addition, the radiosensitivity of B and T lymphocytes changes with their functional state within a wide dose range (6-10 Gy to 50-150 Gy) 22 . Plasma cells in particular, the ultimate evolution step of B lymphocytes, are - according to Biagini 23 – “very radioresistant” and, at least in vitro, are not injured with doses as high as 100 Gy. These experimental data are indirectly confirmed by clinical radiobiology of solitary plasmocytoma. Indeed, from a literature survey on radiation therapy of solitary plasmocytoma up to 1997 24 it emerges that a stable local control of both osseous and extraosseous lesions needs doses as high as 35-40 Gy to 50-70 Gy in five to eight weeks. Therefore, without excluding the direct action on the scanty plasma cells in amyloid areas, it seems unlikely that 20 Gy in two weeks can reduce amyloid deposits uniquely through a plasma cell mechanism, so that other potential mechanisms have been proposed: 1) A radiation effect on the endothelium of local vasculature 17,18,20 with a reduced influx of plasma cells or delivery of distantly produced amyloid precursors; 2) A radiation effect on cells or cofactors, other than plasma cells, leading to changes in local amyloid production or deposition 17,18,20; 3) The induction of an immune response against the deposits activated by local inflammation 18; 4) A free radical mediated radiation effect, modifying and enhancing the degradation of amyloid 17. Radiobiological rationale for an amyloidbased radiation treatment of localized amyloidosis An amyloid-based mechanism, that is a physico-chemical process of amyloid degradation followed by slow reabsorption, can be added to these four mechanisms. 686
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Treatment with ionizing radiation
The universal presence in amyloid of certain glucosaminoglycans (GAGs) such as heparan sulphate and dermatan sulphate, invariably associated with amyloid fibrils 13, has been implicated in the pathogenesis of amyloidosis. Although GAGs are not required for amyloid fibrillogenesis per se, they remain – according to Kisilevsky 25 – a valid therapeutic target. GAGs, highly polymerized polysaccharides, exhibit metachromasia when treated with toluidin blue (just like amyloid) 26. Inflammatory processes induce several changes in their structure leading to GAG depolymerization 27. Moreover, low radiation doses (20 to 100 cGy) depolymerize mucopolysaccarides in connective tissue 28. For this reason, metachromasia decreases and diffusion of liquid injected into the derma increases, as occurs under the enzymatic action of hyaluronidasis 29. From a basic chapter by Alexander in “Summa Radiologica” 30 we learn that irradiated macromolecules undergo scission of the primary chain, intermolecular and intramolecular crosslinking, breaks in hydrogen bonds (H-bonds), and that the action exerted by radiation on protein molecules chiefly consists of breaks in intramolecular Hbonds, with ensuing partial or total molecule unwinding. Relevant to the present research is the action of radiation upon GAGs, associated with amyloid fibrils 13, inasmuch as their depolymerization induced by low radiation doses might indirectly affect the amyloid fibrils. Moreover, the amyloid structure itself might undergo depolymerization because of radiation-induced breaks in intramolecular H-bonds, whose paramount importance in keeping the orderly structure of the fibril-forming beta-sheets is known. Indeed, figure 1 shows the binding sites of H-bonds between parallel beta-sheets inside a protein. Figure 2 schematically shows the mechanism for amyloid fibril formation through the self-association of protein protofilaments along a template of parallel beta-sheets, kept together by H-bonds (not depicted). All this prompts our proposal of an amyloidbased radiation therapy, aiming at depolymerizing amyloid fibrils to allow their subsequent reabsorption. A therapeutic mechanism supported by the knowledge that some degree of amyloid reabsorption has been shown both experimentally and clinically, notwithstanding amyloid fibrils do not undergo phagocytosis or enzymatic degradation 6. In addition, amyloid-based radiation therapy agrees with Dam-
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The Neuroradiology Journal 21: 683-692, 2008
Figure 1 Beta-sheet structure in a globular protein. The planes forming molecular chains are kept together by H-bonds. From B. Alberts et Al (eds.) “Biologia Molecolare della Cellula”. Italian translation by M. Guardo and A. Poyrot. Zanichelli Editore, Bologna, 1984.
macco’s suggestion of studying procedures able to correct the mobilization and elimination of amyloid fibrils 6, as well as with Merlini et Al’s research 11,12 on the use of I-DOX to affect amyloid deposits directly. The postulated molecule degradation through the break in intra-molecular H-bonds is only one of the possible radiation effects on amyloid: should intramolecular crosslinking also occur, it would induce molecule aggregation 31. However, as intramolecular crosslinking reduces the volume of irradiated molecules 30, crosslinking in irradiated amyloid might not be an adverse effect, inasmuch as compression on surrounding tissues would decrease. It follows that even if both effects (H-bond breaks and intramolecular crosslinking) were concurrent, they would not be contrasting factors: crosslinking favouring the volume decrease of amyloid deposits, H-bond breaks favouring amyloid degradation and subsequent reabsorption. Treatment with non-ionizing radiation
In the 1970s Pierre Richand published some papers 32,33,34 on the clinical effects induced by ELF-pulsed 27 MHz short waves (Diapulse). The advantage of the lack of any thermic effect owing to the interpulse relaxation time (ON/ OFF ratio = 1/25) was stressed, together with
Figure 2 Proposed mechanism for amyloid fibril formation (see text). From J.D. Gillmore and P.N. Hawkins: Amyloidosis and the respiratory tract. Thorax 54: 444-451, 1999. Partially modified.
