Efficacy, long-term toxicity, and mechanistic studies of gold nanorods photothermal therapy of cancer in xenograft mice.

Gold nanorods (AuNRs)-assisted plasmonic photothermal therapy (AuNRs-PPTT) is a promising strategy for combating cancer in which AuNRs absorb near-infrared light and convert it into heat, causing cell death mainly by apoptosis and/or necrosis. Developing a valid PPTT that induces cancer cell apoptosis and avoids necrosis in vivo and exploring its molecular mechanism of action is of great importance. Furthermore, assessment of the long-term fate of the AuNRs after treatment is critical for clinical use. We first optimized the size, surface modification [rifampicin (RF) conjugation], and concentration (2.5 nM) of AuNRs and the PPTT laser power (2 W/cm2) to achieve maximal induction of apoptosis. Second, we studied the potential mechanism of action of AuNRs-PPTT using quantitative proteomic analysis in mouse tumor tissues. Several death pathways were identified, mainly involving apoptosis and cell death by releasing neutrophil extracellular traps (NETs) (NETosis), which were more obvious upon PPTT using RF-conjugated AuNRs (AuNRs@RF) than with polyethylene glycol thiol-conjugated AuNRs. Cytochrome c and p53-related apoptosis mechanisms were identified as contributing to the enhanced effect of PPTT with AuNRs@RF. Furthermore, Pin1 and IL18-related signaling contributed to the observed perturbation of the NETosis pathway by PPTT with AuNRs@RF. Third, we report a 15-month toxicity study that showed no long-term toxicity of AuNRs in vivo. Together, these data demonstrate that our AuNRs-PPTT platform is effective and safe for cancer therapy in mouse models. These findings provide a strong framework for the translation of PPTT to the clinic.

Combination of anti-HER3 antibody MM-121/SAR256212 and cetuximab inhibits tumor growth in preclinical models of head and neck squamous cell carcinoma.

The EGFR monoclonal antibody cetuximab is the only approved targeted agent for treating head and neck squamous cell carcinoma (HNSCC). Yet resistance to cetuximab has hindered its activity in this disease. Intrinsic or compensatory HER3 signaling may contribute to cetuximab resistance. To investigate the therapeutic benefit of combining MM-121/SAR256212, an anti-HER3 monoclonal antibody, with cetuximab in HNSCC, we initially screened 12 HNSCC cell lines for total and phosphorylated levels of the four HER receptors. We also investigated the combination of MM-121 with cetuximab in preclinical models of HNSCC. Our results revealed that HER3 is widely expressed and activated in HNSCC cell lines. MM-121 strongly inhibited phosphorylation of HER3 and AKT. When combined with cetuximab, MM-121 exerted a more potent antitumor activity through simultaneously inhibiting the activation of HER3 and EGFR and consequently the downstream PI3K/AKT and ERK pathways in vitro. Both high and low doses of MM-121 in combination with cetuximab significantly suppressed tumor growth in xenograft models and inhibited activations of HER3, EGFR, AKT, and ERK in vivo. Our work is the first report on this new combination in HNSCC and supports the concept that HER3 inhibition may play an important role in future therapy of HNSCC. Our results open the door for further mechanistic studies to better understand the role of HER3 in resistance to EGFR inhibitors in HNSCC.

Honokiol enhances paclitaxel efficacy in multi-drug resistant human cancer model through the induction of apoptosis.

Resistance to chemotherapy remains a major obstacle in cancer therapy. This study aimed to evaluate the molecular mechanism and efficacy of honokiol in inducing apoptosis and enhancing paclitaxel chemotherapy in pre-clinical multi-drug resistant (MDR) cancer models, including lineage-derived human MDR (KB-8-5, KB-C1, KB-V1) and their parental drug sensitive KB-3-1 cancer cell lines. In vitro analyses demonstrated that honokiol effectively inhibited proliferation in KB-3-1 cells and the MDR derivatives (IC50 ranging 3.35 ± 0.13 µg/ml to 2.77 ± 0.22 µg/ml), despite their significant differences in response to paclitaxel (IC50 ranging 1.66 ± 0.09 ng/ml to 6560.9 ± 439.52 ng/ml). Honokiol induced mitochondria-dependent and death receptor-mediated apoptosis in MDR KB cells, which was associated with inhibition of EGFR-STAT3 signaling and downregulation of STAT3 target genes. Combined treatment with honokiol and paclitaxel synergistically augmented cytotoxicity in MDR KB cells, compared with treatment with either agent alone in vitro. Importantly, the combined treatment significantly inhibited in vivo growth of KB-8-5 tumors in a subcutaneous model. Tumor tissues from the combination group displayed a significant inhibition of Ki-67 expression and an increase in TUNEL-positive cells compared with the control group. These results suggest that targeting multidrug resistance using honokiol in combination with chemotherapy drugs may provide novel therapeutic opportunities.

