Trojan-horse nanotube on-command intracellular drug delivery.

A major challenge to nanomaterial-based medicine is the ability to release drugs on-command. Here, we describe an innovative drug delivery system based on carbon nanotubes (CNTs), in which compounds can be released inside cells from within the nanotube "on-command" by inductive heating with an external alternating current or pulsed magnetic field. Without inductive heating the drug remains safely inside the CNTs, showing no toxicity in cell viability tests. Similar to the "Trojan-Horse" in function, we demonstrate the delivery of a combination of chemotherapeutic agents with low aqueous solubility, paclitaxel (Taxol), and C6-ceramide, to multidrug resistant pancreatic cancer cells. Nanotube encapsulation permitted the drugs to be used at a 100-fold lower concentration compared to exogenous treatment yet achieve a comparable ~70% cancer kill rate.

Selective organ preservation in operable locally advanced head and neck squamous cell carcinomas guided by primary site restaging biopsy: long-term results of two sequential brown university oncology group chemoradiotherapy studies.

OBJECTIVES: The long-term outcomes of selective organ preservation in operable, locally advanced head and neck cancers in two sequential chemoradiotherapy (CRT) protocols (HN-53, HN-67) are reported. METHODS: A total of 65 patients were treated with CRT consisting of carboplatin (AUC=1/week) and paclitaxel (60 or 40 mg/m2/week) with radiation (1.8 Gy/day). After 5 weeks of CRT, if primary site biopsies were pathologically negative, then completion CRT to 67-72 Gy was done with neck dissection in node-positive cases. Alternatively, a positive rebiopsy required primary site resection and neck dissection followed by radiotherapy boost as deemed necessary. RESULTS: Pathologic complete responses occurred in 71% patients who then completed CRT; the remaining 29% patients underwent primary site surgery. The 5-year and median overall survival were 47% and 57 months with no statistically significant differences between the two groups. Overall long-term failure rates were: 6% local, 6% regional, and 32% distant. CONCLUSIONS: This strategy of selective organ preservation was effective in 71% patients with CRT, whereas salvage surgery was required in the remainder. Long-term survival was equivalent in both treatment groups.

Factors predictive of the status of sentinel lymph nodes in melanoma patients from a large multicenter database.

BACKGROUND: Numerous predictive factors for cutaneous melanoma metastases to sentinel lymph nodes have been identified; however, few have been found to be reproducibly significant. This study investigated the significance of factors for predicting regional nodal disease in cutaneous melanoma using a large multicenter database. METHODS: Seventeen institutions submitted retrospective and prospective data on 3463 patients undergoing sentinel lymph node (SLN) biopsy for primary melanoma. Multiple demographic and tumor factors were analyzed for correlation with a positive SLN. Univariate and multivariate statistical analyses were performed. RESULTS: Of 3445 analyzable patients, 561 (16.3%) had a positive SLN biopsy. In multivariate analysis of 1526 patients with complete records for 10 variables, increasing Breslow thickness, lymphovascular invasion, ulceration, younger age, the absence of regression, and tumor location on the trunk were statistically significant predictors of a positive SLN. CONCLUSIONS: These results confirm the predictive significance of the well-established variables of Breslow thickness, ulceration, age, and location, as well as consistently reported but less well-established variables such as lymphovascular invasion. In addition, the presence of regression was associated with a lower likelihood of a positive SLN. Consideration of multiple tumor parameters should influence the decision for SLN biopsy and the estimation of nodal metastatic disease risk.

The neoadjuvant therapy of colorectal hepatic metastases and the role of biologic sensitizing and resistance factors.

Liver metastasis represents a common systemic complication of colorectal cancers (CRCs). Partial liver resection has been demonstrated to result in long-term survival in certain well-selected patients with otherwise well-controlled systemic disease. Neoadjuvant therapy has been demonstrated to result in improved resectability and potentially longer survival in patients with liver metastases from CRC. The addition of biologic agents to chemotherapy has been shown to improve response rates and overall survival in patients with metastatic CRC. Here, we are discussing the role of biologic agents in the treatment of patients with liver metastases from CRC. We also discuss the role of biomarkers for response and resistance to such novel therapies.

