David S. Schrump

Surgical Management of Renal Cell Carcinoma With Inferior Vena Cava Tumor Thrombus

BACKGROUND The optimal management of patients with renal cell carcinoma with inferior vena cava tumor thrombus remains unresolved. Traditional approaches have included resection with or without the use of cardiopulmonary bypass. Chemotherapy has played a minor role except for biotherapeutic agents used for metastatic disease. METHODS From January 1989 to January 1996, 37 patients with renal cell carcinoma and inferior vena cava tumor thrombus underwent surgical resection. The 27 men and 10 women had a median age of 57 years (range, 29 to 78 years). Thirty-six patients presented with symptoms; 21 had hematuria. Distant metastases were present in 12 patients. Tumor thrombi extended to the infrahepatic inferior vena cava (n = 16), the intrahepatic inferior vena cava (n = 16), the suprahepatic inferior vena cava (n = 3), and into the right atrium (n = 2). All tumors were resected by inferior vena cava isolation and, when necessary, extended hepatic mobilization and Pringle maneuver, with primary or patch closure of the vena cavotomy. Cardiopulmonary bypass was necessary in only 2 patients with intraatrial thrombus. RESULTS Complications occurred in 11 patients, and 1 patient died 2 days postoperatively of a myocardial infarction (mortality, 2.7%). Twenty patients are alive; overall 2- and 5-year survival rates were 61.7% and 33.6%, respectively. For patients without lymph node or distant metastases (stage IIIa), 2- and 5-year survival rates were 74% and 45%, respectively. The presence of distant metastatic disease (stage IV) at the time of operation did not have a significant adverse effect on survival, as reflected by 2- and 5-year survival rates of 62.5% and 31.3%, respectively. Lymph node metastases (stage IIIc) adversely affected survival as there were no long-term survivors. CONCLUSIONS Resection of an intracaval tumor thrombus arising from renal cell carcinoma can be performed safely and can result in prolonged survival even in the presence of metastatic disease. In our experience, extracorporeal circulatory support was required only when the tumor thrombus extended into the heart.

Epigenomic alterations and gene expression profiles in respiratory epithelia exposed to cigarette smoke condensate

Limited information is available regarding epigenomic events mediating initiation and progression of tobacco-induced lung cancers. In this study, we established an in vitro system to examine epigenomic effects of cigarette smoke in respiratory epithelia. Normal human small airway epithelial cells and cdk-4/hTERT-immortalized human bronchial epithelial cells (HBEC) were cultured in normal media with or without cigarette smoke condensate (CSC) for up to 9 months under potentially relevant exposure conditions. Western blot analysis showed that CSC mediated dose- and time-dependent diminution of H4K16Ac and H4K20Me3, while increasing relative levels of H3K27Me3; these histone alterations coincided with decreased DNA methyltransferase 1 (DNMT1) and increased DNMT3b expression. Pyrosequencing and quantitative RT–PCR experiments revealed time-dependent hypomethylation of D4Z4, NBL2, and LINE-1 repetitive DNA sequences; up-regulation of H19, IGF2, MAGE-A1, and MAGE-A3; activation of Wnt signaling; and hypermethylation of tumor suppressor genes such as RASSF1A and RAR-β, which are frequently silenced in human lung cancers. Array-based DNA methylation profiling identified additional novel DNA methylation targets in soft-agar clones derived from CSC-exposed HBEC; a CSC gene expression signature was also identified in these cells. Progressive genomic hypomethylation and locoregional DNA hypermethylation induced by CSC coincided with a dramatic increase in soft-agar clonogenicity. Collectively, these data indicate that cigarette smoke induces ‘cancer-associated’ epigenomic alterations in cultured respiratory epithelia. This in vitro model may prove useful for delineating early epigenetic mechanisms regulating gene expression during pulmonary carcinogenesis.

