Gemcitabine twice weekly as a radiosensitiser for the treatment of brain metastases in patients with carcinoma: a phase I study

Conventional treatment for brain metastases (BM) is whole-brain radiotherapy (WBRT). Efficacy is poor. It might be increased by a potent radiosensitiser such as gemcitabine which is believed to cross the disrupted blood-brain barrier. Primary objective of this study was to determine the maximum tolerated dose (MTD) of twice weekly gemcitabine given concurrently with WBRT. Patients with BM from carcinoma were included. The dose of WBRT was 30 Gys (10 daily fractions). Gemcitabine was given 2-4 h prior to WBRT on days 1 and 8 for the first cohort of patients and then on days 1, 4, 8 and 11. Starting dose was 25 mg m(-2), escalated by 12.5 mg m(-2) increments. At least three patients were included per level. Dose limiting toxicity (DLT) was defined as grade 4 haematological or grade > or =3 nonhaematological toxicity. A total of 25 patients were included; 74% had a PS 1 (ECOG). In all, 23 had non-small-cell lung cancer, six colorectal, four breast, two renal cell and one oesophageal carcinoma. A total of 92% had concurrent extracranial disease. Six had single BM, 13 had two or three BM and six multiple. Up to 50 mg m(-2) (level 4) no DLT was observed. At 62.5 mg m(-2), one out of six patients developed DLT (thrombocytopenia-bleeding). The next dose level (75 mg m(-2)) was abandoned after grade 4 bone marrow toxicity (fatal neutropenic sepsis) was seen in one out of two patients. So that the dose of 50 mg m(-2) will be taken forward for further study.     

Evaluation of methods for red marrow dosimetry based on patients undergoing radioimmunotherapy

Red marrow dosimetry is essential during radioimmunotherapy and a reliable method is essential in order to find a measure correlated to the toxic effect observed. The aim of this study was to calculate the absorbed dose to red marrow with different methods for the same patients and to compare the results. Patients diagnosed with B-cell lymphoma were treated with ¹³¹I-labelled monoclonal antibodies (LL2, anti-CD22). Blood samples were collected, scintillation camera images were taken and single probe measurements were carried out at different points in time after administration of the radiopharmaceutical. The absorbed dose to red marrow per unit activity administered was calculated using four varieties of the blood method and from activity quantification in the sacrum in the scintillation camera images. The absorbed dose to the total body per unit activity, sometimes used as a measure for determining the toxic effect in red marrow, was calculated from both the scintillation camera images and the single probe measurements. The results from the different methods of calculating the absorbed dose for the same patient and treatment were compared. The ratio of the maximum and the minimum absorbed dose to red marrow calculated using the four variations of the blood method and the sacrum imaging method for one and the same patient varied between 1.8 and 2.8. The correlation coefficients for all the possible combinations of the dosimetry methods, including total body measurements, varied from 0.51 to 0.99. The results show that the variability of the absorbed dose to the bone marrow is dependent on both method and patient.     

Capecitabine as third line therapy in patients with advanced colorectal cancer

This study sought to determine whether third line therapy with capecitabine (cap.) could provide any clinical benefit in patients with advanced colorectal cancer who have progressed on 5-Fu combination therapy with both irinotecan and oxaliplatin. Twenty patients who were pretreated with and had progressed on irinotecan+Nordic FLv (5-Fu/leukovorin) and oxaliplatin+c.i. 5-Fu/leukovorin were studied. Cap. was administered at 1000-1250 mg/m2 bid d1-14 q 3 w. Time to progression (TTP) (either radiological or clinical) and overall survival (OS) were estimated with the Kaplan-Meier actuarial method. The median number of administered cap. courses was four. No radiological or biochemical responses were observed. Three patients were classified as having stable disease at three months. Two of these patients had, however, minor radiological progression and a =100% increase in CEA compared to base line. Seventeen patients were classified as having progressive disease during the first three months period. Median TTP and OS were 2.8 months and 6.1 months, respectively. A response rate of =15% for third line cap. in metastatic CRC can be ruled out. Median PFS was limited in the study population. This observation and the few cases with SD at three months, lead us to believe that little or no clinical benefit can be expected from single drug cap. in patients with irinotecan- and oxaliplatin-combination resistant advanced colorectal cancer.     

Internal microdosimetry for single cells in radioimmunotherapy of B-cell lymphoma

Patients with B-cell lymphoma may have disease manifestations ranging in size from more than a 1000 cm3 down to the volume of a single cell. If targeted radionuclide therapy is to become a curative treatment, all individual tumor cells must also be eliminated. Given the vast differences in particle energy of different electron- emitting radionuclides, one questions whether the mean absorbed dose is a relevant parameter for use in single-cell dosimetry and whether it would not be more accurate to adopt a stochastic approach to dosimetry. Monte Carlo simulations were performed of energy deposition from 1000, 300, 100, or 10 electrons uniformly distributed in a sphere with a radius of 7.7 microm. The simulated electrons were monoenergetic (18 keV, 28 keV, 141 keV, or 935 keV). The absorbed dose per emitted electron, the absorbed fraction, the fraction of the cellular volume in which energy is deposited, and the dose-volume histograms were calculated. Absorbed fractions varied between 0.60 (18 keV) and 0.001 (935 keV), and the absorbed dose to the cell per electron emitted varied by a factor of 10, from 0.898 mGy (18 keV) to 0.096 mGy (935 keV). The specific energy varied between 0 and 46 mGy for the case showing the best uniformity (1000 18-keV electrons). The nonuniformity of the absorbed dose to a cell increases with increasing electron energy and decreases with the number of decays inside the studied volume. The wide distribution of energy deposition should be taken into account when analyzing and designing trials for targeted radionuclide therapy.     