Salvage of pelvic recurrence of colorectal cancer.

Although the incidence of locally recurrent colorectal cancer has been reduced by improved surgical techniques and the frequent use of multimodality therapy, pelvic recurrence remains a significant problem. Radiation or chemotherapy may provide palliation but it is often short-lived. For fit candidates without evidence of extrapelvic disease, surgical resection (anterior resection, abdominoperineal resection, pelvic exenteration, or abdominosacral resection) may be the most appropriate treatment. For patients with unresectable disease, isolated pelvic perfusion may provide effective palliation.

Isolated chemotherapeutic perfusion of pelvis as neoadjuvant or palliative therapy for advanced cancer of the rectum.

INTRODUCTION: Previously irradiated recurrent rectal cancer is a formidable patient threat with limited treatment options. Isolated pelvic perfusion (IPP) by the balloon-occlusion technique provides high-dose regional chemotherapy that may facilitate resection if appropriate or palliate pain and fungating tumor mass in the symptomatic patient. We currently report our results in 49 recurrent rectal cancer patients (26 had neoadjuvant IPP with intent to resect and 23 had IPP for palliation). METHODS: IPP was done for 1 hour with paclitaxel 30 mg/m(2), 5 fluorouracil 1500 mg/m(2), cisplatin/oxaliplatin 60-130 mg/m(2), and mitomycin C 10 to 15 mg/m(2) (the latter three achieving pelvic-to-systemic drug ratios of 6-9:1). RESULTS: Neoadjuvant perfusion in 26 patients achieved a response in 14 patients (made resectable). Seven had R0 resections (clear margins), six by abdominal sacral resection (ABSR), and one by an extended APR. Of seven other patients, one had a complete pathologic response negating planned resection, one had >50% tumor regression in pelvis (but developed distant metastases), and three refused ABSR. Planned ABSR in two patients was aborted because of complicating cardiovascular issues. A variety of medical and cancer issues precluded resection in the remaining 12 of these 26 neoadjuvant patients. Within the neoadjuvant group, median survival was 24 months in the responding (made resectable) group (14 patients) and it was 8 months in the non-resectable group (12 patients), p = 0.0001. In the responding (made resectable) group, seven patients had R0 resections (median survival 26 months) and seven patients were not resected (median survival 18 months), p = 0.0198. In the IPP group for palliation, 17 of 23 patients (74%) had significant relief of pain, and other tumor-related symptoms (mean survival 11 months). CONCLUSION: Isolated pelvic perfusion using a simplified balloon-occlusion technique has promise in palliation of or augmenting resectability of advanced rectal malignancy in patients not amenable to treatment with conventional modalities.

Regional chemotherapy: overview.

Regional chemotherapy was developed in the 1950s and continues to play an integral part in the development of newer therapies for advanced solid malignancies. Regional therapies have evolved in complexity but are still based on the pharmacokinetics of drug delivery to solid malignancies. Newer techniques demonstrate that the combination of regional therapies, hyperthermia, and surgery is essential in promoting improved patient outcomes.

Clinical pharmacokinetics of isolated pelvic perfusion.

The pharmacokinetic results of this study indicate that there is significant enhancement of pelvic drug levels with isolated pelvic perfusion, with the potential of increasing the therapeutic index and clinical efficacy. This system would seem to be ideal for evaluating fast-acting and highly toxic experimental drugs on human pelvic cancers having treatment protocols of limited clinical success. Pharmacokinetic modeling using empiric approximations for the kinetic rate constants is a convenient and novel way of fitting data using relatively simple mathematics and commercially available spreadsheets.

Pelvic perfusion for advanced colorectal cancers.