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the beneficial effect on tissue trophism. What is striking in Richand’s work is the claim of having obtained therapeutic outcomes “in several patients severely affected by hepatic and renal amyloidosis”. The case report of a 55-year-old patient, whose first symptoms appeared in February 1971 merits attention. A diagnosis of hepatorenal amyloidosis and its hopeless prognosis were established at the University Hospital Center in Créteil (France). From Richand and Boulnois’ description we abstract the following 34 : “... in 1953 the patient got a viral hepatitis in the Sahara region, where he was active as a mineral engineer. A long-lasting dental infection affected him during the second world war [from which we can infer the secondary nature of his hepato-renal amyloidosis, FB]. On September 6, 1971 the patient was intensely tired and hardly kept his balance in walking; he complained of pain in both knees and of cramps at night; he was heavily oppressed by windy weather; urine smell was ammoniacal; the liver volume was very increased (8 cm beyond the rib edge). A Diapulse treatment was then instituted (600×6×15’), three days a week, upon the upper abdomen. On October 11 (after four weeks’ treatment) the liver border extended beyond the rib edge no more than 1 cm. On November 3, the liver was no longer palpable. On January 31, 1972 (one month after completion of a four month treatment), hepatic conditions stabilized. The patient’s general status has much improved. Neither bone pains nor cramps are present, arterial pressure is normal. The biological hepatic tests have improved and immunoglobulin values have reached their normal levels. The patient is alive and well in 1978, seven years after treatment” 34. Though difficult to explain, the case report described is undoubtedly exciting, owing to the seven year duration of objective and subjective response to a simple and well tolerated physical treatment. The Diapulse machine generates electromagnetic energy in the 27 MHz band (short waves) with 65 μs pulses separated by 1,600 μs pauses. The 1/25 ON/OFF ratio allows complete dispersion of produced heat between two successive pulses. Clinical research has shown important results, such as quicker induction of healing processes, reduction of connective tissue production, easier and quicker reabsorption of edema, and regeneration of peripheral nerves 35. Diapulse irradiation is considered athermic, in agreement with the fact that heat more 688
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readily induces protein aggregation than degradation. Moreover, we know from physics that to obtain the thermic decomposition of some biological and non-biological polymers (e.g. cellulose, polystyrene, polyvinylchloride) temperatures in the order of 250-400°C are needed 36. Sasahara et Al 37 recently showed that beta-2microglobulin, a protein responsible for dialysis-related amyloidosis, when heated in a cell of a differential scanning calorimeter between 20°C and 100°C, undergoes a sigmoidal transition, representing the conversion of protofibrils into mature amyloid fibrils. It follows that the therapeutic result obtained with low frequency pulsed (600 cps) short waves has to be attributed to non-thermic effects. Among these, a good candidate is the electromagnetic acoustic transduction effect (EMAT), an effect well demonstrated along the electromagnetic spectrum 38 , particularly active when electromagnetic pulse duration falls within the microsecond range. The amyloid fibril degradation factor would then be low frequency mechanical vibration induced by ELF pulsed short waves. This is a sound interpretation, though in competition with the aggregation of beta-2-microglobulin microfibrils into mature amyloid fibrils obtained by Ohhashi et Al 39 through high-frequency mechanical vibration induced by ultrasound. Another physical factor to consider is the amyloid high refraction index 2, due to its highly ordered structure and high εr value, favouring the selective absorption of electromagnetic energy in amyloid deposits. Giving the athermic character of Diapulse emission, it is likely that selective effects in amyloid will be of athermic nature as well, namely of vibrational character. Discussion Is weekly long-term low dose amyloid-based fractionated radiation suitable to treat Alzheimer‘s disease and Creutzfeldt-Jacob‘s encephalopathy?
Degradation of amyloid deposits by chemical or physical means to eliminate their compressive effects is only one, albeit important, component of amyloidosis treatment. On chemical grounds, the innovative approach of Merlini et Al 11,12 with 4’-iodo-4’-deoxydoxorubicin (IDOX) must be emphasized. They observed that weekly fractionated I-DOX can interact directly with amyloid deposits by reducing amyloid load
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without affecting the level of circulating light chains. They used echography, CT scans, MR imaging or scintigraphy with 131I-labeled serum amyloid P component (SAP) to assess the evidence of reduced amyloid load in 14 patients treated in about three years (1995-1997). On physical grounds, high energy radiation beams have been successfully used to reduce localized amyloid deposits in tracheo-bronchial amyloidosis (TBA) with a standard dose of 20 Gy in ten 200 cGy fractions in two weeks (20 Gy/2 w) 17,18,19,20 , a time/dose ratio that will be the central point of discussion. In Neben-Wittich et Al’s series of seven TBA patients 18, the time/dose of 20 Gy/2 w did not appear free from side effects: oesophagitis in five out of seven patients (four grade 1 and one grade 2) and grade 2 pneumonitis in one out of seven patients, which is neither surprising nor difficult to explain. Indeed, according to the dosage system I devised in the 1960s 40 and further developed in the 1990s 24, a tissue dose of 20 Gy in two weeks can induce acutesubacute reactions, characterized by microvascular damage and inflammation (vessel dilatation, increased vessel permeability, interstitial edema), namely: brisk cutaneous erythema followed by dry desquamation (which means oesophagitis at the level of oesophageal mucosa); subacute radiation pneumonitis followed by late lung fibrosis (if large volumes of lung are involved); radiation nephritis (when both kidneys fall in the treated volume). Knowing that radiation can change amyloid protein structure and depolymerize the associated GAG molecules (both of which are DNAindependent mechanisms), the adoption of a definite time/dose ratio loses much of its importance. Therefore to apply radiation to treat amyloid-involved radiosensitive organs (e.g. lungs, kidneys, liver), and, as a last resort, Alzheimer’s disease, the alternative to the 20 Gy/2 w schedule might be a weekly fractionated regimen with very low dose fractions (say 50 to 100 cGy, once a week) and a very long overall treatment time to match the very slow involution rate of irradiated amyloid: hence, a much safer radiotherapeutic approach than those able to induce vascular damage and inflammatory processes. However, the overall dose necessary to induce amyloid degradation with the long-term fractionation regimen cannot as yet be established. Awaiting preliminary clinical trials in TBA patients, we might only guess at an overall dose between 20 and 30 Gy in an