The loss of NKX3.1 expression in testicular--and prostate--cancers is not caused by promoter hypermethylation

BACKGROUND: Recent studies have demonstrated that the NKX3.1 protein is commonly down-regulated in testicular germ cell tumors (TGCTs) and prostate carcinomas. The homeobox gene NKX3.1 maps to chromosome band 8p21, which is a region frequently lost in prostate cancer, but not in TGCT. Mutations have not been reported in the NKX3.1 sequence, and the gene is hypothesized to be epigenetically inactivated. In the present study we examined the methylation status of the NKX3.1 promoter in relevant primary tumors and cell lines: primary TGCTs (n = 55), intratubular germ cell neoplasias (n = 7), germ cell tumor cell lines (n = 3), primary prostate adenocarcinomas (n = 20), and prostate cancer cell lines (n = 3) by methylation-specific PCR and bisulphite sequencing. RESULTS AND CONCLUSIONS: Down-regulation of NKX3.1 expression was generally not caused by promoter hypermethylation, which was only found in one TGCT. However, other epigenetic mechanisms, such as modulation of chromatin structure or modifications of histones, may explain the lack of NKX3.1 expression, which is seen in most TGCTs and prostate cancer specimens.     

The hottest sentinel lymph node is not always the positive node

The technique of identifying the sentinel lymph node (SLN) varies from each individual institution. Generally, the highest isotope count in a lymph node is considered the SLN, whereas other radioactive nodes might also be removed. The purpose of our study was to determine if the hottest node was always the tumor-containing node. Two hundred forty-seven breast cancer patients underwent SLN biopsy from April 1998 to April 2002. Lymphatic mapping involved a radiocolloid injection and lymphoscintigraphy followed by intraoperative assessment with a hand-held gamma probe. All SLN(s) with radioactive counts 10 per cent or more of the ex vivo counts of the most radioactive SLN were removed. The SLN were sliced at 2-mm intervals with 4-microm step-sections (92-microm spacing) and evaluated by microscopy and immunohistochemistry. One hundred twenty (49%) of the 247 patients had 2 or more nodes resected. Of these 120 patients, 33 (28%) had a tumor-bearing node. In 25 (74%) cases, the tumor-bearing node was the most radioactive; however, in 8 (26%) cases, the positive node was a lesser reactive node. Although the most radioactive node in a draining basin is considered the SLN, this is often not the metastatic node. Therefore, all nodes with significant radioactive counts must be removed to ensure accurate staging.     

Increased matrix metalloproteinase-9 concentration and activity after stimulation with interleukin-17 in mouse airways

BACKGROUND: The proteolytic enzyme matrix metalloproteinase (MMP)-9 can degrade structural compounds such as the extracellular matrix and the basement membrane in the airways and lungs. MMP-9 has therefore been implicated in remodelling of the airways and lungs during severe asthma and chronic obstructive pulmonary disease (COPD). METHODS: The effect of the T lymphocyte derived proinflammatory cytokine interleukin (IL)-17 on MMP-9 protein release and activity in the airways was studied in vivo and in vitro. RESULTS: In vivo, intranasal stimulation of mice with IL-17 induced the release of the precursor molecule proMMP-9 in bronchoalveolar lavage (BAL) fluid, associated with a pronounced local accumulation of neutrophils that stained positive for MMP-9. Stimulation with IL-17 also increased the concentration of free soluble MMP-9 that was proteolytically active as determined by a gelatinase substrate assay. The concentration of MMP-9 in BAL fluid had a strong positive correlation with the number of neutrophils; the amount of MMP-9 per neutrophil was not increased by IL-17 stimulation. In vitro, stimulation of mouse neutrophils with IL-17 did not increase the concentration of proMMP-9 in the conditioned medium. CONCLUSION: Local stimulation with IL-17 increases the concentration of biologically active MMP-9 as well as its precursor molecule in mouse airways in vivo. This increase in proteolytic load is probably mainly due to an increased number of neutrophils and not to an increase in the release of MMP-9 from each neutrophil. These findings indicate a link between the T lymphocyte cytokine IL-17 and increased proteolytic load in the airways which may be relevant for chronic inflammatory airway diseases such as severe asthma and COPD.     