Chemoprevention of head and neck cancer with celecoxib and erlotinib: results of a phase ib and pharmacokinetic study.

Epidermal growth factor receptor (EGFR) and COX-2 inhibitors synergistically inhibit head and neck squamous cell carcinoma tumorigenesis in preclinical studies. We conducted a phase I and pharmacokinetic study with the erlotinib and celecoxib combination in patients with advanced premalignant lesions. Thirty-six subjects with oral leukoplakia, mild, moderate, or severe dysplasia, or carcinoma in situ were screened for study participation; 12 consented and received therapy for a median of 5.38 months. Erlotinib was escalated following a standard 3+3 design at 50, 75, and 100 mg orally daily and celecoxib was fixed at 400 mg twice daily for 6 months. Biopsy of lesions and cytobrush of normal mucosa were performed at baseline, 3, 6, and 12 months. Erlotinib pharmacokinetics were analyzed in 10 subjects. The maximum tolerated dose of erlotinib with celecoxib 400 mg BID was 50 mg per day with skin rash being the main observed toxicity. Overall histologic response rate was 63% (complete response, 43%; partial response, 14%; stable disease, 29%; and disease progression, 14%). With median follow-up of 36 months, mean time to progression to higher-grade dysplasia or carcinoma was 25.4 months. Downregulation of EGFR and p-ERK in follow-up biopsies correlated with response to treatment. Larger average erlotinib V/F (approximately 308 L) and CL/F (8.3 L/h) compared with previous studies may be related to relatively large average bodyweights. Average erlotinib t1/2 was 25.6 hours. Encouraging responses to the celecoxib and erlotinib combination correlated with EGFR pathway inhibition. Although erlotinib-related rash was the main limitation to dose escalation, the intervention was well tolerated.

Chemoprevention of head and neck cancer by simultaneous blocking of epidermal growth factor receptor and cyclooxygenase-2 signaling pathways: preclinical and clinical studies.

PURPOSE: We investigated the efficacy and underlying molecular mechanism of a novel chemopreventive strategy combining EGF receptor (EGFR) tyrosine kinase inhibitor (TKI) with cyclooxygenase-2 inhibitor (COX-2I). EXPERIMENTAL DESIGN: We examined the inhibition of tumor cell growth by combined EGFR-TKI (erlotinib) and COX-2I (celecoxib) treatment using head and neck cancer cell lines and a preventive xenograft model. We studied the antiangiogenic activity of these agents and examined the affected signaling pathways by immunoblotting analysis in tumor cell lysates and immunohistochemistry (IHC) and enzyme immunoassay (EIA) analyses on the mouse xenograft tissues and blood, respectively. Biomarkers in these signaling pathways were studied by IHC, EIA, and an antibody array analysis in samples collected from participants in a phase I chemoprevention trial of erlotinib and celecoxib. RESULTS: The combined treatment inhibited head and neck cancer cell growth significantly more potently than either single agent alone in cell line and xenograft models, and resulted in greater inhibition of cell-cycle progression at G1 phase than either single drug. The combined treatment modulated the EGFR and mTOR signaling pathways. A phase I chemoprevention trial of combined erlotinib and celecoxib revealed an overall pathologic response rate of 71% at time of data analysis. Analysis of tissue samples from participants consistently showed downregulation of EGFR, pERK, and pS6 levels after treatment, which correlated with clinical response. CONCLUSION: Treatment with erlotinib combined with celecoxib offers an effective chemopreventive approach through inhibition of EGFR and mTOR pathways, which may serve as potential biomarkers to monitor the intervention of this combination in the clinic. Clin Cancer Res; 19(5); 1244-56. ©2013 AACR.

Pharmacodynamics of DT-IgG, a dual-targeting antibody against VEGF-EGFR, in tumor xenografted mice.