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overall treatment time of about one year (e.g. 50 cGy × 40 - 60 w or 100 cGy × 20-30 w). As far as the oncogenic risk of whole-brain weekly irradiation with 50 (100) cGy is concerned, any assessment is hampered by the atypical fractionation proposed. However, the ten to twenty year latency of the “hypothetical” oncogenic effect will be counter-balanced by the probability of actually improving the miserable life conditions of Alzheimer patients. The treatment proposed, obviously prudenceguided but not lacking a sound rationale, should be first applied to patients with TBA to compare the long-term fractionation results with the standard 20 Gy/2 w results. Should the slowly fractionated dose be equally effective in inducing amyloid degradation without any side effects, the next step would be the low-dose fractionation treatment of Alzheimer’s disease and Creutzfeldt-Jacob’s encephalopathy. As a consequence, a question arises: what is known about brain tolerance to radiation? The brain’s tolerance to ionizing radiation has been analyzed in radiotherapy of human brain tumors, as well as in laboratory animals. Research has privileged the risk assessment of severe radiation encephalopathy and proven the relevance of parameters such as overall dose, overall treatment time, number and magnitude of dose-fractions. Moreover, we know that the tolerance dose definitely decreases as the irradiated brain volume increases from a few cubic centimeters up to the whole brain 24. Radiation treatment of primate brain with conditions similar to those of clinical radiotherapy (2.0 Gy per fraction, five fractions a week, four to eight weeks overall treatment time) with a cumulative dose of 40 Gy in four weeks is followed by negligible damage, whereas a dose of 60 Gy in six weeks is followed by noticeable damage, and a dose of 80 Gy in eight weeks, by severe damage 41. Limitedly to the dose of 40 Gy in four weeks, these results are in good agreement with Cicciarello et Al 42, who irradiated the whole brain of albino rats using 6 MV X-rays, suitable bolus and 20 x 200 cGy (40 Gy) in four weeks. Three weeks after irradiation, signs of low-degree damage were observed, namely: changes in brain metabolism (14C-2DG) and permeability changes in the blood-brain barrier (transcapillary passage of 14 C-alfa aminoisobutyric acid), leading them to conclude that in whole-brain irradiation with daily fractionation time/dose values even lower than those linked to severe bioeffects can determine subclinical, mainly vascular, bioeffects. 689
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Cohen and Creditor 43, in their basic research on severe radiation encephalopathy (radionecrosis, dementia, severe mental retardation in long-term survivors), started from a wealth of clinical and cell kinetic data to arrive at an isoeffect time/dose table for a 5% risk with various dose fractionations. Subsequently, I drew four curves relative to four fractionations (five, three, two and one fraction a week) and overall treatment time of two to ten weeks 24. For comparison with the schedule of TBA radiotherapy (10 x 200 cGy in two weeks), Table 1 shows how the 5% risk of severe brain complications is related to the overall dose and fractionation type within the overall treatment time of two weeks (table 1) Table 1 Five per cent risk of severe brain complications for an overall treatment time of two weeks with four different fractionations (based on Cohen and Creditor’s data) 24,43
33 Gy/2 weeks
with 10 fractions of
330 cGy
25 Gy/2 weeks
w"th 16 frac"ions of
417 cGy
20 Gy/2 weeks
w"th 14 frac"ions of
500 cGy
14.5 Gy/2 weeks
w"th 12 frac"ions of
725 cGy
Last but not least, let us recall that the tolerance limit of irradiated brain is definitely lowered by concomitant chemotherapy, as shown by two papers reporting diffuse, progressive encephalopathy in 10.2% 45 and 45% 44 of patients treated with whole brain radiotherapy (dose fractions 240, 300, 350 cGy) and polychemotherapy for small cell carcinoma of the lung. We conclude that the proposed weekly longterm low dose amyloid-based fractionated radiotherapy of brain amyloidosis may be considered a safe procedure as far as the brain tolerance is concerned, provided no concomitant chemotherapy is implemented. However, as far as its effectiveness on amyloid plaques is concerned, only preliminary trials in TBA cases will be able to afford a definite judgment on its clinical usefulness. Conclusions 1) The dosage schedule 20 Gy/2 w. (10 × 200 cGy in two weeks), whose efficacy in radiotherapy of tracheo-bronchial amyloidosis (TBA) has been proven, represents a time/dose ratio not free from acute side effects, though less toxic than the two week time/dose ratios linked to
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5% of severe radiation encephalopathy reported in table 1. 2) As time/dose values even lower than those linked to 5% of severe complications can induce subclinical, mainly vascular bioeffects 42 in the brain, the 20 Gy/2 w regimen should be avoided in radiation therapy of Alzheimer’s disease. 3) The tolerance limit of irradiated brain is definitely lowered by concomitant chemotherapy 44,45. As current treatment of amyloidosis is largely based on cytotoxic chemotherapy, therefore, radiation therapy of brain amyloidosis should carefully avoid any implementation with chemotherapy. 4) As Alzheimer patients may also be affected by brain atherosclerosis, a factor lowering the brain tolerance to radiation, any radiotherapeutic approach should be extremely cautious. 5) The amyloid-based fractionated radiation treatment we propose for Alzheimer patients is thought to induce the slow degradation of amyloid plaques through two DNA-independent mechanisms. This fact, together with the very slow degradation rate of irradiated amyloid, prompts an innovative whole-brain radiation treatment based on high energy radiation beams, long-term fractionation, low dose fractions (e.g. one 50-100 cGy fraction a week) and a very long overall treatment time (with pauses), under periodic control by means of available imaging techniques able to depict amyloid deposits in the brain (MRl and PET). 6) Given the pathological composition of Alzheimer’s foci, it is likely that radiation therapy is effective on amyloid plaques and congophile angiopathy, but less effective on neurofibrillar interlacing and not at all on cell degeneration. The hope of improving the patient ‘s quality of life will then depend on which functions are impaired by amyloid deposition. 7) The suggested treatment should be preceded by the application of the same therapeutic principle to other cases of non-brain localized amyloidosis, namely TBA cases, to compare the new results of long-term fractionated radiotherapy with the old results of 20 Gy/2 w. 8) Last but not least, as far as the use of nonionizing radiation in the treatment of nonbrain localized amyloidosis is concerned, the only case report to my knowledge on treatment with ELF-pulsed short waves (described in section 4) remains a strong stimulus for further research.
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References 1 Gertz MA: Amyloidosis: Recognition, prognosis, and conventional therapy. Hematology 1999: 339-347. 2 The Merk Manual, Sec. 2, Ch. 18, Amyloidosis. http://www. merck. com/pubs/manual/section2/chapter10/10a.htm. 3 Gertz MA, Lacy MQ, Dispenzieri A: Myeloablative chemotherapy with stem cell rescue for the treatment of primary systemic amyloidosis: a status report. Review. Bone Marrow Transplantation 25: 465-470, 2000. 4 Gertz MA, Lacy MQ, Lust JA et Al: Prospective randomized trial of melphalan and prednisone versus vincristine, carmustine, melphalan, cyclophosphamide, and prednisone in the treatment of primary systemic amyloidosis. J Clin Oncol 17: 262-267, 1999. 5 Blake CC, Serpell LC, Sunde M et Al: A molecular model of the amyloid fibril. Ciba Foundation Symposium, 199: 6-21, 40-45, 1996. 6 Dammacco F, Palumbo A: Amiloidosi e Beta-fibrillosi. In Dammacco F (ed.) “Immunologia in Medicina”. EDI ERMES 1: 181-210, 1989. 6b Bombardieri S: Malattie Reumatologiche, amiloidosi. In: Dammacco F, Paoletti R, Pozza G. “Argomenti di Clinica MedicaPratica”, Nuovo Roversi (Diagnostica e Terapia). Ariete Salute Editore 2: 255-260, 1999. 7 Kyle RA, Gertz MA, Greipp PR et Al: A trial of three regimens for primary amyloidosis: colchicine alone,melphalan and prednisone, and melphalan, prednisone and colchicine. New England J Med 336: 12021207, 1997. 8 Comenzo RL: High-dose therapy for the treatment of primary systemic amyloidosis. Hematology 1999: 347357. 9 Kyle RA, Gertz MA, Greipp PR et Al: Long-term survival (10 years or more) in 30 patients with primary amyloidosis. Blood 93: 1062-1066, 1999. 