Modified High-Dose Melphalan and Autologous Stem Cell Transplantation for Immunoglobulin Light Chain Amyloidosis

High-dose melphalan and autologous stem cell transplantation (HDM/SCT) have been used in patients with immunoglobulin light chain (AL) amyloidosis for over 2 decades now with durable responses, prolonged survival, and decreasing treatment-related mortality. Historically, patients with poorer baseline functional status, advanced age, renal compromise, and cardiac involvement have been treated with a risk-adapted modified conditioning dose of melphalan (mHDM) of 100 to 140 mg/m² before SCT. In part because of these baseline characteristics, patients receiving mHDM/SCT have had poorer outcomes compared with patients receiving full-dose melphalan at 200 mg/m². With the advent of novel therapeutic agents such as proteasome inhibitors, immunomodulatory agents, and monoclonal antibodies for the treatment of AL amyloidosis, it is imperative to understand the long-term effects of mHDM/SCT. Here we report the long-term outcomes of 334 patients with AL amyloidosis treated with mHDM/SCT. Median overall survival was 6.1 years and median event-free survival 4.3 years, with median overall survival reaching 13.4 years for patients who had achieved a hematologic complete response (CR). Overall hematologic response rate was 69%, and treatment-related mortality was 3% after 2010. Thus, mHDM/SCT leads to prolonged survival and favorable outcomes, especially if a hematologic CR is achieved.     

Therapy-induced antitumor vaccination by targeting tumor necrosis factor alpha to tumor vessels in combination with melphalan

Treatment of tumor-bearing mice with mouse (m)TNF-alpha, targeted to tumor vasculature by the anti-ED-B fibronectin domain antibody L19(scFv) and combined with melphalan, induces a therapeutic immune response. Upon treatment, a highly efficient priming of CD4+ T cells and consequent activation and maturation of CD8+ CTL effectors is generated, as demonstrated by in vivo depletion and adoptive cell transfer experiments. Immunohistochemical analysis of the tumor tissue demonstrated massive infiltration of CD4+ and CD8+ T cells 6 days after treatment and much earlier in the anamnestic response to tumor challenge in cured mice. In fact, the curative treatment with L19mTNF-alpha and melphalan resulted in long-lasting antitumor immune memory, accompanied by a mixed Th1/Th2-type response and significant in vitro tumor-specific cytolytic activity. Finally, the combined treatment reduced the percentage and absolute number of CD4+CD25+ regulatory T cells in the tumor-draining lymph nodes of mice responding to therapy, and this was associated with the establishment of protective immunity. These findings pave the way for alternative therapeutic strategies based on the targeted delivery of biological and pharmacological cytotoxic compounds that not only kill most of the tumor cells but, more importantly, trigger an effective and long-lasting antitumor adaptive immune response.     

高效液相色谱法测定美法仑中的有关物质

目的：建立反相高效液相色谱法测定美法仑中的有关物质。方法：采用Diamonsil ODS（4．6mm×150mm,5μm）色谱柱,流动相为0．375％碳酸铵的水溶液：乙腈：冰醋酸（100：35：1．0）,流速为1．0mL／min,检测波长为260nm。结果：美法仑主要成分峰与其有关物质完全分离,在0．0000026～0．002mg／mL范围内线性关系良好,检测限（S／N≥3）为0．8ng／mL。结论：此方法准确、可靠,可用于关法仑有关物质的考察。     

Activity of Hydrolytic Enzymes in Tumour Cells is a Determinant for Anti-tumour Efficacy of the Melphalan Containing Prodrug J1

Recently, we presented a series of melphalan containing di- and tripeptides with high cytotoxic activity and J1 (_l-melphalanyl-p-_l-fluorophenylalanine ethyl ester) was identified as one of the most interesting compounds. It was speculated that the increased activity compared to melphalan itself, demonstrated both in vitro and in vivo, resided in increased transport over the tumour cell membrane and/or hydrolytic cleavage and liberation of melphalan inside the cells. Indeed, overexpression of hydrolytic enzymes like peptidases, esterases and proteases has been described in several types of human malignancies, thus providing a target for selective chemotherapy.

In this work, the details of the increased activity was further investigated and potential tumour selectivity is discussed. The intracellular delivery of melphalan is investigated in detail using peptidase resistant dipeptide derivatives, by enzyme inhibitors and probes for enzymatic activity and by studying the time dependency of drug effect as well as intracellular drug concentrations (cellular pharmacokinetics).

The results show that the activity of the dipeptide mustards is highly dependent on intracellular hydrolysis, which result in rapid intracellular release of the alkylating unit (i.e. free melphalan) in cells with high enzymatic activity. The maximum intracellular melphalan concentration following J1 exposure was reached already after 15 min, thereafter declining with a half-life of approximately 1 h. This rapid intracellular loading resulted in less reduction of activity for J1 than for melphalan and six other standard drugs when human tumour cell lines were exposed to the drugs for a limited time (simulating short half-life in vivo). Peptidase inhibitors inhibited the activity and intracellular release of melphalan, and dipeptide derivatives designed to resist the action of peptidases was less active than the corresponding normal dipeptide.     