Isolated pelvic perfusion (IPP) is a form of intra-arterial local-regional treatment of tumor-bearing organs. IPP using a simplified balloon occlusion technique has shown promise in palliation of resectability of advanced rectal cancer in patients not amenable to treatment with conventional chemoradiation. This article reviews technique criteria and the response to IPP from seven literature studies of isolated pelvic perfusion with colorectal cancer. Current efforts should be directed to improving anti-tumor responses by optimizing chemotherapeutic protocols and modifying perfusion parameters, so that hopefully, this will lead to a more standardized and improved procedure for the isolated pelvic perfusion technique.

Isolated pelvic perfusion: plasma pharmacokinetics depend primarily on drug dosage and not the type of drug.

PURPOSE: Comparison of the pharmacokinetics of four drugs with the isolated pelvic perfusion protocol showed linear relationships between drug dosage and two isolated pelvic plasma parameters, mean AUC (pelvic exposure, microM min) and the mean maximum pelvic drug level (microM). It appears that the pharmacokinetics are sufficiently defined as to predict plasma distribution curves for an additional drug with this protocol. Recent FDA approval of oxaliplatin allowed an evaluation of this premise. METHODS: Linearity of drug dosage with maximum drug levels and exposure (AUC) in the isolated pelvic plasma yields initial estimates of these parameters for additional drugs. Use of an empirical, four-compartment pharmacokinetic model (Wanebo and Belliveau in Cancer Chemother. Pharmacol. 43:427, 1999) allowed the generation of predictive plasma distribution curves. These curves were established by optimizing the initial estimates of maximum drug levels and exposure along with estimates of two additional parameters (half-life of pelvic clearance and pelvic to systemic exposure ratio) from experimental data of the four drugs pharmacokinetically characterized. RESULTS: Calculated plasma distribution curves for oxaliplatin matched the experimental curves from the first three patients receiving oxaliplatin therapy, given the experimental ranges of pharmacokinetic parameters seen with the initial four drugs. CONCLUSION: These results give an overall picture for the plasma pharmacokinetics during the isolation period for the isolated pelvic perfusion protocol. Enough experimental data have been accumulated for five drugs to establish a simple pharmacokinetic model (Wanebo and Belliveau in Cancer Chemother Pharmacol 43:427, 1999) and interdrug relationships (i.e., this report) which can be used to predict reasonable plasma distribution curves for additional drugs with this protocol.

Synergistic effect of interleukin-2 and a vaccine of irradiated melanoma cells transfected to secrete staphylococcal enterotoxin A.

We have previously reported that immunization of mice with melanoma cells transfected to secrete the superantigen, Staphylococcal enterotoxin A (SEA), increased the production of antibodies to the B700 melanoma antigen, stimulated the production of endogenous interleukin 2 (IL-2), activated the expression of CD4, CD8 and CD25 T cell markers and enhanced NK cell activity. Now we show that immunization of mice with a vaccine of irradiated sea-transfected melanoma cells coupled with IL-2 therapy was even more effective in inhibiting the growth of primary melanoma tumors and the development of lung metastases than was the irradiated melanoma cell vaccine alone or IL-2 alone. The morphological and immunological effectiveness of the therapy was dose-dependent on IL-2.

Capacity of murine IL-12 to inhibit the development of primary melanoma tumors and to prevent lung metastases in the melanoma-challenged mice.