10 Comenzo RL, Vosburgh E, Falk RH et Al: Dose-intensive melphalan with blood stem-cell support for the treatment of AL (Amyloid Light Chain) Amyloidosis: survival and response in 25 patients. Blood 91: 36623670, 1998. 11 Gianni L, Bellotti V, Gianni AM et Al: New drug therapy of amyloidoses: Resorption of AL-type deposits with 4’-iodo-4’deoxydoxorubicin. Blood 86: 855, 1995. 12 Merlini GP, Anesi E, Garini P et Al: Treatment of AL Amyloidosis with 4’-iodo-4’-deoxydoxorubicin: An Update. Blood 93: 1112-1113, 1999. 13 Gillmore JD, Hawkins PN: Amyloidosis and the respiratory tract. Thorax 54: 444-451,1999. 14 Higuchi M, Iwata N, Matsuba Y et Al: 19F and 1H MRI detection of amyloid beta plaques in vivo. Nature Neuroscience 8: 527-533, 2005. 15 Giacobini E: Una spia per 1’Alzheimer. è possibile evidenziare gli accumuli della proteina che distrugge le cellule nervose. La Stampa, 18 settembre 2002. Rubrica tSt, tuttoScienzetecnologia. 16 Bresolin N: Malattie Neurologiche. In Dammacco F, Paoletti R, Pozza. G. “Argomenti di Clinica Medica Pratica”. Nuovo Roversi, Diagnostica e Terapia, Ariete Salute Editore 2: 1644, 1999. 17 Kurrus JA, Hayes JK, Hoidal JR et Al: Radiation therapy for tracheobronchial amyloidosis. Chest 114: 14891492, 1998. 18 Neben-Wittich MA, Foote RL, Kaira S: External beam radiation therapy for tracheo- bronchial amyloidosis. Chest 132: 262-267, 2007. 19 Monroe AT, Walia R, Zlotecki RA et Al: Tracheobronchial amyloidosis, a case report of successful treatment with external beam radiation therapy. Chest 125: 784789, 2004. 20 Kaira S, Utz JP, Edell ES et Al: External beam radia-
21 22 23 24 25 26 27
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tion therapy in the treatment of diffuse tracheobronchial amyloidosis. Mayo Clinic Proceedings 76: 853856, 2001. O’Regan A, Fenlon HM, Beamis JF jr et Al: Tracheobronchial amyloidosis. The Boston University Experience from1984 to1999. Medicine 79: 69-79, 2000. Biagini C: Manuale di Radiologia. EMSI, Roma 1980: 473. Biagini C: Radiobiologia e Radioprotezione. Piccin Nuova Libraria, Padova 1999: 292, 369. Bistolfi F: Radiation Oncology. Time-dose charts/ Clinical imaging/Therapeutic management. Edizioni Minerva Medica, Torino 1997: 80-83. Kisilevsky R: Anti-amyloid drugs: potential in the treatment of diseases associated with aging. Drugs & Aging 8: 75-83, 1996. Dragoni G: Elementi di Radiobiologia. CEA, Milano, 1961: 74. Curri SB. Campi ELF: Tessuto connettivo e microvascolarizzazione. In Bistolfi F (ed.) “Campi Magnetici in Medicina. Biologia DiagnosticaTerapia”. Edizioni Minerva Medica, Torino 1986: chapter 36. Mitchell JS: Effetti delle radiazioni sul metabolismo dei tessuti e dei tumori. In Zuppinger A (ed.) “Summa Radiologica, Radiobiologia”. Piccin Editore 2: 385-386, 1972. Miceli and Prodi: quoted by ref. 26. Alexander P: Alterazioni macromolecolari prodotte dalle radiazioni ionizzanti. In: Zuppinger A (ed.) “Summa Radiologica, Radiobiologia” Piccin Editore, Padova 2: 178-211, 1972. Oliveira CL, de la Hoz L, Silva JC et Al: Effect of gamma radiation on beta-lactoglobulin: oligomerization and aggregation. Biopolymers 85: 284-294, 2007. Richand P: Les Micro-Ondes Pulsées en Thérapeutique. Cahiers de l’Electrothérapie 6: 239-243, 1972. Richand P: Les Champs Hertziens en Biologie et en Médecine. Cahiers de Biothérapie 49: 45-49, 1976. Richand P, Boulnois JL: Action des ondee électromagnétiques en biologie et en médecine, des ondes hertziennes au laser. In Wolkowsky ZW (ed.) “Proceed Internat Symp on Wave Therapeutics”. Versailles 1979: 159-175. Gigante G: I Mezzi Fisici in Fisioterapia. © 1983 by AIFB, Roma. first Italian edition 1983 on the first English edition 1982. Original title: Physics in Physiotherapy. Conference Report 35. Editor The Hospital Physicist Association, London 1982. Brandrup J, Immergut EH: Polymer Handbook. Interscience Publishers, John Wiley & Sons, New York, London 1966: V-5. Sasahara K, Yagi IF, Waiki H et Al: Heat-triggered conversion of protofibrils into mature amyloid fibrils of beta 2 - microglobulin. Biochemistry 46: 3286-3293, 2007. Bistolfi F, Brunelli B: On electromagnetic acoustic transduction: A speculative review. Physica Medica 17: 37-66, 2001. Ohhashi Y, Kihara M, Naiki H et Al: Ultrasonicationinduced amyloid fibril formation of beta 2-microglobulin. J Biol Chemistry 280: 32843-8, 2005. Bistolfi F: La cronobiodose in radioterapia. Dose Tempo Volume. Piccin Editore, Padova 1967. Gutin: quoted by ref. 23. Original in Gutin PH et Al: Radiation Injury to the Nervous System. Raven Press, New York 1991. Cicciarello R, Russi E, Albiero F et Al: Metabolismo cerebrale e permeabilità della barriera ematoencefalica in un modello sperimentale di trattamento radioterapico cerebrale. Radiol Med 80: 709-712, l990.
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43 Cohen L, Creditor M: Iso-effect tables for tolerance of irradiated normal human tissues. Int J Rad Oncol Biol Phys 9: 233-241, 1983. 44 Herskovic AM, Orton CG: Elective brain irradiation for small cell anaplastic lung cancer. Int J Rad Oncol Biol Phys 12: 427-29, 1986. 45 Chak LY, Zatz LM, Wasserstein P et Al: Neurological
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dysfunction in patients treated for small cell carcinoma of the lung: a clinical and radiological study. Int J Rad Oncol Biol Phys 12: 385-389, 1986. Prof. F. Bistolfi Via G.B. Riboli, 6/5 16145 Genova. Italy
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Localized Amyloidosis and Alzheimer’s Disease: the Rationale for Weekly Long-Term Low Dose Amyloid-Based Fractionated Radiotherapy F. BISTOLFI Radiotherapy Department, Galliera Hospital; Genoa, Italy
Key words: amyloidosis, Alzheimer, Creutzfeldt-Jacob, fractionated radiotherapy, Diapulse, pulsed short waves
SUMMARY – Amyloidosis, a plasma cell dyschrasia, is characterized by accumulation in the intercellular spaces of fibrillar proteins with a typical beta-sheet pattern. Vascular-cerebral amyloidosis is the hallmark of Alzheimer’s disease and spongiform encephalopathy (Creutzfeldt-Jacob and the like). Current treatment of primary systemic amyloidosis is neither free from complications nor - in some presentations - a mortality rate. Localized tracheo-bronchial amyloidosis (TBA) has been successfully treated with high energy beams of radiation (20 Gy in 10×200 cGy in two weeks). The CT response to radiation takes several months after completion of treatment. As 20 Gy in two weeks are followed by inflammatory reactions, this dosage cannot be suggested in the treatment of amyloidotic radiosensitive organs (e.g. kidneys, liver), or in the hypothetical treatment of Alzheimer’s disease. On the basis of the following points: 1) plasma cells in amyloid deposits are not numerous; 2) plasma cells are radioresistant, both in vitro and in vivo (radiotherapy of solitary plasmocytoma); 3) the effects of radiotherapy (20 Gy/2 w) on TBA localizations cannot be exclusively due to plasma cell killing, this study postulates a biophysical mechanism of radiation-induced H-bond breaks in the beta-sheet structure of amyloid, together with depolymerization of glucosaminoglycans, very radiosensitive molecules invariably associated with amyloid fibrils. As both biophysical effects are DNAindependent, the adoption of a definite time/dose ratio (e.g. 20 Gy/2 w) loses much of its importance. Therefore an innovative alternative might be a weekly long-term low-dose fractionated radiotherapy, matching the very slow response of amyloid to radiation. Before being applied to Alzheimer’s disease, the proposed radiotherapy (RT) schedule should be tried in TBA patients to compare the new results of long-term fractionated RT with the old results of 20 Gy/2 w. Should long-term fractionated RT prove equally (or almost equally) effective, but certainly much less toxic than 20 Gy/2 w, its application to Alzheimer patients might become an effective and safe treatment, provided clinical and objective control by means of current imaging techniques (MRI, PET) can be assured.
