Isolated limb perfusion with biochemotherapy and oncolytic virotherapy combines with radiotherapy and surgery to overcome treatment resistance in an animal model of extremity soft tissue sarcoma

The management of locally advanced or recurrent extremity sarcoma often necessitates multimodal therapy to preserve a limb, of which isolated limb perfusion (ILP) is a key component. However, with standard chemotherapeutic agents used in ILP, the duration of response is limited. Novel agents or treatment combinations are urgently needed to improve outcomes. Previous work in an animal model has demonstrated the efficacy of oncolytic virotherapy when delivered by ILP and, in this study, we report further improvements from combining ILP‐delivered oncolytic virotherapy with radiation and surgical resection. In vitro, the combination of radiation with an oncolytic vaccinia virus (GLV‐1h68) and melphalan demonstrated increased cytotoxicity in a panel of sarcoma cell lines. The effects were mediated through activation of the intrinsic apoptotic pathway. In vivo, combinations of radiation, oncolytic virotherapy and standard ILP resulted in delayed tumour growth and prolonged survival when compared with standard ILP alone. However, local disease control could only be secured when such treatment was combined with surgical resection, the timing of which was crucial in determining outcome. Combinations of oncolytic virotherapy with surgical resection and radiation have direct clinical relevance in extremity sarcoma and represent an exciting prospect for improving outcomes in this pathology.     

The αVβ3/αVβ5 integrin inhibitor cilengitide augments tumor response to melphalan isolated limb perfusion in a sarcoma model

Isolated limb perfusion (ILP) with melphalan and tumor necrosis factor (TNF)‐α is used to treat bulky, locally advanced melanoma and sarcoma. However, TNF toxicity suggests a need for better‐tolerated drugs. Cilengitide (EMD 121974), a novel cyclic inhibitor of alpha‐V integrins, has both anti‐angiogenic and direct anti‐tumor effects and is a possible alternative to TNF in ILP. In this study, rats bearing a hind limb soft tissue sarcoma underwent ILP using different combinations of melphalan, TNF and cilengitide in the perfusate. Further groups had intra‐peritoneal (i.p.) injections of cilengitide or saline 2 hr before and 3 hr after ILP. A 77% response rate (RR) was seen in animals treated i.p. with cilengitide and perfused with melphalan plus cilengitide. The RR was 85% in animals treated i.p. with cilengitide and ILP using melphalan plus both TNF and cilengitide. Both RRs were significantly greater than those seen with melphalan or cilengitide alone. Histopathology showed that high RRs were accompanied by disruption of tumor vascular endothelium and tumor necrosis. Compared with ILP using melphalan alone, the addition of cilengitide resulted in a three to sevenfold increase in melphalan concentration in tumor but not in muscle in the perfused limb. Supportive in vitro studies indicate that cilengitide both inhibits tumor cell attachment and increases endothelial permeability. Since cilengitide has low toxicity, these data suggest the agent is a good alternative to TNF in the ILP setting.     

Hyperthermic isolated limb perfusion in locally advanced limb soft tissue sarcoma: A 24-year single-centre experience

Background: Hyperthermic isolated limb perfusion (HILP) is a locoregional treatment aimed at avoiding amputation in patients with advanced extremity soft tissue sarcomas (STS). Over the last 25 years, HILP procedure has been implemented to maximise its therapeutic ratio. Methods: A retrospective analysis including 117 patients who underwent HILP from 1989 to 2013 was performed. Three different drug schedules were applied: 1) doxorubicin (n?=?47), 2) high dose (3–4?mg) tumour necrosis factor-alpha (TNF-α) plus doxorubicin (n?=?30), 3) low dose (1?mg) TNF-α plus melphalan (L-PAM) (n?=?40). Tumour response was evaluated by MRI or CT and surgical specimens. Toxicity and local progression-free survival (LPFS) were also evaluated. Results: In total 92 (78.6%) patients had primary, 25 (21.4%) had recurrent and 17 (14.5%) had metastatic disease. The subjects in the three groups were homogeneous for clinical-pathological features. Pathological response was complete in 55 patients (47%), partial in 35 (29.9%), regardless of drug schedule (p?=?0.501) and tumour presentation (p?=?0.094). Wieberdink III–V toxicity was registered in 19.1%, 20% and 2.5% of patients, respectively (p?<?0.051). Twenty-eight patients (23.9%) received adjuvant radiotherapy with no relevant toxicity. Five-year LPFS was 81.6% and 74.2% in patients with primary or recurrent disease, respectively (p?=?0.652). After a median follow-up of 36.5 months, the limb sparing rate was 77.8%. Conclusions: HILP performed with different drugs was equally active, either in primary, recurrent or metastatic STS, providing effective limb sparing and durable local control. Low dose TNF-α plus L-PAM had the most favourable toxicity profile. Adjuvant radiotherapy was not associated with relevant toxicity.     

p53 status correlates with histopathological response in patients with soft tissue sarcomas treated using isolated limb perfusion with TNF-alpha and melphalan.