PURPOSE: DT-IgG is a fully humanized dual-target therapeutic antibody being developed to simultaneously target epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF), important signaling molecules for tumor growth. The antitumor pharmacodynamics (PD) of DT-IgG was studied in nude mice bearing human tumor xenografts with different EGFR and VEGF expressions and K-ras oncogene status and compared with bevacizumab, cetuximab and bevacizumab + cetuximab. METHODS: Mice bearing human oral squamous cell carcinoma (Tu212), lung adenocarcinoma (A549), or colon cancer (GEO) subcutaneous xenografts were administered with the antibodies intraperitoneally (i.p.), and tumor volumes were measured versus time. Nonlinear mixed effects modeling (NONMEM) was used to study drug potencies (IC(50)) and variations in tumor growth. RESULTS: The PD models adequately described tumor responses for the antibody dose regimens. In vivo IC(50) values varied with EGFR and K-ras status. DT-IgG had a similar serum t (1/2) as cetuximab (~1.7 vs. 1.5 day), was more rapid than bevacizumab (~6 day), and had the largest apparent distribution volume (DT-IgG > cetuximab > bevacizumab). The efficacy of DT-IgG was comparable to bevacizumab despite lower serum concentrations, but was less than bevacizumab + cetuximab. CONCLUSIONS: A lower IC(50) of DT-IgG partially compensated for lower serum concentrations than bevacizumab and cetuximab, but may require higher doses for comparable efficacy as the combination. The model adequately predicted variations of tumor response at the DT-IgG doses tested and could be used for targeting specific tumor efficacies for future testing.

A dual-targeting antibody against EGFR-VEGF for lung and head and neck cancer treatment.

An antibody simultaneously targeting epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF), two major tumor growth-driving machineries, may provide a novel effective strategy for optimizing tumor targeting and maximizing potential clinical benefits. Human domain antibodies selected against VEGF and EGFR were formatted into a fully human dual-targeting IgG (DT-IgG) to directly target both antigens in a single molecule. We evaluated the efficacy of DT-IgG in comparison with bevacizumab and cetuximab alone and in combination in the lung cancer cell line A549 (low EGFR expression and KRAS mutant) and the head and neck squamous cell carcinoma (HNSCC) cell line Tu212 (high EGFR expression and KRAS wild type) in vitro and in vivo. DT-IgG suppressed Tu212 and A549 cell growth, inhibited EGFR activation and induced apoptosis as effectively as cetuximab, and neutralized VEGF as effectively as bevacizumab. DT-IgG induced EGFR-dependent VEGF internalization, constituting a novel antiangiogenesis mechanism. In xenograft models with lung and head and neck cancer cell lines, DT-IgG displayed efficacy equivalent to bevacizumab in diminishing tumor growth despite its short serum half-life (36 hr in rats) and both agents may constitute preferable alternatives to cetuximab in KRAS-mutant tumors. Immunofluorescence staining revealed that localization of DT-IgG was similar to that of cetuximab, largely associated with EGFR+tumor cells. Our proof of principle study suggests a DT-IgG against EGFR and VEGF as an alternative therapeutic strategy with potentially enhanced clinical benefit.

Detection of circulating tumor cells in human peripheral blood using surface-enhanced Raman scattering nanoparticles.

The detection and characterization of circulating tumor cells (CTC) holds great promise for personalizing medicine and optimizing systemic therapy. However, low specificity, low sensitivity, and the time consuming nature of current approaches have impeded clinical adoption. Here we report a new method using surface-enhanced Raman spectroscopy (SERS) to directly measure targeted CTCs in the presence of white blood cells. SERS nanoparticles with epidermal growth factor peptide as a targeting ligand have successfully identified CTCs in the peripheral blood of 19 patients with squamous cell carcinoma of the head and neck (SCCHN), with a range of 1 to 720 CTCs per milliliter of whole blood. Our technique may provide an important new clinical tool for management of patients with SCCHN and other cancers.

Enhanced anti-tumor activity by the combination of the natural compounds (-)-epigallocatechin-3-gallate and luteolin: potential role of p53.