Modification of human tumour and normal tissue pH during hyperthermic and normothermic antiblastic regional isolation perfusion for malignant melanoma: a pilot study

Human tumour and normal tissue pH were investigated during hyperthermic and normothermic antiblastic regional isolation perfusion and the effects of vascular occlusion, artificially induced hypoxia, hyperglycaemia, haemoglobin level of the perfusate and hyperthermia on tumour and normal tissue pH were evaluated. A pilot study was performed on 10 patients, with locally inoperable recurrent and primary malignant melanoma of the leg. The treatment consisted of a regional isolation perfusion with hyperthermia (120 min at 42–43°C), at femoral level, followed by a normothermic regional isolation perfusion with Melphalan (60 min at 37–38°C), at iliacal level, 7–10 days later. In general, several phases during a regional isolation perfusion can be distinguished: (1) first ischaemic anoxia period; (2) extracorporeal circulation, during which the leg is heated to the desired temperature, after which either hyperthermia or Melphalan is applied; (3) second ischaemic anoxia period. During the anoxia periods the large vessels that supply the leg are temporarily clamped and the effects on tissue pH can be investigated. During extracorporeal circulation, high-dose glucose can be administered to the isolated leg, to acutely decrease tumour tissue pH. Such a decrease is expected to sensitize tumours to hyperthermia, when applied immediately prior to or during heating. At the beginning of the treatment the mean tumour pH was significantly lower than normal tissue pH (7.14, with a mean tumour volume of 39±2 cm³, and 7±38, respectively; p < 0±01). During the perfusions with hyperthermia and Melphalan, tissue pH decreased by -0±41 units and -0±20 for tumour, and -0±11 units for normal tissue, respectively (all statistically significant). The two anoxia periods accounted for approximately half of the net decrease. During these periods tumour pH appeared to decrease more selectively, although there was great variation. The other investigated modalities, such as hyperglycaemia and hyperthermia, also decreased tissue pH, but to a lesser extent. However, a combination of more than one modality caused a larger decrease than a single one, but no preference for tumour could be detected. Before the second perfusion mean tumour pH was significantly increased by 0±14 units, and was no longer significantly different from normal tissue pH in the course of the regional isolation perfusion. This could be the reflection of the reduced tumour volume (by 30%, n.s.). Similar pH changes occurred during this Melphalan perfusion, but they were less pronounced since the total treatment time was snorter. Summarizing, tumour pH can be decreased more than normal tissue pH in the course of the regional isolation perfusion. In particular, vascular occlusion appeared to be tumour pH selective. The observed tumour regression between the first, hyperthermic, and the second perfusion strengthens the hypothesis that acutely lowered tumour pH sensitizes tumour to hyperthermia.     

沙利度胺联合美法仑及VAD方案治疗多发性骨髓瘤24例

 目的　评估沙利度胺联合美法仑及VAD方案治疗多发性骨髓瘤（MM）的疗效及不良反应。方法　治疗组共24例患者,均给予沙利度胺联合美法仑及VAD方案化疗,治疗2个疗程观察疗效。观察项目包括血清M蛋白、肝肾功能、尿蛋白、骨髓象、血象等,记录患者不良反应。疗效标准分为完全缓解（CR）、部分缓解（PR）、未缓解（NR）。结果　治疗组24例患者中CR 13例,PR 6例,NR 5例,有效率79.17 %。结论　沙利度胺联合美法仑及VAD方案治疗MM的临床疗效显著,症状改善明显。     

Unresectable isolated hepatic metastases from solid pseudopapillary neoplasm of the pancreas: A case report of chemosaturation with high-dose melphalan

Background/objectivesSolid pseudopapillary neoplasms of the pancreas (SPN) are rare tumors. For patients with unresectable liver metastases of SPN, no standard treatment has been defined so far. Here we report a case of a 40-year-old woman with SPN and metastases confirmed to the liver, and disease progression in the liver after primary tumor resection and chemotherapy with gemcitabine and cisplatin.MethodsChemosaturation with percutaneous hepatic perfusions is a minimally invasive, repeatable regional therapy which delivers chemotherapy directly to the liver while limiting systemic toxicity. As an individual treatment approach, the patient was treated with chemosaturation with percutaneous hepatic perfusions of melphalan.ResultsThe procedure was performed twice within 8 weeks after which the liver metastases showed a marked reduction in size and vascularization (partial response). Grade 3 leukopenia after the second procedure was managed effectively with granulocyte colony-stimulating factor. No other toxicities were observed. Ten months after initiating treatment, the patient had a good performance status and remained stable.ConclusionsFor SPN with unresectable liver metastases and progression despite systemic treatment, repeat chemosaturation with high-dose melphalan may also offer an effective regional treatment option.     