BACKGROUND: Recombinant tumor necrosis factor-alpha (TNF-alpha) combined to melphalan is clinically administered through isolated limb perfusion (ILP) for regionally advanced soft tissue sarcomas of the limbs. In preclinical studies, wild-type p53 gene is involved in the regulation of cytotoxic action of TNF-alpha and loss of p53 function contributes to the resistance of tumour cells to TNF-alpha. The relationship between p53 status and response to TNF-alpha and melphalan in patients undergoing ILP is unknown. PATIENTS AND METHODS: We studied 110 cases of unresectable limbs sarcomas treated by ILP. Immunohistochemistry was carried out using DO7mAb, which reacts with an antigenic determinant from the N-terminal region of both the wild-type and mutant forms of the p53 protein, and PAb1620mAb, which reacts with the 1620 epitope characteristic of the wild-type native conformation of the p53 protein. The immunohistochemistry data were then correlated with various clinical parameters. RESULTS: P53DO7 was found expressed at high levels in 28 patients, whereas PAb1620 was negative in 20. The tumours with poor histological response to ILP with TNF-alpha and melphalan showed significantly higher levels of p53-mutated protein. CONCLUSIONS: Our results might be a clue to a role of p53 protein status in TNF-alpha and melphalan response in clinical use.     

Outcomes of isolated limb perfusion in the treatment of extremity soft tissue sarcoma: A systematic review

Background

Isolated limb perfusion (ILP) may provide a limb salvage option for locally advanced soft tissue sarcoma (STS) not amenable to local resection.

Methods

A systematic review was performed for studies reporting outcome of ILP for locally advanced STS performed after 1980 in patients aged ≥12 years old. The main endpoints were tumour response and limb salvage rates. Complication and recurrence rates were secondary endpoints.

Results

Eighteen studies were included, providing outcomes for 1030 patients. Tumour necrosis factor-alpha with melphalan was the commonest chemotherapy regime. When reported, 22% of cases achieved a complete tumour response (216/964, 15 studies) with an overall response rate of 72% (660/911, 15 studies). At median follow-up times ranging between 11 and 125 months, the limb salvage rate was 81% in patients who otherwise would have been subjected to amputation. However, 27% of patients suffered local recurrence and 40% suffered distant failure. ILP was associated with severe locoregional reactions in 4% (22/603) of patients. Amputation due to complications within 30 days was necessary in 1.2% of cases (7/586, nine studies). There was insufficient evidence to determine the effect of ILP on survival.

Conclusion

ILP induces a high tumour response rate, leads to a high limb salvage rate but is associated with a high recurrence rate. It provides a limb salvage alternative to amputation when local control is necessary.     

Isolated limb perfusion with melphalan,tumour necrosis factor‐alpha and oncolytic vaccinia virus improves tumour targeting and prolongs survival in a rat model of advanced extremity sarcoma

Isolated limb perfusion (ILP) is a treatment for advanced extremity sarcoma and in‐transit melanoma. Advancing this procedure by investigating the addition of novel agents, such as cancer‐selective oncolytic viruses, may improve both the therapeutic efficacy of ILP and the tumour‐targeted delivery of oncolytic virotherapy. Standard in vitro assays were used to characterise single agent and combinatorial activities of melphalan, tumour necrosis factor‐alpha (TNF‐α) and Lister strain vaccinia virus (GLV‐1h68) against BN175 rat sarcoma cells. An orthotopic model of advanced extremity sarcoma was used to evaluate survival of animals after ILP with combinations of TNF‐α, melphalan and GLV‐1h68. We investigated the efficiency of viral tumour delivery by ILP compared to intravenous therapy, the locoregional and systemic biodistribution of virus after ILP, and the effect of mode of administration on antibody response. The combination of melphalan and GLV‐1h68 was synergistic in vitro. The addition of virus to standard ILP regimens was well tolerated and demonstrated superior tumour targeting compared to intravenous administration. Triple therapy (melphalan/TNF‐α/GLV‐1h68) resulted in increased tumour growth delay and enhanced survival compared to other treatment regimens. Live virus was recovered in large amounts from perfused regions, but in smaller amounts from systemic organs. The addition of oncolytic vaccinia virus to existing TNF‐α/melphalan‐based ILP strategies results in survival advantage in an immunocompetent rat model of advanced extremity sarcoma. Virus administered by ILP has superior tumour targeting compared to intravenous delivery. Further evaluation and clinical translation of this approach is warranted.     