Phase I Study of Decitabine-Mediated Gene Expression in Patients with Cancers Involving the Lungs, Esophagus, or Pleura

Purpose: The DNA methylation paradox, manifested as derepression of cancer-testis antigens, and silencing of tumor suppressors during malignant transformation, provides the rationale for the utilization of chromatin remodeling agents for cancer therapy. A phase I trial was done to examine pharmacokinetics, toxicities, and gene expression mediated by 5-aza-2′-deoxycytidine (DAC) in patients with thoracic malignancies. Experimental Design: Thirty-five patients with cancers refractory to standard therapy received continuous 72-hour DAC infusions using a phase I dose-escalation schema. Each full course of therapy consisted of two identical 35-day cycles. Plasma DAC levels were evaluated by liquid chromatography-mass spectrometry techniques. Quantitative reverse transcription-PCR, methylation-specific PCR, and immunohistochemical techniques were used to evaluate NY-ESO-1, MAGE-3, and p16 expression in tumor biopsies. Long oligonucleotide arrays were used to evaluate gene expression profiles in laser-captured tumor cells before and after DAC exposure. Results: Thirty-five patients were evaluable for toxicities; 25 were evaluable for treatment response. Myelosuppression constituted dose-limiting toxicity. The maximum tolerated dose of DAC was 60 to 75 mg/m2 depending on the number of prior cytotoxic chemotherapy regimens. No objective responses were observed. Plasma DAC concentrations approximated thresholds for gene induction in cultured cancer cells. Target gene induction was observed in 36% of patients. Posttreatment antibodies to NY-ESO-1 were detected in three patients exhibiting NY-ESO-1 induction in their tumor tissues. Complex, heterogeneous gene expression profiles were observed in pretreatment and posttreatment tissues. Conclusion: Prolonged DAC infusions can modulate gene expression in primary thoracic malignancies. These findings support further evaluation of DNA-demethylating agents alone or in combination with other regimens targeting induced gene products for the treatment of these neoplasms.

Sequential 5-Aza-2 deoxycytidine-depsipeptide FR901228 treatment induces apoptosis preferentially in cancer cells and facilitates their recognition by cytolytic T lymphocytes specific for NY-ESO-1.

Global alterations in chromatin structure profoundly influence gene expression in thoracic neoplasms, silencing tumor suppressors while facilitating the expression of various cancer testis antigens such as NY-ESO-1. Although recent studies have shown that histone deacetylase inhibitors can potentiate tumor suppressor gene induction mediated by demethylating agents in cancer cells, the ability of these agents to augment cancer testis antigen expression have not been fully defined. The authors designed the current study to determine whether the histone deacetylase inhibitor, depsipeptide FR901228 (DP), could enhance NY-ESO-1 induction mediated by the DNA demethylating agent 5-Aza-2′-deoxycytidine (DAC) in cell lines established primarily from thoracic cancers. Quantitative reverse-transcriptase polymerase chain reaction analysis revealed that, under exposure conditions potentially achievable in clinical settings, DAC dramatically induced NY-ESO-1 expression in cultured cancer lines. DP alone mediated negligible target gene induction but significantly augmented DAC-mediated induction of NY-ESO-1. After DAC or sequential DAC–DP treatment, HLA-A*0201 cancer cells were recognized by an HLA-A*0201 CTL specific for NY-ESO-1. Although sequential DAC/DP exposure did not uniformly enhance immune recognition of target cells compared with DAC alone, this treatment mediated profound induction of apoptosis in cancer cells but not normal human bronchial epithelia. The apoptotic effects of DAC, DP, or sequential DAC–DP did not correlate in an obvious manner with histology, or the magnitude of NY-ESO-1 induction in cancer cells. Although the mechanisms have not been fully defined, sequential DAC–DP treatment may be a novel strategy to augment antitumor immunity in cancer patients.

