Improving the efficacy of cancer immunotherapy.

A series of cancer vaccines have been evaluated in clinical trials with encouraging results, but the demonstration of clinical benefit in confirmatory studies has so far proven to be difficult. The development of cancer vaccines is hampered by a range of issues particular to this field of research. On 12th March 2008, the Biotherapy Development Association convened a workshop to discuss issues faced by scientists and clinicians involved in the development of cancer vaccines. This paper is a review of the field, based on discussions held at the BDA workshop, and describes biological barriers encountered in generating effective immune responses to tumours, methodological obstacles encountered in the improvement of immunological monitoring which aims to improve inter-laboratory and inter-trial comparisons, challenges in clinical trial design and problems posed by the lack of specific regulation for cancer vaccines and the impact on their development. Ultimately, a number of general solutions are posed: (1) better patient selection, (2) use of multi-modal treatments that affect several aspects of the immune system at once, (3) a requirement for the development of good biomarkers to stratify patients for selection prior to trial and as surrogates for clinical response and (4) harmonisation of SOPs for immunological monitoring of clinical trials.

Dendritic cells for active immunotherapy: optimizing design and manufacture in order to develop commercially and clinically viable products.

Dendritic cell (DC) active immunotherapy is potentially efficacious in a broad array of malignant disease settings. However, challenges remain in optimizing DC-based therapy for maximum clinical efficacy within manufacturing processes that permit quality control and scale-up of consistent products. In this review we discuss the critical issues that must be addressed in order to optimize DC-based product design and manufacture, and highlight the DC based platforms currently addressing these issues. Variables in DC-based product design include the type of antigenic payload used, DC maturation steps and activation processes, and functional assays. Issues to consider in development include: (a) minimizing the invasiveness of patient biological material collection; (b) minimizing handling and manipulations of tissue at the clinical site; (c) centralized product manufacturing and standardized processing and capacity for commercial-scale production; (d) rapid product release turnaround time; (e) the ability to manufacture sufficient product from limited starting material; and (f) standardized release criteria for DC phenotype and function. Improvements in the design and manufacture of DC products have resulted in a handful of promising leads currently in clinical development.

Secretory type II phospholipase A2 (PLA2G2A) expression status in colorectal carcinoma derived cell lines and in normal colonic mucosa.

There is good evidence now that the secretory type II phospholipase A2 (Pla2g2a) gene represents the Mom1 locus, a genetic modifier of tumor resistance in the multiple intestinal neoplasia (Min) mouse. Previously we have mapped the human homolog PLA2G2A to 1p35-36.1 within a region that is the target of frequent deletions in sporadic colorectal tumors. Here we show 64% loss of heterozygosity (LOH) at the PLA2G2A locus in primary tumors. We studied PLA2G2A expression in both colorectal tumor cell lines and normal mucosa. Most of the lines lacked detectable PLA2G2A transcripts by Northern analysis. Large differences in expression were seen among normal mucosa of different patients with sporadic tumors. We analysed the coding region of PLA2G2A in eight colorectal cancer cell lines with hemizygous deletion at 1p35-36/PLA2G2A, in none we did detect a mutation. Biallelic expression of PLA2G2A was observed in a cell line heterozygous for an exon 3 polymorphism, rendering unlikely that imprinting is a pathway participating in the loss of PLA2G2A function. It remains uncertain if PLA2G2A, in particular its apparent lack of expression in tumor cells, might be a factor in human colorectal tumorigenesis.

Expression of CD44 standard and isoforms in human breast cancer xenografts and shedding of soluble forms into serum of nude mice.

Standard CD44 (CD44s) and variant isoforms (CD44v) are expressed on different malignant cells and tissues. Their upregulation has been implicated, in the progression and metastasis of malignomas. In this work we addressed the question of whether these molecules are also expressed on xenografted human breast carcinomas and if certain expression patterns are correlated with biological parameters like tumour size, hormone receptor status, histology, growth rate, chemoresistance and microenvironment. Additionally, we were interested in the shedding of soluble CD44 (sCD44) into the blood circulation of tumour bearing nude mice. The human breast carcinomas MCF-7, MCF-7/ADR, 4296 and MDA-MB435, 4134 and 4151 were transplanted subcutaneously (sc.) or into the mammary fat pad (mfp.) of nude mice. The expression of the CD44s, -v6, and -v9 isoforms was determined at different time points on tissue samples by immunohistochemistry or RT-PCR employing human-specific antibodies or primers, respectively. The serum concentration of CD44s and -v6 was measured by human specific ELISAs. All tumours expressed CD44s. The lowest level was observed in the MCF-7 cancer. The CD44v6 and -v9 sequences and epitopes were distinctly expressed in MCF-7/ADR. MDA-MB435, 4134, 4151 and 4296, while MCF-7 lacked these isoforms. The highest serum concentration of the v6 isoform was detected in mice bearing the tumour 4296 with a high tendency for lymphogenic metastasis. The serum levels of sCD44 were in 5/6 xenografts linearly correlated with the tumour size. Interestingly, there was a remarkable difference between the two sublines MCF-7 and MCF-7/ADR: Both the tissue and serum levels of CD44 isoforms indicated that the development of multidrug resistance is accompanied by an alteration in the expression of membrane proteins discussed to be involved in metastasis. There was no relation of tissue expression with the transplantation site and the hormone receptor status of the tumour lines. CD44s and its variant isoforms are expressed in human xenotransplanted breast cancers in very different levels and patterns. The highest expression in the tumour 4296 is related to lymphogenic metastasis while the absence of isoforms in the model MCF-7 is related to non-metastatic behaviour. CD44 is shed into the serum and can be used for monitoring of tumour growth.