重组改构人肿瘤坏死因子治疗恶性肿瘤的随机对照研究

目的:观察重组改构人肿瘤坏死因子(Recombinant mutant human tumor necrosis factor,rmhTNF)治疗晚期恶性肿瘤的有效性和安全性。方法:92例患者随机纳入试验组和对照组。试验组在化疗的同时,rmhTNF 400万u/m2,肌注,第1～7天和第11～17天;对照组仅给联合化疗;两组均每21天重复,连用二个周期。结果:①试验组和对照组的有效率分别为11.11％和2.78％,两组疗效无显著性差异(P>0.05);②试验组和对照组治疗前后KPS评分及其差值无显著性差异(P>0.05);③rmhTNF的主要不良反应为一过性轻度注射局部疼痛63.0％、骨骼肌痛38.9％、发热24.1％、感冒样症状22.0％和局部红肿硬结13.0％。结论:受病例数等因素影响rmhTNF联合化疗药物治疗晚期肺癌、头颈癌、胃肠道癌、肾癌及恶性黑色素瘤的疗效不优于单纯化疗。rmhTNF联合化疗的耐受性和安全性良好,为进一步扩大病例临床研究提供了可靠的保证。     

Histological response assessment following neoadjuvant isolated limb perfusion in patients with primary,localised, high-grade soft tissue sarcoma

Purpose: Histological response assessment following neoadjuvant treatment can help identify patients at a higher risk for systemic disease progression. Our goal was to evaluate whether mitotic count and the amount of viable tumour following neoadjuvant isolated limb perfusion (ILP) for primary, locally advanced, non-metastatic, high-grade extremity soft tissue sarcoma correlate with prognosis. Patients and methods: This study is a retrospective analysis of 61 patients who underwent neoadjuvant ILP followed by surgical resection with curative intent between 2001 and 2011. Non-parametric analyses were carried out with the Mann-Whitney U and the Wilcoxon signed-rank test. Survival curves were calculated with the Kaplan-Meier method and compared with the log-rank test. Results: The median follow-up was 44 months for all patients and 55 months for survivors. The amount of viable tumour after ILP had no correlation with overall (OS) (P?=?0.227) or event-free (EFS) (P?=?0.238) survival probability. Patients with a low mitotic count after ILP had a significantly higher OS (P?<?0.001), EFS (P?=?0.002) and post-relapse survival probability (P?=?0.030) compared to patients with an intermediate or high mitotic count. Conclusions: The mitotic count following ILP for primary, high-grade, locally advanced, non-metastatic soft tissue sarcoma appears to significantly correlate with prognosis. If these results are validated in a prospective setting, they could provide a rationale for the design of adjuvant systemic chemotherapy trials with the goal of improving the prognosis of patients with an intermediate or high mitotic count after ILP.     

Tumor vasculature‐targeted delivery of tumor necrosis factor‐α*

BACKGROUND: Recently, considerable efforts have been directed toward antivascular therapy as a new modality to treat human cancers. However, targeting a therapeutic gene of interest to the tumor vasculature with minimal toxicity to other tissues remains the objective of antivascular gene therapy. Tumor necrosis factor‐α (TNF‐α) is a potent antivascular agent but has limited clinical utility because of significant systemic toxicity. At the maximum tolerated doses of systemic TNF‐α, there is no meaningful antitumor activity. Hence, the objective of this study was to deliver TNF‐α targeted to tumor vasculature by systemic delivery to examine its antitumor activity. METHODS: A hybrid adeno‐associated virus phage vector (AAVP) was used that targets tumor endothelium to express TNF‐α (AAVP‐TNF‐α). The activity of AAVP‐TNF‐α was analyzed in various in vitro and in vivo settings using a human melanoma tumor model. RESULTS: In vitro, AAVP‐TNF‐α infection of human melanoma cells resulted in high levels of TNF‐α expression. Systemic administration of targeted AAVP‐TNF‐α to melanoma xenografts in mice produced the specific delivery of virus to tumor vasculature. In contrast, the nontargeted vector did not target to tumor vasculature. Targeted AAVP delivery resulted in expression of TNF‐α, induction of apoptosis in tumor vessels, and significant inhibition of tumor growth. No systemic toxicity to normal organs was observed. CONCLUSIONS: Targeted AAVP vectors can be used to deliver TNF‐α specifically to tumor vasculature, potentially reducing its systemic toxicity. Because TNF‐α is a promising antivascular agent that currently is limited by its toxicity, the current results suggest the potential for clinical translation of this strategy. Cancer 2009. Published 2008 by the American Cancer Society.     