Natural dietary agents have drawn a great deal of attention toward cancer prevention because of their wide safety margin. However, single agent intervention has failed to bring the expected outcome in clinical trials; therefore, combinations of chemopreventive agents are gaining increasingly popularity. In the present study, we investigated a combinatorial approach using two natural dietary polyphenols, luteolin and EGCG, and found that their combination at low doses (at which single agents induce minimal apoptosis) synergistically increased apoptosis (3-5-fold more than the additive level of apoptosis) in both head and neck and lung cancer cell lines. This combination also significantly inhibited growth of xenografted tumors in nude mice. The in vivo findings also were supported by significant inhibition of Ki-67 expression and increase in TUNEL-positive cells in xenografted tissues. Mechanistic studies revealed that the combination induced mitochondria-dependent apoptosis in some cell lines and mitochondria-independent apoptosis in others. Moreover, we found more efficient stabilization and ATM-dependent Ser(15) phosphorylation of p53 due to DNA damage by the combination, and ablation of p53 using shRNA strongly inhibited apoptosis as evidenced by decreased poly(ADP-ribose) polymerase and caspase-3 cleavage. In addition, we observed mitochondrial translocation of p53 after treatment with luteolin or the combination of EGCG and luteolin. Taken together, our results for the first time suggest that the combination of luteolin and EGCG has synergistic/additive growth inhibitory effects and provides an important rationale for future chemoprevention trials of head and neck and lung cancers.

Role of cyclooxygenase-2 in tumor progression and survival of head and neck squamous cell carcinoma.

Inhibition of cyclooxygenase-2 (COX-2) pathways may have significant implications for the prevention and treatment of head and neck squamous cell carcinoma (HNSCC). COX-2 is overexpressed in both premalignant lesions and invasive HNSCC. We examined COX-2 expression by immunohistochemistry in normal tissues, different stages of premalignant lesions, and carcinoma in situ (CIS). We also evaluated the correlation between COX-2 expression and clinical characteristics of HNSCC patients. Tissue specimens were obtained from the following: premalignant lesions from 25 subjects enrolled in a biochemoprevention trial, tumor samples collected at diagnosis from 38 HNSCC patients enrolled in an induction chemotherapy trial, and normal control tissues from 10 noncancer, nonsmoking subjects. COX-2 was expressed in early and intermediate stages of premalignant lesions, increasing first in the basal and parabasal layers, then lower spinous, and upper spinous layers. This correlation was noted in normal epithelium (P < 0.0001), histologically normal in-field samples (P < 0.0001), low-grade dysplasia (P = 0.024), and moderate-grade dysplasia (P = 0.009), but was lost in the majority of high-grade dysplasia/CIS (P = 0.896). COX-2 expression was also noted to increase progressively through the early stages of premalignancy, and to decrease in severe/CIS stage and invasive carcinoma. COX-2 expression in tumors from patients treated with induction chemotherapy was correlated with overall survival after controlling for clinical variables. These findings elucidate the differential expression pattern of COX-2 in stages of head and neck premalignant lesions and invasive carcinoma, supporting the rationale for COX-2 inhibition as an important strategy for cancer chemoprevention. Further validation of COX-2 expression is needed in prospective ongoing chemoprevention trials.

HFT-T, a targeting nanoparticle, enhances specific delivery of paclitaxel to folate receptor-positive tumors.

Nonspecific distribution of chemotherapeutic drugs (such as paclitaxel) is a major factor contributing to side effects and poor clinical outcomes in the treatment of human head and neck cancer. To develop novel drug delivery systems with enhanced efficacy and minimized adverse effects, we synthesized a ternary conjugate heparin-folic acid-paclitaxel (HFT), loaded with additional paclitaxel (T). The resulting nanoparticle, HFT-T, is expected to retain the antitumor activity of paclitaxel and specifically target folate receptor (FR)-expressing tumors, thereby increasing the bioavailability and efficacy of paclitaxel. In vitro experiments found that HFT-T selectively recognizes FR-positive human head and neck cancer cell line KB-3-1, displaying higher cytotoxicity compared to the free form of paclitaxel. In a subcutaneous KB-3-1 xenograft model, HFT-T administration enhanced the specific delivery of paclitaxel into tumor tissues and remarkably improved antitumor efficacy of paclitaxel. The average tumor volume in the HFT-T treatment group was 92.9 +/- 78.2 mm(3) vs 1670.3 +/- 286.1 mm(3) in the mice treated with free paclitaxel. Furthermore, paclitaxel tumors showed a resurgence of growth after several weeks of treatment, but this was not observed with HFT-T. This indicates that HFT-T could be more effective in preventing tumors from developing drug resistance. No significant acute in vivo toxicity was observed. These results indicate that specific delivery of paclitaxel with a ternary structured nanoparticle (HFT-T) targeting FR-positive tumor is a promising strategy to enhance chemotherapy efficacy and minimize adverse effects.

Phase I clinical and pharmacodynamic evaluation of oral CI-1033 in patients with refractory cancer.

