Britta Wahren

Preoperative short‐term radiotherapy in rectal carcinoma. A preliminary report of a prospective randomized study

Between 1980 and 1983, 373 patients with clinically resectable rectal adenocarcinoma entered a prospective randomized study aimed to evaluate the effect of short‐term preoperative radiotherapy. Protocol violations were identified in 21 instances. Of the remaining 352 patients, 182 were randomized to surgical treatment only (S‐group). Immediately, before surgery, 170 patients were irradiated to the pelvic region with 25 Gy (2500 rad) during a 5‐day period (RT‐group). Of these patients, 59% underwent abdominoperineal excision, 38% anterior resection, and 3% laparotomy only. At surgery distant metastases were discovered in 32 patients (9%). There were no significant differences between the groups in the distribution of age, sex, operative methods, and tumor stage according to the original Dukes classification. During the follow‐up time, ranging between 6 months and 3 years, tumor recurrence occurred in 35 patients, 19 in the S‐group and 16 in the RT‐group. Fifteen patients in the S‐group had pelvic recurrence compared to 10 patients in the RT‐group. Distant metastases occurred in six and eight patients, respectively. Two patients in each group had both pelvic and distant recurrence. There was no correlation between tumor recurrence and type of operation. Median time interval from diagnosis to pelvic recurrence was 10 months in the S‐group and 16 months in the RT‐group. Postoperative complications in the form of wound sepsis were slightly more common in the RT‐group. In summary, the applied treatment regimen, is well‐tolerated and apparently does not affect the Dukes stage of the tumor. Although there is no statistically significant difference, there is a trend of less pelvic recurrence in patients receiving preoperative radiotherapy. Cancer 55:1182‐1185, 1985.

Measurement of urinary cea‐like substance. An aid in management of patients with bladder carcinoma

Urine and serum samples from patients with bladder carcinomas were studied for the occurrence of and variations in CEA content before, during, and after radiation therapy. The concentration of CEA‐like substances in urine increased with a more advanced clinical stage of the tumor, although there were large intercase variations. In serum, slightly increased values were noted only in advanced cases. During radiation therapy, high CEA values were found at around mid‐course. This could be related to a breakdown of tumor tissue. Judging from data for urine from radiation‐treated prostatic carcinomas without known tumors in the bladder, radiation alone was not responsible for the elevation of CEA. Urinary infections contributed to raised levels of CEA‐like substances in some cases. At the end of successful radiation therapy (as verified by cystoscopy, cytology, and clinical examination), 25 patients had CEA values in the urine comparable to normal values (14 ± 7 ng CEA/ml). The decrease was significant from the initial values to those after radiotherapy (p < 0.01). Four patients whose tumors persisted had high values (68 ± 46 ng CEA/ml). In patients who had previously received radiation treatment for bladder carcinomas, CEA values were high in 20 with recurrences (58 ± 36 ng CEA/ml) while they were lower in 13 who were free of recurrence (14 ± 6 ng CEA/ml). These findings indicate that urinary CEA determinations may be used in the immediate followup and management of patients treated for bladder carcinoma. It also appears to be of prognostic significance.

Assessment of serial CEA determinations in urine of patients with bladder carcinoma

Carcinoembryonic antigen (CEA) levels in urine and serum from 294 patients with bladder cancer in varying stages have been clinically evaluated. All urine samples were obtained from patients with intact renal function and without bacterial infection in the bladder. The samples were collected before, during, and at follow‐up examination after radiotherapy. They were perchloric acid extracted before being assayed in a double‐antibody radioimmunoassay. The geometric mean of urine CEA levels for patients with primary tumors of Stage T1 or T2 was significantly lower than that for those with Stage T3 or T4 disease. The urine CEA levels for patients with tumors of various histologic grades did not differ. The urine CEA levels decreased from before to after radiation treatment of the primary tumor. Patients with recurrence within six months after undergoing primary treatment had higher initial mean urine CEA levels than did those without evidence of recurrence. The prognostic information for recurrence was limited to the more advanced tumors. Differences were also found between the means of samples taken before recurrence and after treatment of recurrent tumors; with regression of the tumor, a lower mean urine CEA level was found; with progression, a higher value. Urine CEA levels before any treatment were higher when the patients had a short survival time. Serum CEA levels were not related to stage or grade of the primary bladder tumor but levels were slightly elevated with metastases. The determination of urine CEA levels seems to be useful in the follow‐up of patients with bladder carcinoma because when initially high, it adds to the information of the T classification and predicts early recurrence, and the monitoring of individual patients after primary treatment is useful for detecting recurrence.