Amyloidosis Amyloidosis 1,2,3,4,5,6,6b is accumulation in the tissues of various insoluble fibrillar proteins in an amount sufficient to impair normal functions. The classical clinical classification 6b recognizes five forms: primary amyloidosis, myeloma-associated amyloidosis, secondary amyloidosis, localized and pseudotumoral amyloidosis and familial-hereditary amyloidosis.
Primary systemic amyloidosis (AL, immunoglobulin light chain amyloid) results from the extracellular deposition of insoluble immunoglobulin light (AL) or heavy chains (AH), which disrupt organ function and ultimately lead to the death of the patient. The heart, lung, skin, tongue, thyroid gland and intestinal tract may be involved. Localized amyloid ‘tumors’ may be found in the tracheo-bronchial system or other sites. Hepatomegaly, nephrotic 683
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proteinuria and congestive cardiomyopathy are common, as well as painful peripheral neuropathy. Secondary amyloidosis (AA) is associated with chronic diseases (tuberculosis, bronchiectasis, osteomyelitis, leprosy, rheumatoid arthritis, granulomatous ileitis) and shows a predilection for the spleen, liver, kidney, adrenals and lymphnodes. Amyloid associated with multiple myeloma has the same distribution as primary systemic amyloidosis. Familial forms often exhibit distinctive types of neuropathy, nephropathy and cardiopathy. Other forms of amyloidosis are the hemodialysis-related amyloidosis and two brain diseases: Alzheimer’s disease and spongiform encephalopathies. The current classification of amyloidosis is based on the composition of the subunit fibril protein and recognizes the following associations 1,6 b,13: Primary systemic amyloidosis and Myeloma
monoclonal immunoglobulin light chains (AL) or heavy chains (AH)
Secondary amyloidosis
AA
serum amyloid A protein
Hemodialysis related amyloidosis
A β2 M
β2 -microglobulin
Familial and senile amyloidosis
ATTR
transthyretin
Alzheimer
A β PP
β-amyloid precursor protein
Spongiform encephalopathies
prion protein.
Under light microscopy, amyloid is a homogeneous highly refractile metachromatic substance with an affinity for Congo red. Under electron microscopy, amyloid consists of linear, nonbranching fibrils of about 5-15 nm×800 nm. Under X-ray diffraction, the heterogeneous fibril protein chains show a typical beta-sheet pattern, on which both the optical and tintorial properties of amyloid depend. Blake et Al 5, using high resolution electron microscopy and X-ray diffraction, concluded that all beta-sheets composing the protofilaments have a helical arrangement, and that the beta-sheet helix may be the generic core structure of amyloid. Therefore, all proteins in amyloid deposits share fibrillar structure and beta-sheet conformation: hence the comprehensive term of beta-fibrillosis to indicate all clinical forms of amyloidosis 6. Treatment of primary systemic amyloidosis is directed first to the blood marrow plasma 684
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cell dyschrasia. Current treatment 2,3,4,7,8,9,10,11,12 includes melphalan/prednisone or melphalan/ prednisone/colchicine 2,7, sometimes with the addition of other alkylating agents. However, therapy with multiple alkylating agents did not result in a higher response rate or longer survival time 4. Since 1995 autologous stem cell transplantation has been incorporated in the high-dose treatment of primary systemic amyloidosis. The response rates are substantially better than those for patients treated with lowdose traditional melphalan and prednisone 3,8,10, but the related mortality is very high, reaching 44% in some series. As a consequence, patients eligible to receive stem cell transplantation must represent a highly selected population 3. In cases of severe single organ involvement, organ transplantation such as heart, kidney or liver transplantation may have a successful outcome 2. An innovative treatment, aimed at directly reducing the amyloid deposits has been proposed by Merlini et Al 11,12 using I-DOX (4’-iodo4’-deoxydoxorubicin). This will be discussed below in view of its conceptual links with the proposed amyloid-based radiation treatment. Independently on the clinical response in terms of survival time and quality of life 2,3,4,7,8,9,10, what deserves attention is the fact that amyloid deposition is not an irreversible process inasmuch as amyloid deposits can be substantially reduced by therapy. Therefore, any new method of treating localized amyloid involvement of single organs must be welcome. Alzheimer’s disease and CreutzfeldtJacob’s encephalopathy as brain localized amyloidosis Amyloid-related diseases now include Alzheimer’s disease and transmissible spongiform encephalopathies 13. Alzheimer’s disease, now considered the most common form of amyloidosis, is due to vascular-cerebral amyloidosis and shows a series of pathological components: neuritic plaques, neurofibrillar interlacing, cell degeneration and congophilic angiopathy 6. Formation of senile plaques composed of amyloid β protein is a pathological hallmark of Alzheimer’s disease in human brain and precedes the onset of symptoms by many years. According to Higuchi et Al 14, noninvasive detection of such plaques could afford a presymptomatic diagnosis by MRI with the intravenous administration of a 19F containing amyloido-
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philic compound specifically labeling amyloid beta plaques in the brain. When injected in the vascular system, another β-amyloidophilic compound, the anylin-derived phenyl-hydrobenzothiazole (PHB), crosses the brain barrier and stains the amyloid senile plaques. By labeling PHB with 14C, the plaques may be demonstrated in the brains of living Alzheimer patients using positron emission tomography (PET) 15. A joint clinical study involving Swedish and USA institutions showed very impressive PET images in the brains of nine Alzheimer patients in comparison with five normal subjects. 14C PHB-PET will certainly become a useful method to control the objective effects of therapy in Alzheimer patients 15. It has also been claimed that some infective agents named prions (protein infective particles) can give rise to some forms of amyloidosis associated with Creutzfeldt-Jacob’s disease, a severe spongiform encephalopathy characterized by the presence of amyloid plaques in the brain and cerebellum. Prions are neither viruses nor bacteria but protein molecules able to polymerize in rod-like filaments, whose crystallographic presentation at the X-ray diffraction is that typical of beta-sheets forming amyloid fibrils. While Creutzfeldt-Jacob’s encephalopathy affects 50-70-year-old subjects, the atypical new variant (bovine spongiform encephalitis, BSE) commonly known as “mad cow disease” affects younger patients with atypical signs and is thought to be caused by the consumption of prion-infected beef 6,16. Localized amyloidosis in the tracheobronchial system: and external beam radiation therapy Tracheo-bronchial amyloidosis (TBA) is characterized by deposition of amyloid in the trachea and bronchi as submucosal plaques and/or pseudotumoral masses 17. In both cases various degrees of lumen narrowing ensue. The condition is not common and the severity of the clinical course depends on its respiratory and infective complications. Over the years, multimodal therapeutic approaches have been adopted, including bronchoscopic methods (laser resection, balloon dilatation, temporary placement of a silicon stent). Since the publication by Kurrus et Al in 1998 17 on an effective high energy radiation treatment in one 67-year-old patient, a few other papers appeared in the following