Bortezomib and Depsipeptide Sensitize Tumors to Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand: A Novel Method to Potentiate Natural Killer Cell Tumor Cytotoxicity

The proteasome inhibitor, bortezomib, and the histone deacetylase inhibitor, depsipeptide (FK228), up-regulate tumor death receptors. Therefore, we investigated whether pretreatment of malignant cells with these agents would potentiate natural killer (NK)-mediated tumor killing. NK cells isolated from healthy donors and patients with cancer were expanded in vitro and then tested for cytotoxicity against tumor cell lines before and after exposure to bortezomib or depsipeptide. In 11 of 13 (85%) renal cell carcinoma cell lines and in 16 of 37 (43%) other cancer cell lines, exposure to these drugs significantly increased NK cell-mediated tumor lysis compared with untreated tumor controls (P < 0.001). Furthermore, NK cells expanded from patients with metastatic renal cell carcinoma were significantly more cytotoxic against autologous tumor cells when pretreated with either bortezomib or depsipeptide compared with untreated tumors. Tumors sensitized to NK cell cytotoxicity showed a significant increase in surface expression of DR5 [tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-R2; P < 0.05]; in contrast, surface expression of MHC class I, MIC-A/B, DR4 (TRAIL-R1), and Fas (CD95) did not change. The enhanced susceptibility to NK cell killing was completely abolished by blocking TRAIL on NK cells, and partially abolished by blocking DR5 on tumor cells. These findings show that drug-induced sensitization to TRAIL could be used as a novel strategy to potentiate the anticancer effects of adoptively infused NK cells in patients with cancer.

A Spectrum of Severe Familial Liver Disorders Associate with Telomerase Mutations

Background Telomerase is an enzyme specialized in maintaining telomere lengths in highly proliferative cells. Loss-of-function mutations cause critical telomere shortening and are associated with the bone marrow failure syndromes dyskeratosis congenita and aplastic anemia and with idiopathic pulmonary fibrosis. Here, we sought to determine the spectrum of clinical manifestations associated with telomerase loss-of-function mutations. Methodology/Principal Findings Sixty-nine individuals from five unrelated families with a variety of hematologic, hepatic, and autoimmune disorders were screened for telomerase complex gene mutations; leukocyte telomere length was measured by flow fluorescence in situ hybridization in mutation carriers and some non-carriers; the effects of the identified mutations on telomerase activity were determined; and genetic and clinical data were correlated. In six generations of a large family, a loss-of-function mutation in the telomerase enzyme gene TERT associated with severe telomere shortening and a range of hematologic manifestations, from macrocytosis to acute myeloid leukemia, with severe liver diseases marked by fibrosis and inflammation, and one case of idiopathic pulmonary fibrosis but not with autoimmune disorders. Additionally, we identified four unrelated families in which loss-of-function TERC or TERT gene mutations tracked with marrow failure, pulmonary fibrosis, and a spectrum of liver disorders. Conclusions/Significance These results indicate that heterozygous telomerase loss-of-function mutations associate with but are not determinant of a large spectrum of hematologic and liver abnormalities, with the latter sometimes occurring in the absence of marrow failure. Our findings, along with the link between pulmonary fibrosis and telomerase mutations, also suggest a common pathogenic mechanism for fibrotic diseases in which defective telomere repair plays important role.

Cytotoxicity mediated by histone deacetylase inhibitors in cancer cells: mechanisms and potential clinical implications.

Aberrant expression of epigenetic regulators of gene expression contributes to initiation and progression of cancer. During recent years, considerable research efforts have focused on the role of histone acetyltransferases (HATs) and histone deacetylases (HDACs) in cancer cells, and the identification of pharmacologic agents that modulate gene expression via inhibition of HDACs. The following review highlights recent studies pertaining to HDAC expression in cancer cells, the plieotropic mechanisms by which HDAC inhibitors (HDACi) mediate antitumor activity, and the potential clinical implications of HDAC inhibition as a strategy for cancer therapy.