Intracerebral xenotransplantation of GFP mouse bone marrow stromal cells in intact and stroke rat brain: graft survival and immunologic response

The present study characterized survival and immunologic response of bone marrow stromal cells (BMSCs) following transplantation into intact and stroke brains. In the first study, intrastriatal transplantation of BMSC (60,000 in 3 microl) or vehicle was performed in normal adult Sprague-Dawley male rats that subsequently received daily cyclosporin A (CsA, 10 mg/kg, IP in 3 ml) or vehicle (olive oil, similar volume) starting on day of surgery up to 3 days posttransplantation. Animals were euthanized at 3 or 30 days posttransplantation and brains were processed either for green fluorescent protein (GFP) microscopy or flow cytometry (FACS). Both GFP epifluorescence and FACS scanning revealed GFP+ BMSCs in both groups of transplanted rats with or without CsA, although significantly increased (1.6- to 3-fold more) survival of GFP+ BMSCs was observed in the immunosuppressed animals. Further histologic examination revealed widespread dispersal of BMSCs away from the graft core accompanied by many long outgrowth processes in non-CsA-transplanted animals, whereas a very dense graft core, with cells expressing only sporadic short outgrowth processes, was observed in CsA-transplanted animals. There were no detectable GFP+ BMSCs in nontransplanted rats that received CsA or vehicle. Immunologic response via FACS analysis revealed a decreased presence of cytotoxic cells, characterized by near complete absence of CD8+ cells, and lack of activation depicted by low CD69 expression in CsA-treated transplanted animals. In contrast, elevated levels of CD8+ cells and increased activation of CD69 expression were observed in transplanted animals that received vehicle alone. CD4+ helper cells were almost nondetectable in transplanted rats that received CsA, but also only minimally elevated in transplanted rats that received vehicle. Nontransplanted rats that received either CsA or vehicle displayed very minimal detectable levels of all three lymphocyte markers. In the second study, a new set of male Sprague-Dawley rats initially received bilateral stereotaxic intrastriatal transplantation of BMSCs and 3 days after were subjected to unilateral transient occlusion of middle cerebral artery. The animals were allowed to survive for 3 days after stroke without CsA immunosuppression. Epifluorescence microscopy revealed significantly higher (5-fold more) survival of transplanted GFP+ BMSCs in the stroke striatum compared with the intact striatum. The majority of the grafts remained within the original dorsal striatal transplant site, characterized by no obvious migration in intact striatum, but with long-distance migration along the ischemic penumbra in the stroke striatum. Moreover, FACS scanning analyses revealed low levels of immunologic response of grafted BMSCs in both stroke and intact striata. These results, taken together, suggest that xenotransplantation of mouse BMSCs into adult rats is feasible. Immunosuppression therapy can enhance xenograft survival and reduce graft-induced immunologic response; however, in the acute phase posttransplantation, BMSCs can survive in intact and stroke brain, and may even exhibit long-distance migration and increased outgrowth processes without immunosuppression.     

Dose-fractionated radioimmunotherapy in non-Hodgkin's lymphoma using DOTA-conjugated, 90Y-radiolabeled, humanized anti-CD22 monoclonal antibody, epratuzumab.

PURPOSE: Fractionated radioimmunotherapy may improve therapeutic outcome by decreasing heterogeneity of the dose delivered to the tumor and by decreasing hematologic toxicity, thereby allowing an increased amount of radionuclide to be administered. Because humanized anti-CD22 epratuzumab can be given repeatedly, a single-center study was conducted to establish the feasibility, safety, optimal dosing, and preliminary efficacy of weekly administrations of 90Y-labeled 1,4,7,10-tetra-azacyclodecane-N,N',N',N'-tetraacetic acid-conjugated epratuzumab. EXPERIMENTAL DESIGN: Cohorts of three to six patients with B-cell lymphoma received 185 MBq/m2 [90Y]epratuzumab with unconjugated epratuzumab (total protein dose 1.5 mg/kg) once weekly for two to four infusions, with [(111)In]epratuzumab coadministered at first infusion for scintigraphic imaging and dosimetry. RESULTS: Sixteen patients received treatment without significant infusional reactions. The overall objective response rate was 62% (95% confidence interval, 39-86%) in both indolent (75%) and aggressive disease (50%). Complete responses (CR/CRu) occurred in 25% of patients and were durable (event-free survival, 14-41 months). Two patients receiving four infusions had hematologic dose-limiting toxicity. Serum epratuzumab levels increased with each weekly dose. Of 13 patients with tumor cell CD22 expression determined by flow cytometry, seven of eight with strongly positive results had objective responses, versus one of five with negative or weakly positive results (P = 0.032). CONCLUSIONS: Radioimmunotherapy with weekly 185 MBq/m2 [90Y]epratuzumab achieved a high objective response rate (62%) across lymphoma subtypes, including durable CRs. The findings that three weekly infusions (555 MBq/m2, total dose) can be administered safely with only minor toxicity, that antibody levels increased during treatment weeks, and that therapeutic response predominantly occurs in patients with unequivocal CD22 tumor expression provide guidance for future studies.