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ten years, confirming the usefulness of external beam radiation therapy (EBRT) in nine adult patients with TBA 18,19,20,21. CT scans are essential to show the extension of lesions prior to treatment planning and during follow-up. Amyloid deposits may be displayed as superficial plaques, submucosal infiltration at the level of the trachea, carina, mainstem bronchi, or bulky masses narrowing the bronchial lumen. Amyloid-induced collapse of one single lobe may also occur. The irradiation technique relies upon 4 MV, 6 MV, 10 MV x-ray beams through two parallel AP-PA opposing fields, sometimes 18 with an added right and left lateral field arrangement. Customized field techniques aim at limiting the dose to lung parenchyma. The given dose is a standard time/dose ratio of 20 Gy in ten daily 200 cGy fractions in two weeks (20 Gy/2 w.). In one case report 19 the dose was 24 Gy in 12 daily 200 cGy fractions and in another case report 17 the same schedule (20 Gy/2 w) was applied six months later because of a distal extension of the primary lesion treated with the same dose. Good symptomatic and objective results are described: the median time to a subjective response was four months (range 1-7 months) in Neben-Wittich’s series of seven patients 18. However, the objective results at bronchoscopy and CT scans are much slower, taking several months after completion of treatment (range 10-16 months) 17,20. One case report 19 found a resolution of objective signs starting one month after treatment and continuously progressing over the subsequent 16 months. Side effects are mainly grade 1 and 2 oesophagitis, but grade 2 pneumonitis was also observed 18. The median follow-up in Neben-Wittich’s series was 40 months (range 10-69 months). Follow-up in other case-reports reached 18 months 17,19 and 21 months 20. As EBRT can provide symptomatic as well as objective improvement, it becomes a treatment option for localized tracheo-bronchial amyloidosis causing airway obstruction. The mechanism of treatment: does EBRT affect only plasma cells?
The mechanism by which EBRT can reduce TBA deposits is unclear 18. What has been established is that TBA, like AL amyloidosis, arises from immunoglobulins 21. Therefore, it has been postulated that amyloid deposits are the result of light chain deposition by local plasma cells 18. According to O’Reagan et Al 21. “as plasma cells are radiosensitive” (yet, see further) low-dose radiation may offer an alternative approach to 685
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the pharmacological treatment of TBA. Monroe et Al 19 also based their treatment plan and dose (24 Gy) on the association of monoclonal plasma cell Ig light chain deposits in both multiple myeloma and amyloidosis, presuming that the successful low-dose radiation treatment in both cases depends on the inactivation of monoclonal plasma cells. However, available pathological data do not show large numbers or large clusters of plasma cells in areas of amyloidosis 17,18,20 , which somewhat weakens the hypothesis of TBA improvement exclusively through plasma cell injury leading to decreased amyloidogenic protein production and deposition 20. In addition, the radiosensitivity of B and T lymphocytes changes with their functional state within a wide dose range (6-10 Gy to 50-150 Gy) 22 . Plasma cells in particular, the ultimate evolution step of B lymphocytes, are - according to Biagini 23 – “very radioresistant” and, at least in vitro, are not injured with doses as high as 100 Gy. These experimental data are indirectly confirmed by clinical radiobiology of solitary plasmocytoma. Indeed, from a literature survey on radiation therapy of solitary plasmocytoma up to 1997 24 it emerges that a stable local control of both osseous and extraosseous lesions needs doses as high as 35-40 Gy to 50-70 Gy in five to eight weeks. Therefore, without excluding the direct action on the scanty plasma cells in amyloid areas, it seems unlikely that 20 Gy in two weeks can reduce amyloid deposits uniquely through a plasma cell mechanism, so that other potential mechanisms have been proposed: 1) A radiation effect on the endothelium of local vasculature 17,18,20 with a reduced influx of plasma cells or delivery of distantly produced amyloid precursors; 2) A radiation effect on cells or cofactors, other than plasma cells, leading to changes in local amyloid production or deposition 17,18,20; 3) The induction of an immune response against the deposits activated by local inflammation 18; 4) A free radical mediated radiation effect, modifying and enhancing the degradation of amyloid 17. Radiobiological rationale for an amyloidbased radiation treatment of localized amyloidosis An amyloid-based mechanism, that is a physico-chemical process of amyloid degradation followed by slow reabsorption, can be added to these four mechanisms. 686
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Treatment with ionizing radiation
The universal presence in amyloid of certain glucosaminoglycans (GAGs) such as heparan sulphate and dermatan sulphate, invariably associated with amyloid fibrils 13, has been implicated in the pathogenesis of amyloidosis. Although GAGs are not required for amyloid fibrillogenesis per se, they remain – according to Kisilevsky 25 – a valid therapeutic target. GAGs, highly polymerized polysaccharides, exhibit metachromasia when treated with toluidin blue (just like amyloid) 26. Inflammatory processes induce several changes in their structure leading to GAG depolymerization 27. Moreover, low radiation doses (20 to 100 cGy) depolymerize mucopolysaccarides in connective tissue 28. For this reason, metachromasia decreases and diffusion of liquid injected into the derma increases, as occurs under the enzymatic action of hyaluronidasis 29. From a basic chapter by Alexander in “Summa Radiologica” 30 we learn that irradiated macromolecules undergo scission of the primary chain, intermolecular and intramolecular crosslinking, breaks in hydrogen bonds (H-bonds), and that the action exerted by radiation on protein molecules chiefly consists of breaks in intramolecular Hbonds, with ensuing partial or total molecule unwinding. Relevant to the present research is the action of radiation upon GAGs, associated with amyloid fibrils 13, inasmuch as their depolymerization induced by low radiation doses might indirectly affect the amyloid fibrils. Moreover, the amyloid structure itself might undergo depolymerization because of radiation-induced breaks in intramolecular H-bonds, whose paramount importance in keeping the orderly structure of the fibril-forming beta-sheets is known. Indeed, figure 1 shows the binding sites of H-bonds between parallel beta-sheets inside a protein. Figure 2 schematically shows the mechanism for amyloid fibril formation through the self-association of protein protofilaments along a template of parallel beta-sheets, kept together by H-bonds (not depicted). All this prompts our proposal of an amyloidbased radiation therapy, aiming at depolymerizing amyloid fibrils to allow their subsequent reabsorption. A therapeutic mechanism supported by the knowledge that some degree of amyloid reabsorption has been shown both experimentally and clinically, notwithstanding amyloid fibrils do not undergo phagocytosis or enzymatic degradation 6. In addition, amyloid-based radiation therapy agrees with Dam-
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Figure 1 Beta-sheet structure in a globular protein. The planes forming molecular chains are kept together by H-bonds. From B. Alberts et Al (eds.) “Biologia Molecolare della Cellula”. Italian translation by M. Guardo and A. Poyrot. Zanichelli Editore, Bologna, 1984.