A Multidisciplinary Approach to Therapy for Unresectable Malignant Thymoma

Malignant thymoma is a rare mediastinal tumor [1]. The important prognostic factors in this condition are disease stage and completeness of surgical resection [2-4]. In the early stages, the tumor can be completely resected. However, complete resection of advanced-stage tumors has been difficult [4], and surgery in such cases has not substantially changed the biology of the tumor because gross residual disease or microinvasion of surrounding structures, including the pleura and the pericardium, has led to a high incidence of eventual recurrence. Ki-67 has been used as an important indicator of the biological behavior of tumor cells [5, 6]. In cases of invasive thymoma, expression of Ki-67 has correlated with proliferating activity of tumor cells and clinical stage. There is also a general correlation between the labeling index of Ki-67 and both invasiveness and histologic subtype [6]. We investigated whether such correlations were valid in tissue samples obtained from our patients. Radiation therapy has been important in the management of patients with advanced thymoma. Postoperative irradiation of tumors graded stage II or beyond has been shown to reduce the risk for recurrence in patients with invasive thymoma who have had complete resection [7, 8]. However, in cases of incomplete resection, postoperative irradiation has not substantially changed overall survival rates; disease tends to recur both locally and distantly [9, 10]. Chemotherapy has shown significant antitumor activity against unresectable, recurrent, or metastatic thymomas [11, 12]. Our experience and that of others has indicated that overall major response rates with combination chemotherapy based on cisplatin and doxorubicin were between 50% and 90% in chemotherapy-naive patients [11-14]. To improve tumor resectability and to determine the disease-free and overall survival times of patients with locally advanced unresectable thymoma, we designed a prospective study of a multimodal treatment regimen. Methods Patients Patients with Masaoka stage III or IVA tumors [2] were eligible for this study. The resectability of disease was determined by the thoracic surgeons before patients entered the protocol. Patients had a performance status of less than 2 on the Zubrod scale, bidimensional measurable disease, adequate bone marrow (absolute granulocyte counts > 1500 cells/mm3 and platelet counts > 100 000 cells/mm3), adequate hepatic function (serum total bilirubin level < 1.5 mg/dL [25.65 mol/L]), and adequate renal function (serum creatinine level < 1.5 mg/dL [132.6 mol/L]). Left ventricular ejection fraction in all participants was examined by using two-dimensional echocardiography before treatment. Signed informed consent was obtained, and the protocol was approved by the institutional review board at the M.D. Anderson Cancer Center. Protocol We designed this prospective study for patients with pathologically confirmed malignant thymoma. The study schema consisted of three courses of induction chemotherapy, surgical resection, radiation therapy, and three courses of consolidation chemotherapy (Figure 1). Figure 1. Treatment schema and outcome for patients with locally advanced unresectable thymoma (stage III or IVA). Induction chemotherapy consisted of cyclophosphamide, 500 mg/m2, on day 1; continuous infusion of doxorubicin, 20 mg/m2 per day, on days 1 to 3 (total, 60 mg/m2); cisplatin, 30 mg/m2 per day, on days 1 to 3 (total, 90 mg/m2); and prednisone (100 mg/d for 5 days). This cycle was repeated three times at 3- to 4-week intervals. Prophylactic granulocyte colony-stimulating factor was not used. After induction chemotherapy, we assessed clinical response by measuring tumor size on computed tomography [13]. Within 3 to 4 weeks after the last chemotherapy cycle, we used computed tomography to assess tumor resectability. Resection was done during this exploration, and the pathologic specimen was assessed for the degree of tumor necrosis. Within 3 to 6 weeks of surgery, patients who had had complete resection and whose tumors were at least 80% necrotic began radiation therapy with a total dose of 50 Gy. Patients were irradiated with a total dose of 60 Gy if resection was incomplete or if less than 80% of the tumor was necrotic. Consolidation chemotherapy with 80% doses of cyclophosphamide, doxorubicin, and cisplatin and a 100% dose of prednisone was repeated every 3 to 4 weeks for three courses. Ki-67 Expression After induction chemotherapy, 11 patients had surgical resection (1 patient did not have surgery) and 16 tissue samples were obtained. All samples underwent immunohistochemical analysis so that we could determine whether Ki-67 expression correlated with tumor necrosis after chemotherapy. Anti-Ki-67 antibody (clone MIB1) was obtained from Zymed Laboratory, Inc. (San Francisco, California), and immunohistochemistry was done by using the standard procedure [6]. Statistical Analysis Overall survival was