Expression of endogenous markers of hypoxia for the HIF-1 and HIF-2 pathway is strongly associated with radiotherapy failure. Using immunohistochemical methods it is possible to identify subgroups of HNSCC patients who are highly curable with radiotherapy, or who are excellent candidates for clinical trials on hypoxia-targeting drugs in two distinct pathways.

CD44 is a cell surface HA-binding glycoprotein that is overexpressed to some extent by almost all tumors of epithelial origin and plays an important role in tumor initiation and metastasis. CD44 is a compelling marker for cancer stem cells of many solid malignancies. In addition, interaction of HA and CD44 promotes EGFR-mediated pathways, consequently leading to tumor cell growth, tumor cell migration, and chemotherapy resistance in solid cancers. Accumulating evidence indicates that major HA-CD44 signaling pathways involve a specific variant of CD44 isoforms; however, the particular variant almost certainly depends on the type of tumor cell and the stage of the cancer progression. Research to date suggests use of monoclonal antibodies against different CD44 variant isoforms and targeted inhibition of HA/CD44-mediated signaling combined with conventional radio/chemotherapy may be the most favorable therapeutic strategy for future treatments of advanced stage malignancies. Thus, this paper briefly focuses on the association of the major CD44 variant isoforms in cancer progression, the role of HA-CD44 interaction in oncogenic pathways, and strategies to target CD44-overexpressed tumor cells.

Various methods are available for the measurement of proliferation rates in tumours, including mitotic counts, estimation of the fraction of cells in S-phase of the cell cycle and immunohistochemistry of proliferation-associated antigens. The evidence, advantages and disadvantages for each of these methods along with other novel approaches is reviewed in relation to breast cancer. The potential clinical applications of proliferative indices are discussed, including their use as prognostic indicators and predictors of response to systemic therapy. IntroductionThe development and continued growth of cancers involves altered rates of cell proliferation. In early breast cancer, measurement of proliferation can be used in conjunction with tumour size, grade, nodal status and steroid receptor status as a prognostic indicator [1,2]. Proliferation rates can provide useful information on prognosis and aggressiveness of individual cancers and can be used to guide treatment protocols in clinical practice. Adjuvant chemotherapy has been shown to improve survival in patients with breast cancer, but has potentially serious side effects. The potential of prognostic factors is to determine which patients are at higher risk of recurrence such that patients who stand to benefit more from adjuvant treatment can be identified. In the future, changes in proliferation rates during or after systemic therapy may be utilized as predictors of response and allow further tailoring of therapy. Information on proliferation rates is also necessary for the development of therapeutic agents, some of which may be targeted directly at specific points in the cell division pathway.Various techniques have been developed to evaluate and quantify proliferation rates in the laboratory. Mitotic count estimates are widely used as a simple measure of cellular proliferation and are often incorporated into tumour grading systems [3]. Other methods have been developed, such as the detection of cells undergoing DNA synthesis using assays for thymidine uptake [4], flow cytometry to estimate the percentage of cells in S phase of the cell cycle or the detection of antigens associated with proliferation. This review will discuss current and developmental methods for assessing proliferation and the potential applications of such knowledge in the treatment of breast cancer. Table 1 summarises these methods and highlights their individual advantages and limitations. Mitotic indexCellular proliferation involves several defined phases. Cells in the resting (G0) phase are stimulated to enter the active cycle at the first gap (G1) phase. During this period of time, the cell prepares for DNA synthesis (the S phase), which is followed by a second phase of relative inactivity (G2) and preparation for the separation of the chromatids in the mitotic (M) phase. Cells can then recycle by entering the G1 phase or return to the resting G0 phase. Proliferation was first measured by counting mitotic bodies on paraffin-embedded tumour specimens stained using haematoxylin-eo...

Glucose transporter-1 protein (GLUT1) and carbonic anhydrase IX (CAIX) are regulated by hypoxia inducible factor-1 (HIF-1) and have been studied as putative intrinsic cellular markers for hypoxia. This study directly compares CAIX and GLUT1 with pimonidazole binding in a prospective series of bladder cancer patients and also studies the prognostic significance of the markers, in combination with vascularity and proliferation, in a retrospective series of bladder cancer patients treated in a phase II trial of radical radiotherapy with carbogen and nicotinamide (ARCON). A total of 21 patients with a diagnosis of transitional cell carcinoma of the bladder received 0.5 g m À2 pimonidazole. Serial tumour sections were stained for pimonidazole, GLUT1 and CAIX and compared. Tissue sections obtained from a series of 64 patients previously treated for invasive bladder cancer using ARCON were stained for GLUT1 and CAIX together with Ki-67 and CD31/34. There was a good geographical colocalisation of both intrinsic markers with pimonidazole and a highly significant agreement in individual patients; correlation coefficients were 0.82 (P ¼ 0.0001) for GLUT1 and 0.74 (Po0.0001) for CAIX. In both series of patients, the intrinsic hypoxia markers were highly correlated with each other and a correlation with proliferation was also evident in the retrospective study. In univariate and multivariate analyses, GLUT1 and CAIX were independent predictors for overall and cause specific survival. The hypoxia markers did not predict for local control or metastases-free survival although higher Ki-67 indices showed a trend towards local failure. The data suggest that both hypoxia modification and accelerated treatment may be valid treatment options in bladder cancer.

In vivo bromodeoxyuridine (BrdUrd) labelling of the human large bowel was performed and a detailed histochemical localisation of label in' sections of crypts was undertaken using a monoclonal antibody to BrdUrd containing DNA. Flow cytometric studies on extracted nuclei were also performed (data presented elsewhere). The average crypt in the human large bowel (excluding the rectum) was 82 cells in height and 41 cells in circumference, with a total of about 2000 cells (assuming a topographical correction factor of 0.6). Ten per cent ofthe cells were replicating their DNA -that is, were in the S phase of the cell cycleand 0-4% were in mitosis. The median position for the labelling index versus cell position frequency plot is at the 20th cell position -at a quarter of the crypt height. The lower and upper limits of the cell proliferation are given by the 5th and 95th percentiles at cell positions 4 and 43 respectively. The peak labelling index is about 30% and it occurs at cell position 15. The labelling index at the crypt base, the probable stem cell zone, is about 14%, suggesting that these cells have a longer cell cycle.Taking a value of 8*6 hours for the duration of the S phase (deduced from the flow cytometric data) and assuming a growth fraction of 1 0 for the mid-crypt, these data provide an estimate of about 30 hours for the cell cycle time. The rectal crypts are about the same size but contain about 30% fewer S phase cells. The data also yielded a per cent BrdUrd labelled mitosis curve.