macco’s suggestion of studying procedures able to correct the mobilization and elimination of amyloid fibrils 6, as well as with Merlini et Al’s research 11,12 on the use of I-DOX to affect amyloid deposits directly. The postulated molecule degradation through the break in intra-molecular H-bonds is only one of the possible radiation effects on amyloid: should intramolecular crosslinking also occur, it would induce molecule aggregation 31. However, as intramolecular crosslinking reduces the volume of irradiated molecules 30, crosslinking in irradiated amyloid might not be an adverse effect, inasmuch as compression on surrounding tissues would decrease. It follows that even if both effects (H-bond breaks and intramolecular crosslinking) were concurrent, they would not be contrasting factors: crosslinking favouring the volume decrease of amyloid deposits, H-bond breaks favouring amyloid degradation and subsequent reabsorption. Treatment with non-ionizing radiation
In the 1970s Pierre Richand published some papers 32,33,34 on the clinical effects induced by ELF-pulsed 27 MHz short waves (Diapulse). The advantage of the lack of any thermic effect owing to the interpulse relaxation time (ON/ OFF ratio = 1/25) was stressed, together with
Figure 2 Proposed mechanism for amyloid fibril formation (see text). From J.D. Gillmore and P.N. Hawkins: Amyloidosis and the respiratory tract. Thorax 54: 444-451, 1999. Partially modified.
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the beneficial effect on tissue trophism. What is striking in Richand’s work is the claim of having obtained therapeutic outcomes “in several patients severely affected by hepatic and renal amyloidosis”. The case report of a 55-year-old patient, whose first symptoms appeared in February 1971 merits attention. A diagnosis of hepatorenal amyloidosis and its hopeless prognosis were established at the University Hospital Center in Créteil (France). From Richand and Boulnois’ description we abstract the following 34 : “... in 1953 the patient got a viral hepatitis in the Sahara region, where he was active as a mineral engineer. A long-lasting dental infection affected him during the second world war [from which we can infer the secondary nature of his hepato-renal amyloidosis, FB]. On September 6, 1971 the patient was intensely tired and hardly kept his balance in walking; he complained of pain in both knees and of cramps at night; he was heavily oppressed by windy weather; urine smell was ammoniacal; the liver volume was very increased (8 cm beyond the rib edge). A Diapulse treatment was then instituted (600×6×15’), three days a week, upon the upper abdomen. On October 11 (after four weeks’ treatment) the liver border extended beyond the rib edge no more than 1 cm. On November 3, the liver was no longer palpable. On January 31, 1972 (one month after completion of a four month treatment), hepatic conditions stabilized. The patient’s general status has much improved. Neither bone pains nor cramps are present, arterial pressure is normal. The biological hepatic tests have improved and immunoglobulin values have reached their normal levels. The patient is alive and well in 1978, seven years after treatment” 34. Though difficult to explain, the case report described is undoubtedly exciting, owing to the seven year duration of objective and subjective response to a simple and well tolerated physical treatment. The Diapulse machine generates electromagnetic energy in the 27 MHz band (short waves) with 65 μs pulses separated by 1,600 μs pauses. The 1/25 ON/OFF ratio allows complete dispersion of produced heat between two successive pulses. Clinical research has shown important results, such as quicker induction of healing processes, reduction of connective tissue production, easier and quicker reabsorption of edema, and regeneration of peripheral nerves 35. Diapulse irradiation is considered athermic, in agreement with the fact that heat more 688
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readily induces protein aggregation than degradation. Moreover, we know from physics that to obtain the thermic decomposition of some biological and non-biological polymers (e.g. cellulose, polystyrene, polyvinylchloride) temperatures in the order of 250-400°C are needed 36. Sasahara et Al 37 recently showed that beta-2microglobulin, a protein responsible for dialysis-related amyloidosis, when heated in a cell of a differential scanning calorimeter between 20°C and 100°C, undergoes a sigmoidal transition, representing the conversion of protofibrils into mature amyloid fibrils. It follows that the therapeutic result obtained with low frequency pulsed (600 cps) short waves has to be attributed to non-thermic effects. Among these, a good candidate is the electromagnetic acoustic transduction effect (EMAT), an effect well demonstrated along the electromagnetic spectrum 38 , particularly active when electromagnetic pulse duration falls within the microsecond range. The amyloid fibril degradation factor would then be low frequency mechanical vibration induced by ELF pulsed short waves. This is a sound interpretation, though in competition with the aggregation of beta-2-microglobulin microfibrils into mature amyloid fibrils obtained by Ohhashi et Al 39 through high-frequency mechanical vibration induced by ultrasound. Another physical factor to consider is the amyloid high refraction index 2, due to its highly ordered structure and high εr value, favouring the selective absorption of electromagnetic energy in amyloid deposits. Giving the athermic character of Diapulse emission, it is likely that selective effects in amyloid will be of athermic nature as well, namely of vibrational character. Discussion Is weekly long-term low dose amyloid-based fractionated radiation suitable to treat Alzheimer‘s disease and Creutzfeldt-Jacob‘s encephalopathy?