measured from the date of registration for induction chemotherapy to the date of last follow-up or death. Disease-free survival was measured from the date of last treatment to the date of last follow-up or recurrence. We estimated survival curves by using the method of Kaplan and Meier [15]. We calculated the Pearson correlation coefficient between Ki-67 expression and percentage of tumor necrosis, using the average if a participant had multiple observations. All calculations were done by using SAS software (SAS Institute, Inc., Cary, North Carolina). Results From February 1990 to December 1996, a total of 13 patients were consecutively enrolled in the study. One of these patients was later deemed ineligible because a final pathologic diagnosis of thymic carcinoma was made. Patient characteristics are shown in Table 1. Table 1. Patient Characteristics Induction chemotherapy produced three complete responses, eight partial responses, and one minor response, for an overall major response rate of 92%. Eleven patients had surgical exploration; 1 patient refused surgical resection. Tumors were completely resected in all 3 patients whose disease responded completely and in 6 of the 8 patients whose disease responded partially; thus, 9 of 11 patients (82%) were responders. Resection was incomplete in 2 patients (18%), including 1 of the patients with partial response and the 1 patient with a minor response. All pathologic specimens obtained during surgery were evaluated for extent of tumor necrosis. Two of the three complete responders had 100% tumor necrosis, and one complete responder and one partial responder had tumor necrosis greater than 80%. Seven other patients had tumor necrosis less than 80%. All 12 patients received radiation therapy. The 4 patients (36%) whose tumors had more than 80% necrosis on complete resection received 50 Gy; the 7 patients who had less than 80% necrosis or had incomplete resection received 60 Gy. The patient who refused surgery received only 54 Gy of the planned 60 Gy because of insufficient compliance. All 11 patients who underwent surgery had consolidation chemotherapy (Figure 1). Overall, after completion of the planned therapy, 10 patients remain disease free at a median follow-up period of 43 months (disease-free survival rate at 7 years, 73%). Two patients who had incomplete tumor resection had locoregional recurrent disease but are still alive (overall survival rate at 7 years, 100%). The major side effect during induction and consolidation chemotherapy was myelosuppression. One patient required a prophylactic platelet transfusion but had no bleeding. Other hematologic side effects were modest. The most common nonhematologic side effects were fatigue, nausea and vomiting, and decreased appetite. Two patients developed neutropenic fever during induction chemotherapy but recovered fully with intravenous antibiotics. One patient developed radiation-induced mild pneumonitis and esophagitis. No patients developed cardiac toxicity. No surgical morbidity or mortality occurred. We correlated the degree of tumor necrosis with Ki-67 expression in the samples after induction chemotherapy. The samples with tumor necrosis greater than 80% expressed a minimal degree of Ki-67 (mean labeling index, 0.02 [range, 0.01 to 0.03]). The overall correlation between tumor necrosis and Ki-67 expression was high (Pearson r = 0.88). Discussion Complete surgical resection is an important prognostic factor for locally advanced malignant thymoma [16]. It is critical to convert locally advanced unresectable tumors (stage III and IVA) to resectable tumors. Complete resection of these advanced tumors is often unfeasible because the tumors invade adjacent mediastinal structures, including major blood vessels and the pericardium. Preoperative (neoadjuvant or induction) chemotherapy may enhance tumor resectability. With this goal in mind, we administered induction chemotherapy to all patients and achieved a major response rate of 92%. Our results and those of others [17-19] suggest that malignant thymoma is highly responsive to chemotherapy. More important, disease in all of our patients became resectable with this preoperative chemotherapy. Another striking finding was the degree of tumor necrosis in tissue specimens. Almost half of the patients whose tumors were completely resected had tumor necrosis greater than 80% in resected specimens. All patients received postoperative radiation therapy. Komaki and Cox [7] summarized data from the literature showing that recurrence (local or distant) was found in 25% of patients who received postoperative radiation therapy and 57% of patients who did not receive this therapy. These data clearly show that postoperative radiation therapy for tumors graded stage II or higher further reduces risk for recurrence in patients who have had even complete resection. The total dose of radiation therapy in the postoperative setting has not been well established in thymoma; we used doses similar to those that palli