TNF-alpha and melphalan-based isolated limb perfusion: no evidence supporting the early destruction of tumour vasculature

Background:

Isolated limb perfusion with TNF-alpha and melphalan (TM-ILP) is a highly effective treatment for locally advanced tumours of the extremities. Previous research suggests an almost immediate disintegration of the blood supply of the tumour. The aim of the present study was to verify this hypothesis using non-invasive measurements of microvascular perfusion and tissue oxygenation.

Methods:

A total of 11 patients were included in the study. TM-ILP was performed under mildly hyperthermic conditions (39 °C) in the extremities via proximal vascular access. Capillary-venous microvascular blood flow, haemoglobin level (Hb) and oxygen saturation (SO₂) were determined using laser Doppler and white-light spectroscopy, respectively, before TM-ILP and at 30 min, 4 h, 1 day, 4 days, 1 week, 2 weeks and 6 weeks after TM-ILP from tumour and healthy muscle tissues.

Results:

Blood flow and Hb were mostly higher, whereas SO₂ was lower, in tumour tissue compared with muscle tissue. In both tumour and muscle tissues, blood flow significantly increased immediately after TM-ILP and remained elevated for at least 2 weeks, followed by a return to the initial values 6 weeks after the procedure.

Conclusion:

No signs were found of early destruction of the tumour vasculature. The observations suggest that an inflammatory reaction is one of the key elements of TM-ILP.     

Results of limb-salvage surgery with vascular reconstruction for soft tissue sarcoma in the lower extremity: comparison between only arterial and arterovenous reconstruction

BACKGROUND: Reports on vascular reconstruction in conjunction with limb salvage techniques for soft tissue sarcomas in the lower extremity have been published. The aim of this study was to investigate the necessity for venous reconstruction by comparing the results of arterial reconstruction alone and those of arteriovenous reconstruction. METHODS: Twenty-five (6.7%) of 373 patients underwent vascular resections for lower limb soft tissue sarcomas. Only arterial reconstruction was performed for 12 patients. Arteriovenous reconstruction was performed for 13 patients. The clinical results, complications, and postoperative function were compared between only arterial reconstruction group and arteriovenous reconstruction group. RESULTS: Limb salvage was achieved in 92% of all cases. No local recurrence has been observed. Nineteen patients (76%) are continuously free of disease. Histopathologic examination of the specimens showed that blood vessels had been infiltrated in 24%. The mean Musculoskeletal Tumor Society score was 70%. There were no significant differences in complication rate and postoperative function between two groups. CONCLUSIONS: Wide resection with vascular reconstruction for patients of soft tissue sarcomas involving major vessels in the lower limb provide long term local control and limb salvage. This study could not indicate the usefulness of additional venous reconstruction after vascular resection in the lower extremity.     

A new limb salvage surgery in cases of high-grade soft tissue sarcoma using photodynamic surgery, followed by photo- and radiodynamic therapy with acridine orange

BACKGROUND: To maintain excellent limb function after tumor resection in patients with high-grade malignant sarcomas, we developed and established a new surgical adjuvant therapy using acridine orange (AO) after intra-lesional or marginal resection while sparing normal tissues of major nerves, vessels or bones adjacent to the tumor. METHOD: Our AO therapy procedure was combined with photodynamic surgery (PDS), photodynamic therapy (PDT) and radiodynamic therapy (RDT). In this study, 26 patients with primary high-grade soft tissue sarcomas were treated with AO therapy. RESULT: Results showed a low local recurrence rate (7.7%) and good local recurrence-free rate (88%) after AO therapy. Limb function of all patients was maintained at 100% of ISOLS criteria. CONCLUSION: Based on these results, we concluded that AO therapy is useful for local control after margin-positive tumor resection and for preserving excellent limb function in patients with high-grade soft tissue sarcomas.     

Phosphorylated form of pyruvate dehydrogenase α1 mediates tumor necrosis factor α-induced glioma cell migration

Cell migration is an important factor influencing the treatment outcomes of high-grade glioma (World Health Organization grades III–IV). Using immunohistochemical staining, the present study demonstrated that the protein levels of phosphorylated pyruvate dehydrogenase α1 (p-PDHA1) were increased according to the grade of glioma. Moreover, p-PDHA1 mediated tumor necrosis factor-α (TNF-α)-induced cell migration in glioma cells. Phalloidin staining and western blot analysis were used to detect the protein level of p-PDHA1 in U251 glioma cells stimulated by TNF-α at different time points. Phalloidin staining was used to observe the cytoskeletal structure. The effects on the expression of specific migration markers and on the cytoskeletal structure were also detected. Dichloroacetic acid is an inhibitor of PDK. These results indicated that p-PDHA1 served an important role in the migration of glioma cells, and consequently in the development of glioma.