Interleukin-12 (IL-12) has the capacity to activate cytotoxic lymphocytes, stimulate natural killer cells, induce the production of INF-gamma, and be synergistic with IL-2. We have evaluated this cytokine in an experimental model for metastatic melanoma that approximates the major clinical stages of metastatic dissemination. To develop primary melanoma tumors, mice were injected subcutaneously with 5 x 10(5) cells in a volume of 25 microliters into the middle of the tail (11). In a month, mice were started to be treated for 4 weeks with recombinant murine IL-12 (R mIL-12) at the following doses: 0, 0.5, 2.5, 5.0, 15.0, and 50 micrograms/kg. Diameters of the primary melanoma tumors were measured at weekly intervals. At the end of 13 weeks (9 weeks from the start of treatment with R mIL-12), all surviving mice were sacrificed. Pathological examination of lung metastases (macroscopy) was done with all dead or sacrificed mice. Treatment of mice bearing melanoma at a dose of 300 ng/mouse (15 micrograms/kg) inhibited development of primary tumors in 40% of mice. The primary tumor diameters were significantly lower in the group treated with 300 ng/mouse (15 micrograms/kg) in comparison to controls. At the end of the observation period, groups treated with 0.5, 2.5, 15.0, and 50 micrograms/kg had mean primary tumor diameters smaller than the control group. Evaluation of IL-12 therapy on primary tumor growth, mean diameters of primary tumors, survival rate, and development of lung metastases showed that the best results were observed using 300 ng/mouse (15 micrograms/kg) R mIL-12.

An effective in vitro antitumor response against human pancreatic carcinoma with paclitaxel and daunorubicin by induction of both necrosis and apoptosis.

The highly metastatic human pancreatic cell line L3.6 was used to study mechanisms for antitumor activity with various chemotherapeutic drug combinations. The most effective drugs were daunorubicin (IC50 0.4 microM), doxorubicin (IC50 22 microM), paclitaxel (IC50 5.3 microM) and 5-fluorouracil (IC50 5.4 microM). The most effective drug combination was equitoxic concentrations of paclitaxel and daunorubicin. Kinetic analysis demonstrated that both paclitaxel and daunorubicin had to be added simultaneously for maximum cytotoxicity. Daunorubicin treatment alone demonstrated ROS (reactive oxygen species) induction and cellular morphological changes more consistent with chemical damage in a total of 93% of the cells and apoptotic changes in 20% of the cell population. The apoptosis induced by daunorubicin does not appear to be caspase-dependent, as demonstrated by the lack of conversion of the procaspases 8 and 3. Within 24 h of treatment with paclitaxel, Bcl-2 formed a doublet at 26 kilodaltons and the expression was abrogated with daunorubicin and the combination of the two drugs as determined by Western blots. These data suggest that the human pancreatic cell line L3.6 is more effectively killed following treatment with chemotherapeutic agents that cause death through at least two pathways, a caspase-dependent and caspase-independent apoptosis and necrosis.

Mucosal melanomas.

Cutaneous malignant melanoma is a common malignancy and has been increasing at an alarming rate in the United States. Sun exposure is a well-known risk factor related to this disease and much is understood regarding the etiology and epidemiology of cutaneous melanomas. In contrast, primary mucosal melanomas represent an extremely rare malignancy and do not have the same risk factors or behavior patterns. They occur in areas that have no sun exposure and solid predisposing risk factors have not been identified, making this disease very difficult to diagnose or screen for. It is usually diagnosed at a later stage and carries a poor prognosis. Identifying the differences between a primary lesion and a metastatic melanoma is often challenging, because of the lack of definitive criteria, both pathologically and clinically. The rich vascular and lymphatic network surrounding these lesions may be responsible for their aggressive behavior and poor prognosis. In addition, the obscure locations where mucosal melanomas occur are an obvious reason why these lesions often go unnoticed until symptoms develop. Recent literature has raised significant questions regarding recommended treatment strategies. Earlier reports advocated radical surgery as the mainstay of therapy; however, local recurrence and survival were unchanged whether radical surgery or local excision was performed, and the most recent data are favoring the conservative approach when appropriate. Unfortunately, a multitude of adjuvant therapies have been tried without any success. Adjuvant radiotherapy plays a role when combined with surgery, particularly in the head and neck region and female genitalia, but this is reserved for nodal and locoregionally advanced disease and has had no effect when used as a prophylactic method. It is difficult to make significant advances in treatment strategies because of the rarity of the disease. As an example, one in 75 persons born in the year 2000 will develop cutaneous melanoma in his lifetime, compared with four cases per ten million people diagnosed with mucosal melanoma per year in the United States. Possible new therapies based on new biologic and immunologic findings may have future promise on being able to impact this disease. Until then, this aggressive tumor continues to have a poor prognosis, and surgical resection continues to be the mainstay of primary therapy.