PURPOSE: To determine the tolerability and pharmacokinetics of CI-1033 given daily for 7 days of a 21-day cycle. Tumor response and changes in erbB receptor tyrosine kinase activity in tumor and skin tissue were examined, and modulation of potential biomarkers in plasma was explored. DESIGN: This was a dose-finding phase I study in patients with advanced solid malignancies. Patients were evaluated for safety, pharmacokinetics, and tumor response. Pharmacodynamic markers, such as Ki67, p27, and erbB receptor status, were assessed in tumor and skin tissue using immunohistochemical and immunoprecipitation methodologies. Plasma biomarkers HER2, vascular endothelial growth factor, interleukin-8, and matrix metalloproteinase-9 were evaluated using immunologic techniques. RESULTS: Fifty-three patients were enrolled in the study. Dose-limiting toxicity (emesis, persistent rash, and mouth ulcer) was observed at 750 mg. The maximum tolerated dose was 650 mg. There were no confirmed objective responses. CI-1033 treatment showed down-regulation of epidermal growth factor receptor, HER2, and Ki67 in a variety of tumor tissues and up regulation of p27 in skin tissue. Plasma HER2 was reduced following CI-1033 administration, but no consistent change in vascular endothelial growth factor, interleukin-8, or matrix metalloproteinase-9 was noted. CI-1033 plasma concentrations were proportional to dose. CONCLUSION: The safety and pharmacokinetic profile of CI-1033 was favorable for multidose oral administration. Evidence of modulation of erbB receptor activity in tumor and skin tissue was accompanied by changes in markers of proliferation and cell cycle inhibition. Additional clinical trials are warranted in defining the role of CI-1033 in the treatment of cancer and further assessing the utility of antitumor markers.

Interaction between epidermal growth factor receptor- and cyclooxygenase 2-mediated pathways and its implications for the chemoprevention of head and neck cancer.

Head and neck squamous cell carcinoma is a well-known model for chemoprevention studies because of its field cancerization effect, its multistep carcinogenesis process, and the easy accessibility of biopsies to target lesions. With new understandings of head and neck carcinogenesis and the development of molecular targeted therapy, chemoprevention trials for head and neck squamous cell carcinoma have been rapidly updated. Cyclooxygenase-2 (COX-2) and epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors are gaining significant attention as potential chemopreventive agents. Both COX-2 and EGFR are involved in head and neck carcinogenesis. Targeting COX-2 and EGFR separately has shown promising antitumor activity. Recently, combinations of COX-2 and EGFR tyrosine kinase inhibitors have been reported to show synergistic/additive effects in preclinical studies. Because COX-2 and EGFR tyrosine kinase inhibitors are toxic as single agents in clinical trials, the combination of COX-2 and EGFR tyrosine kinase inhibitors used at lower doses seems more promising than monotherapy with either as a novel strategy in head and neck cancer chemoprevention.

Tumor growth inhibition by simultaneously blocking epidermal growth factor receptor and cyclooxygenase-2 in a xenograft model.

PURPOSE: Our previous study revealed that simultaneously targeting epidermal growth factor receptor (EGFR) tyrosine kinase and cyclooxygenase-2 (COX-2) additively or synergistically inhibited growth of squamous cell carcinoma of the head and neck (SCCHN) in vitro. However, an in vivo efficacy of this combined treatment in SCCHN has not been studied. EXPERIMENTAL DESIGN: Nude mice were pretreated with control (1% Tween 80), ZD1839 (50 mg/kg) alone, celecoxib (50 mg/kg) alone, or a combination of ZD1839 and celecoxib at the same dosages for 7 days before injection of a human SCCHN cell line Tu212. The animals were continuously treated with the agents 5 days a week for about 11 weeks. RESULTS: Tumor growth in the combined treatment was significantly inhibited compared with the control (P < 0.001), ZD1839 (P = 0.005), or celecoxib (P < 0.001). At the same time, a dramatic delay of tumor progression was observed in the combined treatment compared with all other three groups. Molecular analysis showed that the combined treatment significantly decreased prostaglandin E metabolite production. The cooperative effect of these two agents in combination was also associated with down-regulation of phosphorylated EGFR, phosphorylated extracellular signal-regulated kinase, and phosphorylated signal transducers and activators of transcription 3 levels and reduction of vascular endothelial growth factor and Ki-67 expression. Specifically, gene silencing of both EGFR and COX-2 by small interfering RNA further confirmed the cooperative antitumor effect. CONCLUSION: The current results strongly suggest that a cooperative effect of the combined treatment on tumor progression is mediated through blocking both EGFR- and COX-2-related pathways. This combination regimen may provide a promising strategy for cancer therapy and chemoprevention in SCCHN.