Intracompartmental pressure during hyperthermic isolated limb perfusion for melanoma and sarcoma.

Side effects of isolated limb perfusion (ILP) include rhabdomyolysis, paresthesia, or nerve palsy. The increase in intracompartmental pressure during ILP is thought to be linked to neuro- and muscular toxicity, and fasciotomy is recommended for protection. In 24 patients, intracompartmental pressure was measured. A flexible 5 F probe was placed into the non-tumour-bearing compartment of the perfused limb. Interstitial fluid pressure was measured using a piezoresistant tip. Compartmental pressure values were continuously recorded during and after ILP. The drugs used were a combination of doxorubicin, cisplatinum and melphalan or rhTNF-alpha combined with melphalan. The median overall compartmental pressure prior to ILP was 13 mmHg (range: 11-21 mmHg); during the heat-up phase the median pressure rose to 28 mmHg. During therapeutic perfusion a further increase could be documented and the maximum pressure measured was 90 mmHg; the median of the pressure maxima of all patients was 34 mmHg. During wash-out, at the end of the perfusion, a clear reduction in compartment pressures could be observed and the median dropped to a value of 27 mmHg. In all patients a continuous decrease in compartmental pressure could be recorded, reaching the pre-ILP values by 48 h post-operatively. A dramatic increase in compartmental pressure during ILP can be observed by continuous monitoring. Because of our observation that during the wash-out phase elevated compartmental pressures return to normal, there is no general indication for a fasciotomy. However, for patients maintaining a peak compartmental pressure above a critical threshold of 35 to 40 mmHg fasciotomy may be indicated.

[Expression of CD44 isoforms--a new histopathologic parameter in colorectal carcinoma?]. / Expression von CD44 Isoformen-ein neuer histopathologischer Parameter beim kolorektalen Karzinom?

Expression of CD44H and CD44-isoforms were examined by immunohistochemistry in 102 patients with colorectal tumors in correlation to histological grading, staging and clinical outcome. From normal mucosa to colorectal tumors we found an upregulation of CD44H and CD44-v9 and a de-novo expression of CD44-v6. When compared to histological grading it was found that CD44-v6 expression indicates more differentiated tumors in contrast to less differentiated colorectal carcinomas showing decreasing CD44-v6 expression. Further we came to the result that increasing CD44-v6 expression correlates with progressive tumor stage. In the metastases we found a significant decrease in expression of CD44H, CD44-v9 and-v6 when compared to corresponding primary colorectal tumors. Wether the differential CD44 expression can be used as a prognostic histopathological marker for the progression of colorectal cancer has to be further validated. For final interpretation, our prospective study has to be continued further.

Deletion mapping defines different regions in 1p34.2-pter that may harbor genetic information related to human colorectal cancer.

Cytogenetic and molecular analyses of colorectal cancer cells have revealed deletions at 1p as prominent alterations, suggesting that genetic information on the short arm of chromosome 1 has a role in tumorigenesis. In this study we have used 33 microsatellite markers to fine map deletions at 1p in primary colorectal carcinomas. We found 1p-deletions in 84% of the cases (31/37). High frequencies of loss of heterozygosity (LOH), often the result of small independent interstitial deletions in the same tumor, defined three regions, that may harbor genetic information relevant for colorectal cancer: (i) region A between D1S243 and D1S468 (7cM; 1p36.3); (ii) region B between D1S436 and D1S199 (7cM; 1p35.1-36.31) and (iii) region C between D1S496 and D1S255 (1cM; 1p34.2-35). In addition we identified seven cell lines with LOH at 1p, all of which have deletions that span at least from the distal border of region A to the proximal border of region C.