Changing patterns of failure of head and neck cancer.

BACKGROUND: With the increased use of neoadjuvant therapy for advanced stage squamous cell carcinoma of the head and neck, we have observed an apparent change in the pattern of failure from predominantly locoregional sites to distant metastases. We reviewed the patterns of failure in cancers of the oral cavity, oropharynx, and larynx at our institution during the last decade. OBJECTIVE: To determine whether there has been a significant change in the patterns of recurrence from the historical locoregional failure to distant sites, and whether this change is associated with the increased use of multimodality therapy. METHODS: We reviewed cancer registry data on patients with squamous cell carcinoma of the head and neck diagnosed between January 1, 1988, and December 31, 1999. Sites included the oral cavity and oropharynx (including the tongue, floor of mouth, retromolar trigone, gingiva, tonsil, and lip) and larynx. RESULTS: Among 432 patients with squamous cell carcinoma of the head and neck, 280 (65%) had oral cavity and oropharyngeal cancers, and 152 (35%) had laryngeal cancers. Overall, 19% developed locoregional recurrence, and 8% developed distant failure. Although locoregional failure for oral cavity and oropharyngeal squamous cell carcinoma decreased from 26% to 16% from 1988-1993 to 1994-1999, distant failure increased significantly from 3% to 8%. During these periods, multimodality therapy was used in 62% of oral cavity and oropharyngeal cancers, and this rate remained essentially unchanged. For laryngeal cancer, locoregional and distant failure remained stable at 18% and 9%, respectively. In these laryngeal cancers, the use of multimodality therapy decreased from 60% to 46%, but this difference was not statistically significant (P =.43). CONCLUSIONS: Although locoregional control in oral cavity and oropharyngeal cancers has improved significantly with the use of multimodality therapy, the incidence of distant failure has doubled. In laryngeal squamous cell carcinoma, the patterns of failure have not changed significantly.

Prognostic biomarkers in phase II trial of cetuximab-containing induction and chemoradiation in resectable HNSCC: Eastern cooperative oncology group E2303.

PURPOSE: We sought to evaluate the correlation between tissue biomarker expression (using standardized, quantitative immunofluorescence) and clinical outcome in the E2303 trial. EXPERIMENTAL DESIGN: Sixty-three eligible patients with operable stage III/IV head and neck squamous cell cancer (HNSCC) participated in the Eastern Cooperative Oncology Group (ECOG) 2303 phase II trial of induction chemotherapy with weekly cetuximab, paclitaxel, and carboplatin followed by chemoradiation with the same regimen. A tissue microarray (TMA) was constructed and EGF receptor (EGFR), ERK1/2, Met, Akt, STAT3, ß-catenin, E-cadherin, EGFR Variant III, insulin-like growth factor-1 receptor, NF-κB, p53, PI3Kp85, PI3Kp110a, PTEN, NRAS, and pRb protein expression levels were assessed using automated quantitative protein analysis (AQUA). For each dichotomized biomarker, overall survival (OS), progression-free survival (PFS), and event-free survival (EFS) were estimated by the Kaplan-Meier method and compared using log-rank tests. Multivariable Cox proportional hazards models were used to estimate HRs and test for significance. RESULTS: Forty-two of 63 patients with TMA data on at least one biomarker were included in the biomarker analysis. Tumor extracellular signal-regulated kinase (ERK)1/2 levels were significantly associated with PFS [HR (low/high), 3.29; P = 0.026] and OS [HR (low/high), 4.34; P = 0.008]. On multivariable Cox regression analysis, ERK1/2 remained significantly associated with OS (P = 0.024) and PFS (P = 0.022) after controlling for primary site (oropharynx vs. non-oropharynx) and disease stage (III vs. IV), respectively. Clustering analysis revealed that clusters indicative of activated RAS/MAPK/ERK and/or PI3K/Akt pathways were associated with inferior OS and/or PFS and maintained significance in multivariable analysis. CONCLUSIONS: These results implicate PI3K/Akt and RAS/MAPK/ERK pathways in resistance to cetuximab-containing chemoradiation in HNSCC. Large prospective studies are required to validate these results.