Mechanism of melphalan crosslink enhancement by misonidazole pretreatment

Sensitization of Chinese hamster ovary cells to melphalan (L-PAM) toxicity by prior treatment with misonidazole (MISO, 5 mM, 2 hr, hypoxic conditions, 37 degrees C) is associated with increased levels of DNA crosslinks believed to be the critical lesion for bifunctional alkylating agent toxicity. Enhanced L-PAM crosslinking of DNA could occur by a variety of mechanisms in MISO-pretreated cells including: (1) increased transport or binding of L-PAM, (2) decreased repair of L-PAM monoadducts which would allow more time for their conversion to crosslinks, (3) decreased crosslink repair (unhooking of one arm), or (4) chemical modification of the DNA structure, presumably by bound MISO derivatives, such that crosslink formation is facilitated. Previous studies have eliminated mechanisms (1) and (3). Mechanism (4) was investigated by following MISO-pretreatments of whole cells with L-PAM treatments of the isolated DNA from these cells. This was accomplished by using a modification of the alkaline elution assay for DNA crosslink measurement in which a 1 hr treatment with L-PAM (0-12 micrograms/ml) was inserted between the cell lysis steps and DNA elution procedure. Treatment of bare DNA with L-PAM modeled very well the crosslinking behavior in whole cells although it was somewhat more efficient (more crosslinks at a given L-PAM dose). In the presence of double stranded DNA and absence of repair systems during and after the L-PAM exposure, it was determined that MISO-pretreatments did not increase the crosslinking efficiency of L-PAM (mechanism [4] above). Inhibition of repair of L-PAM monoadducts (mechanism [2] above) still remains as a possible means for crosslink enhancement by MISO-pretreatment.     

Modification of alkylating agent cytotoxicity by cisplatin

The effect of cisplatin on the cytotoxicity of alkylating agents in the RIF-1 tumor both in vivo and in vitro was investigated. A single dose of cisplatin (1 mg/kg) enhanced the in vivo tumor killing by melphalan (L-PAM; 8 mg/kg). The effect was maximal when cisplatin was given between 3 hours before and 1 hour after injecting L-PAM; the enhancement was lost as the time interval increased. Similar results were obtained with cyclophosphamide (CYT; 75 mg/kg) and CCNU (50 mg/kg) although the enhancement was much less than that seen with L-PAM. The enhancement by cisplatin was constant at all L-PAM doses as measured by both cloning assay and regrowth delay. Measurements of white blood cell counts four days after drug administration suggests that a therapeutic gain can be achieved. Exponential and plateau phase RIF-1 cells grown in monolayer culture were also exposed to various L-PAM doses. A 1 hour pre-exposure to cisplatin (0.5 micrograms/ml) under aerobic conditions increased the cell killing by L-PAM. The results are discussed with reference to the possible reasons for the therapeutic gain.     

Potentiation of the cytostatic effect of melphalan on colorectal cancer hepatic metastases by infusion of buthionine sulfoximine (BSO) in the rat Enhanced tumor glutathione depletion by infusion of BSO in the hepatic artery

Purpose: Glutathione (GSH) plays an important role in the resistance of tumors to cytostatics. Therefore, depletion of GSH by the GSH synthesis inhibitor buthionine sulfoximine (BSO) has been proposed to enhance the efficacy of certain anticancer agents. We studied the effect of BSO in rats bearing intrahepatically implanted tumors of the CC531 colorectal cancer cell line on the antitumor activity of melphalan (L-PAM). Since these liver tumors tend to derive most of their blood supply from the hepatic artery, we evaluated whether delivery of BSO into the hepatic artery would more selectively decrease GSH levels in the implanted tumor tissue as compared with normal liver and extrahepatic tissues. Methods: Tumor-bearing rats were treated with a 24-h continuous infusion of 0.375 mmol/kg BSO via the jugular vein, immediately followed by a bolus L-PAM (15 μmol/kg; 4.5 mg/kg) infusion via the hepatic artery. Laparotomy was performed on day 14 and 28 after treatment for measurement of the liver tumors. For the evaluation of locoregional administration of BSO, a 24-h continuous infusion of 0.375 mmol/kg BSO was delivered into either the hepatic artery, the portal vein, or the jugular vein in freely moving rats and GSH levels in the tumor, liver, kidney, lung, heart, bone marrow, and blood were measured. Results: BSO infusion via the jugular vein increased the antitumor efficacy of L-PAM injected into the hepatic artery 2-fold as determined at 14 days after treatment. Although infusion of BSO via the hepatic artery depleted GSH more severely in the tumor as compared with jugular vein or portal vein administration, the additional effect was only slight (10%). No difference was observed in any other tissue. Conclusion: GSH depletion increased the cytostatic efficacy of L-PAM 2-fold in vivo as determined at 14 days after treatment. Hepatic artery infusion of BSO translated into a statistically significant, but probably not therapeutically relevant, increase in tumor GSH depletion as compared with the other routes of BSO administration. Received: 21 September 1998 / Accepted: 4 January 1999