Carcinoembryonic antigen in serum, urine and cells of patients with bladder carcinoma

SummaryA raised level of CEA-like substance has been demonstrated by radioimmunoassay in the urine of patients with bladder carcinoma, in concentrations which increase with a more advanced stage, and in serum of patients with advanced disease. In a 2-year follow-up of patients receiving chemotherapy, a correlation of raised urinary CEA to local recurrence was seen, as well as rising and high serum values with metastases. In the patients who responded to treatment, CEA values became normal. CEA was also located in carcinoma cells from bladder washings in 24–61 % of the cases. Combined studies of CEA in serum, urine and cells may be used to study the biology of the tumor and perhaps also in the monitoring of patients with urothelial carcinoma.

Cellular content of carcinoembryonic antigen in urothelial carcinoma

CEA containing cells can be demonstrated in 25–60% of urothelial carcinomas. No staining was seen with benign urothelial cells. Using microfluorometry of single cells, mean fluorescence intensity with anti‐CEA antisera was 3–6 times that of preparations stained in parallel with nonimmune sera. A comparison of the IF results with RIA implies a possibility to detect <1 pg CEA/ cell in unfixed specimens. The cells which stained with anti‐CEA antisera did not have any apparent morphologic properties differentiating them from neighboring nonstained cells but it was mainly in the cell populations from well‐ and moderately well‐differentiated tumors that CEA containing cells were seen. Therefore quantitative measurements of CEA amount/cell may be a parameter, in addition to morphologic differentiation, to study new properties of tumor cell populations.

Tumour markers in urology: Aids in cancer diagnosis and management

In recent years substances have been discovered which occur in increased amounts in tumour cells as opposed to normal cells, or in raised concentrations in the blood of patients with advanced tumours. New techniques and the availability of purer reagents have permitted us to characterise these marker substances for diagnostic and prognostic use. Already several substances are being used to monitor disease activity. The present summary does not attempt to cover the established literature, but discusses some recent findings of potential interest to clinicians.

Potent cellular and humoral immunity against HIV-1 elicited in mice by a DNA-prime/MVA-boost vaccine regimen intended for human use

In an experimental vaccine model, mice were primed three times with plasmids encoding multiple subtypes (A, B and C) of gag, envelope (env) and reverse transcriptase (RT) and adjuvant rGM-CSF followed by modified vaccinica Ankara (MVA) with the recombinant form A_E, in theory providing protection against HIV subtypes A-E. The cellular responses, as measured by IFN-gamma secretion gave up to 2000 gag-specific SFC/million PBMC. The humoral and cellular responses were further increased by the MVA-boosts with env and gag specific ELISpot responses above 3500 secreting/106 cells. Intracellular cytokine staining showed remarkably high numbers (~15%) of gag and env specific CD8+ T cells. This paved the way for our clinical trial with the multigene/multisubtype DNA plasmids boosted with the MVA construct. Conclusions: This preclinical study clearly shows the potential of combining these particular DNA and MVAs in a prime/boost regimen and that it is possible to induce a strong and broad humoral and cellular response directed against several parts of HIV as well as to several subtypes of the virus.

DNA immunization targeting carcinoembryonic antigen in colorectal cancer patients