Meeting the biologic challenge of colorectal metastases.

An overview of colorectal cancer discussed (Philip Paty) the good outcome after primary management with local control in 90-95 % of colon and 85 % in rectal cancer patients with major progression to metastases and to death related to hematogenous dissemination. The major disease pathways include the APC, aneuploid pathway involving mutations of P53, KRAS, SMAD 4, or the CMP/MSI pathway, mismatched repair defect as characterized by Lynch syndrome, the major hereditary form which may also have KRAS and P53 mutations. The common sporadic colorectal cancers are MS1 high, with many patients having BRAF and KRAS mutations. The sentinel node biopsy in colorectal cancer surgery may provide more definitive staging and perhaps modification of the extent of resection with better outcome as suggested by Dr. Saha. The identification of sentinel lymph nodes outside of the planned bowel resection may increase the resection biologically indicated by the sentinel lymph node location leading to better outcome. In a small study by Dr. Saha, the operation was enhanced in 21 % by extending the length of bowel resection, which increased node recovery to 18.5 nodes versus 12 nodes with the more conventional resection, increasing nodal recovery, and positivity to 60 % with reduction to five year recurrence rate to 9 % versus 27 % with the conventional resection. A new (Swiss) technique for pathologic node examination, the OSNA (the One Step Nucleic Acid diagnostic system), was presented which demonstrated increased detection of micro-metastases in a focused pathology study of 22 patients (Zuber) to 11 out of 15 patients versus the 7 micro-metastases identified by the standard single slide per node, and compared to 14 out of 15 with an intensive multi-slide technique. This suggests value in pursuing OSNA study by other centers with relevant clinical trials to establish its true value. An analysis of liver resection for metastatic colorectal cancer (CRC) emphasized the value of 10-year follow-up (DeAngelica). The 10-year survival of 102 patients among 612 patients was 17 % (Memorial Sloan Kettering data). At the five-year point 99 of 102 survivors were NED and 86 have been free of disease since the resection. The usual five-year figure after hepatic resection reveals that one-third of five-year survivors die from recurrence of distant disease suggesting the value of longer term follow-up in these patients. An additional question reviewed related to the role of neoadjuvant systemic chemotherapy (with response rates in the 50 % range) to produce down staging of the hepatic metastases and allow one to retrieve these patients with possible residual disease. In a series of 116 patients who had hepatic resection of CRC metastases in presence of regional node metastases, post neoadjuvant chemotherapy (normally not candidates for resection) these patients were demonstrated to have a 95 % recurrence at median time of 9 months. This raises a cautionary note to the literature report of five-year survivals in the 20-30 % range for hepatic metastases in presence of extra hepatic disease. Such may reflect patient selection rather than a true measure of the biology of disease, and warrant clinical trial evaluation. Lastly, regional therapy and overall systemic therapy were addressed by Dr. Kemeny. The CALGB study of hepatic artery infusion (HAI) with FUDR, dexamethasone versus 5FU leucovorin showed an overall survival of 24.4 months with HAI versus 20 months with systemic therapy (P = 0.0034). An adjuvant trial of HAI at MSK in 156 patients showed an overall survival benefit at 2 year and recent long term 10yr follow-up showing a significant overall survival of 41 % with HAI versus 27 % with systemic therapy (5FU leucovorin). In the neoadjuvant Nordlinger trial for hepatic metastases, there was a significant outcome differences-the preoperative therapy group had 9.2 % increase of progression free survival versus the surgery alone group which suggests the value of combining neoadjuvant surgery in good risk liver resection candidates. Conclude the final lesson from this well presented mini symposium confirms the need for continued evaluation of the numerous discussion points by clinical trial.