Use of mailed urine specimens in diagnosing urothelial carcinoma by cytology and DNA image analysis.

OBJECTIVE: To evaluate the usefulness of urine specimens collected via a mailer system and analyzed by cytology and DNA ploidy for the detection of urothelial carcinoma (UC). STUDY DESIGN: We retrospectively reviewed the diagnoses of 91 mailed urine specimens received from 72 patients, 67% of whom had a history of UC. The specimens were fixed in an equal volume of 50% ethanol solution before being mailed. The cytologic findings were interpreted in conjunction with DNA ploidy image analysis. We compared these initial diagnoses with those of follow-up examinations, including biopsies, cystoscopic findings and urinary cytology/DNA ploidy analyses. In addition, to examine the quality of the mailed samples, 3 cytopathologists performed a blinded assessment of cytologic slides of 20 mailed and 17 fresh urinary samples for bacterial overgrowth, urothelial degeneration, and presence of proteinaceous material and crystals. RESULTS: Follow-up was available for 68 of the 91 mailed specimens. The sensitivity for detecting UC using mailed urine specimens that were analyzed by both cytology and DNA ploidy was 61%, while specificity was 92%. The levels of bacterial overgrowth and urothelial degeneration in the mailed specimens were not significantly greater than in the fresh specimens (p>0.05). The levels of proteinaceous material and crystals were significantly higher in the mailed specimens (p<0.05). CONCLUSION: The results of combined cytology and DNA ploidy image analysis by using mailed urine samples were comparable to those of fresh urine specimens for the detection of UC reported in previous publications. The increase in crystals and proteinaceous material did not impede diagnostic interpretation. The mailing system is a reliable and convenient method of monitoring and triaging patients with UC or related symptoms.

Phase II study of a multidisciplinary approach with induction chemotherapy, followed by surgical resection, radiation therapy, and consolidation chemotherapy for unresectable malignant thymomas: final report.

PURPOSE: To evaluate tumor resectability after induction chemotherapy and to determine disease-free and overall survival rates of patients with locally advanced unresectable thymoma that received a multimodal treatment regimen. PATIENTS AND METHODS: Twenty-two patients (9 men, 13 women) with histologically confirmed invasive thymoma were treated with a multidisciplinary regimen consisting of three courses of induction chemotherapy, surgical resection, and radiation therapy, followed by three courses of consolidation chemotherapy. The median age was 47 years (range, 25-70). Eleven patients had stage III disease, 10 patients, stage IVA, and one patient, IVB. The most common histologic type was lymphocytic. Induction chemotherapy consisted of 500 mg/m(2) of cyclophosphamide on day 1; doxorubicin (20 mg/m(2) per day) on days 1-3 via continuous infusion (a total of 60 mg/m(2)); cisplatin (30 mg/m(2) per day) on days 1-3 (a total of 90 mg/m(2)); and prednisone (100 mg per day) on days 1-5. This cycle was repeated three times at 3-4-week intervals. Patients then underwent surgery for tumor resection and received radiotherapy. Consolidation chemotherapy given at 80% of the induction chemotherapy doses of cyclophosphamide, doxorubicin, and cisplatin and 100% of the dose of prednisone was then repeated every 3-4 weeks for a total of three courses. RESULTS: Induction chemotherapy produced major responses in 17 (77%) of the 22 patients including 3 (14%) complete responses (CR) and 14 (63%) partial responses (PR). Twenty-one patients underwent surgical exploration: 16 (76%) had complete resection and 5 (24%) had incomplete resection; one patient refused surgery. All 22 patients received radiation therapy. Nineteen of 22 patients completed the planned therapy, and all but one had completed consolidation chemotherapy at the time of analysis. With a median follow-up time of 50.3 months, 18 of the 19 patients who completed the multidisciplinary approach were disease-free. Of the 22 patients originally registered, 20 were alive at the time of analysis (one patient died of endocarditis, and one died of recurrent disease). The overall survival rate was 95% at 5 years (95% confidence interval (CI), 0.87-1.0) and 79% at 7 years (95% CI, 0.55-1.0). The progression-free survival rates were 77% at 5 years (95% CI, 0.58-1.0) and 77% at 7 years (95% CI, 0.58-1.0). The major side effect from induction and consolidation chemotherapy was myelosuppression. Nine patients experienced grade III/IV neutropenia, which included neutropenic fever in two patients, and grade III thrombocytopenia in two patients. The most common nonhematologic side effects were fatigue, nausea and vomiting, and decreased appetite. One patient experienced acute respiratory distress syndrome after surgical resection and required a prolonged hospitalization. No patients developed cardiac toxic effects, and no surgical mortality occurred. CONCLUSIONS: The use of induction chemotherapy to optimize surgical resectability of thymoma followed by radiation therapy and consolidation chemotherapy lead to good control of residual disease and high overall survival rates. We believe that this combined multidisciplinary approach prolongs lives and may cure locally advanced unresectable malignant thymomas. Future prospective multi-institutional studies are needed to further verify or define the best treatment for this patient population.