A TCR Targeting the HLA-A*0201–Restricted Epitope of MAGE-A3 Recognizes Multiple Epitopes of the MAGE-A Antigen Superfamily in Several Types of Cancer

Adoptive immunotherapy using TCR-engineered PBLs against melanocyte differentiation Ags mediates objective tumor regression but is associated with on-target toxicity. To avoid toxicity to normal tissues, we targeted cancer testis Ag (CTA) MAGE-A3, which is widely expressed in a range of epithelial malignancies but is not expressed in most normal tissues. To generate high-avidity TCRs against MAGE-A3, we employed a transgenic mouse model that expresses the human HLA-A*0201 molecule. Mice were immunized with two HLA-A*0201–restricted peptides of MAGE-A3: 112–120 (KVAELVHFL) or MAGE-A3: 271–279 (FLWGPRALV), and T cell clones were generated. MAGE-A3–specific TCR α- and β-chains were isolated and cloned into a retroviral vector. Expression of both TCRs in human PBLs demonstrated Ag-specific reactivity against a range of melanoma and nonmelanoma tumor cells. The TCR against MAGE-A3: 112–120 was selected for further development based on superior reactivity against tumor target cells. Interestingly, peptide epitopes from MAGE-A3 and MAGE-A12 (and to a lesser extent, peptides from MAGE-A2 and MAGE-A6) were recognized by PBLs engineered to express this TCR. To further improve TCR function, single amino acid variants of the CDR3 α-chain were generated. Substitution of alanine to threonine at position 118 of the α-chain in the CDR3 region of the TCR improved its functional avidity in CD4 and CD8 cells. On the basis of these results, a clinical trial is planned in which patients bearing a variety of tumor histologies will receive autologous PBLs that have been transduced with this optimized anti–MAGE-A3 TCR.

Clinical and molecular responses in lung cancer patients receiving Romidepsin

Purpose: Our preclinical experiments indicated that Romidepsin (Depsipeptide FK228; DP) mediates growth arrest and apoptosis in cultured lung cancer cells. A phase II trial was done to examine clinical and molecular responses mediated by this histone deacetylase inhibitor in lung cancer patients. Experimental Design: Nineteen patients with neoplasms refractory to standard therapy received 4-h DP infusions (17.8 mg/m2) on days 1 and 7 of a 21-day cycle. Each full course of therapy consisted of two identical 21-day cycles. Plasma DP levels were evaluated by liquid chromatography–mass spectrometry techniques. A variety of molecular end points were assessed in tumor biopsies via immunohistochemistry techniques. Long oligo arrays were used to examine gene expression profiles in laser-captured tumor cells before and after DP exposure, relative to lung cancer cells and adjacent normal bronchial epithelia from patients undergoing pulmonary resections. Results: Nineteen patients were evaluable for toxicity assessment; 18 were evaluable for treatment response. Myelosuppression was dose limiting in one individual. No significant cardiac toxicities were observed. Maximum steady-state plasma DP concentrations ranged from 384 to 1,114 ng/mL. No objective responses were observed. Transient stabilization of disease was noted in nine patients. DP enhanced acetylation of histone H4, increased p21 expression in lung cancer cells, and seemed to shift global gene expression profiles in these cells toward those detected in normal bronchial epithelia. Conclusion: Although exhibiting minimal clinical efficacy at this dose and schedule, DP mediates biological effects that may warrant further evaluation of this histone deacetylase inhibitor in combination with novel-targeted agents in lung cancer patients.