Active specific immunotherapy targeting carcinoembryonic antigen (CEA) may induce antigen-specific humoral and cellular responses in cancer patients. Plasmid DNA, encoding tumor antigens, represents a novel approach of delivering conformational antigens. Here we report immune data of an explorative study using CEA66-DNA (non-glycosylated cytoplasmic CEA) and tetwtCEA-DNA (wild type glycosylated and secreted CEA) for immunization in combination with cyclophosphamide and GM-CSF in the adjuvant setting of radically operated colorectal cancer (CRC). 10 patients received intradermal (id) or intramuscular (im) CEA66-DNA delivered by needle-free Biojector at weeks 0, 2, 6 (part 1). 10 patients received tetwtCEADNA id by needle injection and electroporation at weeks 0 and 12 (part 2). In part 3, 6 patients were primed with CEA66-DNA and boosted with tetwtCEA-DNA. A significant increase of CD4+ effector memory, CD8+ effector and CD8+ effector memory T cells was seen in part 1. An immune response against CEA atleast one time point was noted in 15/20 (75%) patients in parts 1 and 2 together. The frequency of patients mounting a CEA-specific cellular immune responses was significantly higher in part 1 (100%) than in part 2 (50%) (p=0.03). In part 3, 5/6 (83%) patients showed a CEA-specific immune response after a prime-boost protocol. The higher CEA-specific T cell responses seen in part 1, may indicate reduced immunological tolerance induced by the non-glycosylated intracellularly produced CEA66-DNA immunogen. Humoral responses determined by ELISA were low. Further studies are warranted to optimize vaccination schedules to induce both cellular and humoral anti-CEA responses of clinical significance. Clinical trials.gov.identification number (parts 2/3) is NCT01064375. Abbrevations CEA: Carcinoembryonic Antigen; CRC: Colorectal Carcinoma; DFS: Disease free survival; ELISA: Enzyme-linked immunosorbent assay; ELISPOT: Enzyme-linked immunospot; EP: Electroporation; GM-CSF: Granulocyte-macrophage colony-stimulating factor; HRP: Horseradish peroxidase; id: intradermal; IFN-γ: interferon γ; IL-4: Interleukin-4; IL-10: Interleukin-10; im: Intramuscular; OS: Overall survival; PBMC: Peripheral blood mononuclear cells; PBS: Phosphate buffered saline; PHA: Phytohemagglutinin; PPD: Purified protein derivative of tuberculin; sc: Subcutaneous; SI: Stimulation index; SIIR: Sustained induced immune response; STIIR: Single time point induced immune response; TAA: Tumor associated antigens; TCV: Therapeutic cancer vaccines; TNF-α: Tumor necrosis factor-α Introduction Colorectal carcinoma (CRC) is a major cause of cancer-related mortality. Despite introduction of new drugs, a large proportion of patients remain incurable. Adjuvant chemotherapy is standard for the treatment of stage III colon cancer and increases the 5-year survival rate to more than 70% and is routinely also used in stage II colon cancer patients with high-risk of relapse. Several chemotherapeutic agents approved for metastatic CRC have however failed to improve the prognosis for patients with stages II and III CRC [1]. New therapeutic approaches are needed and immunotherapy may offer a novel targeted therapeutic option [1]. The goal of therapeutic cancer vaccines (TCV) is to induce a robust long-lasting immune response with limited toxicity [2]. Most tumor cells express tumor-associated antigens (TAA), which might act as targets for the immune system [3]. A commonly expressed TAA in gastrointestinal cancer is the carcinoembryonic antigen (CEA) which has been explored in immunotherapy trials [4-11]. Vaccination targeting CEA in humans was shown to induce antigen-specific humoral, CD4+ helper as well as CD8+ cytotoxic T-cell (CTL) responses [8,11-14]. Immunisation with proteins comprising multiple CD4+ and CD8+ T cell epitopes, have failed to induce a robust CD8+ response which may partly be explained by defect peptide processing of exogenous proteins not entering the MHC class I antigen presentation pathway. DNA contains immunostimulatory CpG sequences that stimulate cytosolic DNA-sensing pathways, interferon regulatory factors and Fas-FasL Correspondence to: Håkan Mellstedt, MD, PhD, Prof, Department of Oncology and Pathology (Radiumhemmet), Cancer Centre Karolinska, Karolinska University Hospital Solna, SE-171 76 Stockholm, Sweden, Tel: +46851774641; E-mail: hakan.mellstedt@karolinska.se

Do circulating HIV vaccine plasmids contribute to immunogenicity in humans

Results Two months after the third immunization, an HIV RNA quantitative PCR was performed to confirm the noninfected status of the participants. In half of the vaccines, in total receiving 3–12 mg plasmid DNA, we noted reactions of plasmid DNA or values corresponding to 20– 1200 HIV RNA copies in the Roche Amplicor assay. Env and/or gag encoding plasmids were detected in the plasma or serum of the vaccines, but no HIV. A PCR for HIV protease (the protease gene is not included in the vaccine) was negative in all cases. Antigen and antibody assays have confirmed that the individuals were not infected. The study is still blinded, but a total of over 90% have responded very well by T-cell assays. Conclusion HIV vaccine plasmids given id or im were identified as late as two months after immunization. A relation between immunogenicity and circulating vaccine plasmids will be sought. DNA plasmids may give positive signals in a standard HIV RNA quantative assay. from 2006 International Meeting of The Institute of Human Virology Baltimore, USA. 17–21 November, 2006

A unique amino acid deletion in the chimpanzee Cyclin T1 does not affect Tat trans-activation of HIV.

We have cloned and sequenced the chimpanzee (Pan troglodytes) cyclin T1 cDNA and performed functional HIV-1 Tat trans-activation studies. A unique codon deletion leading to a deleted asparagine residue in the N-terminal region of the first cyclin domain was discovered. This mutation does not significantly change the trans-activation of HIV-1, suggesting that Tat-Cyclin T1 mediated transcription is not a major barrier to HIV replication in the chimpanzee.