Cytomorphologic spectrum of small lymphocytic lymphoma in patients with an accelerated clinical course.

BACKGROUND: Patients with transformed large cell lymphoma (TLCL), or Richter syndrome, and patients with small lymphocytic lymphoma/leukemia (SLL) in accelerated phase (SLL-AP) frequently undergo fine-needle aspiration (FNA) to elucidate the cause of massive lymphadenopathy and a worsening clinical condition. It is well known that patients with Richter syndrome have poor clinical outcomes as a result of the biologic aggressiveness of the transformed large cells that often are refractory to therapy. The objective of this study was to evaluate reliable criteria for recognizing SLL-AP and SLL in transformation that are needed to determine the appropriate clinical management of individual patients. METHODS: FNA specimens from 20 patients with SLL, 26 patients with SLL-AP, and 13 patients with TLCL or Richter syndrome were identified based on the reported diagnosis. Patients were included only if they had histologically confirmed SLL that also satisfied the immunophenotypic criteria of CD5/CD23 coexpression with negative CD10 and < 55% prolymphocytes. On the basis of an initial blind review of routinely stained slides from the FNA specimens, the authors defined four groups based on a consensus review diagnosis: Group 1: typical SLL; Group 2: low-grade SLL-AP; Group 3: high-grade SLL-AP; and Group 4: TLCL. Cytomorphologic features, including the proportion of intermediate-to-large cells with prominent nucleoli and intermediate-sized plasmacytoid lymphoid cells, increased numbers of mitotic figures, the presence of apoptotic bodies and necrosis, and a myxoid/dirty background, were considered features that indicated an accelerated phase. Specimens that had more than two grades of diagnostic discrepancy compared with the original reported diagnosis were reexamined using a Ki-67 immunostaining labeling index to reach a final review diagnosis. RESULTS: On the basis of the review diagnosis, the specimens were recategorized as follows: Group 1: 20 patients with typical SLL; Group 2: 13 patients with low-grade-SLL-AP; Group 3: 16 patients with high-grade SLL-AP; and Group 4: 10 patients with TLCL. The mean Ki-67 labeling index (%) was correlated with the morphologic progression of SLL as follows: Group 1: 11%; Group 2: 16%; Group 3: 34%; and Group 4: 48%. The follow-up by subsequent FNA or biopsy demonstrated large cell transformation in 25% of patients in Group 1, 25% of patients in Group 2, and 100% of patients in Group 3. Among the clinical parameters, increased value of serum beta2-microglobulin and lactic dehydrogenase (LDH) levels were found to be well correlated with the cytomorphologic progression of SLL/chronic lymphocytic leukemia. CONCLUSIONS: Aspirates from patients who had signs and symptoms clinically suspicious for SLL-AP demonstrated a spectrum of cytomorphologic features, ranging from low-grade SLL-AP and high-grade SLL-AP to TLCL. The results showed that the Ki-67 labeling index, as determined by immunohistochemical studies in FNA specimens, and the levels of serum beta2-microglobulin and LDH are valuable diagnostic adjuncts for recognizing a subset of patients with SLL-AP or SLL in transformation who may require more aggressive therapy.

Detection of numerical aberrations of chromosomes 7 and 9 in cytologic specimens of pleural malignant mesothelioma.