Degradation of amyloid deposits by chemical or physical means to eliminate their compressive effects is only one, albeit important, component of amyloidosis treatment. On chemical grounds, the innovative approach of Merlini et Al 11,12 with 4’-iodo-4’-deoxydoxorubicin (IDOX) must be emphasized. They observed that weekly fractionated I-DOX can interact directly with amyloid deposits by reducing amyloid load
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without affecting the level of circulating light chains. They used echography, CT scans, MR imaging or scintigraphy with 131I-labeled serum amyloid P component (SAP) to assess the evidence of reduced amyloid load in 14 patients treated in about three years (1995-1997). On physical grounds, high energy radiation beams have been successfully used to reduce localized amyloid deposits in tracheo-bronchial amyloidosis (TBA) with a standard dose of 20 Gy in ten 200 cGy fractions in two weeks (20 Gy/2 w) 17,18,19,20 , a time/dose ratio that will be the central point of discussion. In Neben-Wittich et Al’s series of seven TBA patients 18, the time/dose of 20 Gy/2 w did not appear free from side effects: oesophagitis in five out of seven patients (four grade 1 and one grade 2) and grade 2 pneumonitis in one out of seven patients, which is neither surprising nor difficult to explain. Indeed, according to the dosage system I devised in the 1960s 40 and further developed in the 1990s 24, a tissue dose of 20 Gy in two weeks can induce acutesubacute reactions, characterized by microvascular damage and inflammation (vessel dilatation, increased vessel permeability, interstitial edema), namely: brisk cutaneous erythema followed by dry desquamation (which means oesophagitis at the level of oesophageal mucosa); subacute radiation pneumonitis followed by late lung fibrosis (if large volumes of lung are involved); radiation nephritis (when both kidneys fall in the treated volume). Knowing that radiation can change amyloid protein structure and depolymerize the associated GAG molecules (both of which are DNAindependent mechanisms), the adoption of a definite time/dose ratio loses much of its importance. Therefore to apply radiation to treat amyloid-involved radiosensitive organs (e.g. lungs, kidneys, liver), and, as a last resort, Alzheimer’s disease, the alternative to the 20 Gy/2 w schedule might be a weekly fractionated regimen with very low dose fractions (say 50 to 100 cGy, once a week) and a very long overall treatment time to match the very slow involution rate of irradiated amyloid: hence, a much safer radiotherapeutic approach than those able to induce vascular damage and inflammatory processes. However, the overall dose necessary to induce amyloid degradation with the long-term fractionation regimen cannot as yet be established. Awaiting preliminary clinical trials in TBA patients, we might only guess at an overall dose between 20 and 30 Gy in an
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overall treatment time of about one year (e.g. 50 cGy × 40 - 60 w or 100 cGy × 20-30 w). As far as the oncogenic risk of whole-brain weekly irradiation with 50 (100) cGy is concerned, any assessment is hampered by the atypical fractionation proposed. However, the ten to twenty year latency of the “hypothetical” oncogenic effect will be counter-balanced by the probability of actually improving the miserable life conditions of Alzheimer patients. The treatment proposed, obviously prudenceguided but not lacking a sound rationale, should be first applied to patients with TBA to compare the long-term fractionation results with the standard 20 Gy/2 w results. Should the slowly fractionated dose be equally effective in inducing amyloid degradation without any side effects, the next step would be the low-dose fractionation treatment of Alzheimer’s disease and Creutzfeldt-Jacob’s encephalopathy. As a consequence, a question arises: what is known about brain tolerance to radiation? The brain’s tolerance to ionizing radiation has been analyzed in radiotherapy of human brain tumors, as well as in laboratory animals. Research has privileged the risk assessment of severe radiation encephalopathy and proven the relevance of parameters such as overall dose, overall treatment time, number and magnitude of dose-fractions. Moreover, we know that the tolerance dose definitely decreases as the irradiated brain volume increases from a few cubic centimeters up to the whole brain 24. Radiation treatment of primate brain with conditions similar to those of clinical radiotherapy (2.0 Gy per fraction, five fractions a week, four to eight weeks overall treatment time) with a cumulative dose of 40 Gy in four weeks is followed by negligible damage, whereas a dose of 60 Gy in six weeks is followed by noticeable damage, and a dose of 80 Gy in eight weeks, by severe damage 41. Limitedly to the dose of 40 Gy in four weeks, these results are in good agreement with Cicciarello et Al 42, who irradiated the whole brain of albino rats using 6 MV X-rays, suitable bolus and 20 x 200 cGy (40 Gy) in four weeks. Three weeks after irradiation, signs of low-degree damage were observed, namely: changes in brain metabolism (14C-2DG) and permeability changes in the blood-brain barrier (transcapillary passage of 14 C-alfa aminoisobutyric acid), leading them to conclude that in whole-brain irradiation with daily fractionation time/dose values even lower than those linked to severe bioeffects can determine subclinical, mainly vascular, bioeffects. 689
Localized Amyloidosis and Alzheimer’s Disease: the Rationale for Weekly Long-Term Low Dose Amyloid-Based ...
Cohen and Creditor 43, in their basic research on severe radiation encephalopathy (radionecrosis, dementia, severe mental retardation in long-term survivors), started from a wealth of clinical and cell kinetic data to arrive at an isoeffect time/dose table for a 5% risk with various dose fractionations. Subsequently, I drew four curves relative to four fractionations (five, three, two and one fraction a week) and overall treatment time of two to ten weeks 24. For comparison with the schedule of TBA radiotherapy (10 x 200 cGy in two weeks), Table 1 shows how the 5% risk of severe brain complications is related to the overall dose and fractionation type within the overall treatment time of two weeks (table 1) Table 1 Five per cent risk of severe brain complications for an overall treatment time of two weeks with four different fractionations (based on Cohen and Creditor’s data) 24,43
33 Gy/2 weeks
with 10 fractions of
330 cGy
25 Gy/2 weeks
w"th 16 frac"ions of
417 cGy
20 Gy/2 weeks
w"th 14 frac"ions of
500 cGy
14.5 Gy/2 weeks
w"th 12 frac"ions of
725 cGy
Last but not least, let us recall that the tolerance limit of irradiated brain is definitely lowered by concomitant chemotherapy, as shown by two papers reporting diffuse, progressive encephalopathy in 10.2% 45 and 45% 44 of patients treated with whole brain radiotherapy (dose fractions 240, 300, 350 cGy) and polychemotherapy for small cell carcinoma of the lung. We conclude that the proposed weekly longterm low dose amyloid-based fractionated radiotherapy of brain amyloidosis may be considered a safe procedure as far as the brain tolerance is concerned, provided no concomitant chemotherapy is implemented. However, as far as its effectiveness on amyloid plaques is concerned, only preliminary trials in TBA cases will be able to afford a definite judgment on its clinical usefulness. Conclusions 1) The dosage schedule 20 Gy/2 w. (10 × 200 cGy in two weeks), whose efficacy in radiotherapy of tracheo-bronchial amyloidosis (TBA) has been proven, represents a time/dose ratio not free from acute side effects, though less toxic than the two week time/dose ratios linked to
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5% of severe radiation encephalopathy reported in table 1. 2) As time/dose values even lower than those linked to 5% of severe complications can induce subclinical, mainly vascular bioeffects 42 in the brain, the 20 Gy/2 w regimen should be avoided in radiation therapy of Alzheimer’s disease. 3) The tolerance limit of irradiated brain is definitely lowered by concomitant chemotherapy 44,45. As current treatment of amyloidosis is largely based on cytotoxic chemotherapy, therefore, radiation therapy of brain amyloidosis should carefully avoid any implementation with chemotherapy. 4) As Alzheimer patients may also be affected by brain atherosclerosis, a factor lowering the brain tolerance to radiation, any radiotherapeutic approach should be extremely cautious. 5) The amyloid-based fractionated radiation treatment we propose for Alzheimer patients is thought to induce the slow degradation of amyloid plaques through two DNA-independent mechanisms. This fact, together with the very slow degradation rate of irradiated amyloid, prompts an innovative whole-brain radiation treatment based on high energy radiation beams, long-term fractionation, low dose fractions (e.g. one 50-100 cGy fraction a week) and a very long overall treatment time (with pauses), under periodic control by means of available imaging techniques able to depict amyloid deposits in the brain (MRl and PET). 6) Given the pathological composition of Alzheimer’s foci, it is likely that radiation therapy is effective on amyloid plaques and congophile angiopathy, but less effective on neurofibrillar interlacing and not at all on cell degeneration. The hope of improving the patient ‘s quality of life will then depend on which functions are impaired by amyloid deposition. 7) The suggested treatment should be preceded by the application of the same therapeutic principle to other cases of non-brain localized amyloidosis, namely TBA cases, to compare the new results of long-term fractionated radiotherapy with the old results of 20 Gy/2 w. 8) Last but not least, as far as the use of nonionizing radiation in the treatment of nonbrain localized amyloidosis is concerned, the only case report to my knowledge on treatment with ELF-pulsed short waves (described in section 4) remains a strong stimulus for further research.
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