BACKGROUND: Accurate diagnosis of malignant mesothelioma (MM) in effusion fluid specimens can be challenging. Previous cytogenetic studies demonstrated that chromosomal aneusomy is commonly associated with MM. The authors attempted to determine whether the detection of numerical chromosomal aberrations in the effusion fluid specimens could be a useful diagnostic adjunct in morphologically inconclusive cases for MM. METHODS: The authors analyzed 17 cytologic specimens (15 pleural fluids, 2 fine-needle aspirates) of histologically confirmed MM obtained from 16 patients using fluorescent in situ hybridization (FISH) with centromeric chromosome 7 and 9 probes. Seventeen benign pleural effusion fluid specimens containing reactive/benign mesothelial (RM) cells were used as controls. After destaining of Diff-Quik-stained smears, the slides were hybridized using a standard FISH protocol. One hundred cells/probe/case were counted manually. RESULTS: For chromosome 7, polysomy (defined as >/= 3 signals/nucleus in >10% of the cells) was present in 88% and 0% of the MM and the RM cases, respectively. Trisomy (defined as 3 signals/nucleus in > 5% of the cells) was present in 77% and 0% of the MM and the RM cases, respectively. Tetrasomy (defined as 4 signals/nucleus in > 10% of the cells) was present in 29% and 0% of the MM and the RM cases, respectively. The mean values of positive cells showing polysomy 7 and trisomy 7 were markedly different in MM and RM cases (39% vs. 4% and 31% vs. 1%, respectively). The frequency of polysomy 9 and trisomy 9 in MM and RM cases was 69% vs. 13% and 62% vs. 6%, respectively. CONCLUSIONS: These results suggest that polysomy of chromosome 7 or 9 is a common finding in MM but uncommon in RM. Therefore, in conjunction with cytomorphologic and clinical suspicion of MM, detection of numerical chromosomal aberrations of chromosome 7 using FISH with routine cytologic smears can be a valuable diagnostic adjunct.

Detection of a subset of CD30+ anaplastic large cell lymphoma by interphase fluorescence in situ hybridization.

T/null-cell anaplastic large cell lymphoma (ALCL) is a morphologically and clinically heterogeneous group of non-Hodgkin's lymphoma; to date several morphologic variants have been described on histologic specimens. However, the cytologic features of these variants in the fine-needle aspiration (FNA) specimens have not been well evaluated. The t(2;5)(p23;q35) has been identified in a subset of T/null-ALCL and is known to be associated with a favorable prognosis. We reviewed the cytomorphologic characteristics in 24 FNA specimens of ALCL. In all cases, the diagnosis was confirmed on histologic specimens, and immunohistochemical studies for anaplastic lymphoma kinase (ALK) protein expression were performed on the aspirates. The presence of ALK breakpoints were evaluated in nine cases, using a DNA break-apart probe on chromosome 2 covering the ALK gene by fluorescence in situ hybridization (FISH) techniques. Two hundred cells per case were examined. The results were expressed as the percentage of cells containing more than two signals of chromosome 2 to the total number of cells counted. FNA sites included lymph nodes (20), lung (2), breast (1), and soft tissue (1). The median age of the patients was 56 yr (range, 17-75 yr). Twenty cases had systemic involvement; in four cases, skin was the primary site with secondary involvement of the lymph nodes. All cases were CD30(+) by immunohistochemistry; 20 were of T-cell phenotype and 4 were null cell type. The cytologic evaluation revealed typical anaplastic morphology (common type) with many "hallmark cells" in 16 (67%) cases. Other morphologic variants identified were small cell pattern in five cases, monomorphic pattern in two cases, and lymphohistiocytic pattern in one case. FISH studies showed that six (66.7%) of nine cases had at least two signals of chromosome 2, consistent with ALK breakpoints. With careful cytomorphologic evaluation in conjunction with appropriate immunohistochemical studies, a diagnosis of ALCL can be confidently made in the FNA specimens in the cellular aspirates and its morphologic variants also can be recognized. Furthermore, the FNA specimen is suitable in detecting ALK breakpoints by FISH study, permitting rapid identification of a subset of patients with ALCL, who may have a favorable prognosis. Using a commercially available probe, detection of ALK breakpoints in the FNA specimens is simple and can be a useful diagnostic adjunct in cases where distinction from other lymphomas or lymphoid lesions is morphologically difficult.

Advances in the management of malignant mesothelioma.

Malignant mesotheliomas are very aggressive tumors that originate from mesothelial cells, which form the serosal lining of the pleura, pericardial, and peritoneal cavities. Finding effective chemotherapeutic treatment for malignant mesothelioma is a challenge. There is no standard treatment because this tumor is relatively resistant to therapy. A resurgence of interest has been expressed in novel therapies and conventional treatments used in different ways. Several treatment modalities have been studied, including chemotherapy, radiotherapy, surgery, and immunotherapy. Chemotherapy can be administered systemically or directly into the pleura. This review presents the results of the most recent trials and highlights the most promising advances in the battle against this aggressive disease.