No abstract

Novel antibodies have been generated by immunizing with bacterially expressed fragments of the repetitive motif of the Ki-67 gene. One such antibody, MIB1, recognizes a fixation and embedding resistant epitope on the Ki-67 protein if sections are previously microwaved in a citrate buffer. We have investigated the utility of this antibody as a marker of cell proliferation in archival material. The microwave technique is simple but requires careful monitoring since different tissues and fixatives require different irradiation times. Strong nuclear immunoreactivity was detected with all fixatives studied. Cytoplasmic staining was not identified. In a wide range of normal tissues the distribution and number of MIB1 immunoreactive cells matched that of cryostat sections stained with Ki-67. In nude mouse xenografts in which the growth fraction had been defined using a fraction of labelled mitosis method, the labelling index with MIB1 matched that previously determined for Ki-67 and correlated well with the growth fraction. Other markers of proliferation (e.g. proliferating cell nuclear antigen) have been shown to be expressed in DNA repair, thus we investigated expression of MIB1 immunoreactivity in situations of DNA repair in vivo--ultraviolet irradiated human skin. MIB1 staining correlated with semi-conservative DNA synthesis rather than excision repair DNA synthesis. Finally, the morphological and cell cycle distribution of MIB1 expression is identical to that of Ki-67. Thus, MIB1 represents a new anti-Ki-67 antibody which appears to be a robust marker of cell proliferation easily applicable to archival material.

The last 2 years have seen a flood of reports in Histopathology and other pathology journals describing the application of antibodies that recognize proliferating cell nuclear antigen (PCNA) as a marker of cell proliferation in histological material. This interest is a consequence of the widely held belief that such markers may prove to be useful objective indicators of biological behaviour of at least some forms of tumour. This is coupled with the resistance of some PCNA epitopes to conventional fixation and embedding. The literature relating to PCNA is already rather confused and some rather extravagant claims and suggestions have been made, sometimes without a clear understanding of the biology of the PCNA molecule and the complexities of the processes in which it is involved. We will briefly review the history of PCNA and attempt to clarify our current understanding of this player in the processes of DNA replication.In 1978 Miyachi et al.' described an autoimmune serum from patients with systemic lupus erythematosus which recognized a nuclear antigen distributed in proliferating cells which was consequently termed proliferating cell nuclear antigen (PCNA). PCNA is a 36 kDa acidic non-histone nuclear protein which functions as an auxiliary protein for DNA polymerase 6 and is an absolute requirement for DNA synthesis24. Immunofluorescent studies have shown the existence of two populations of PCNA during S phase of the cell cycle, one that is nucleoplasmic as in quiescent cells and is easily extracted by detergent and another that is associated to specific nuclear structures5. Detergent resistant PCNA immunoreactivity is co-localized with bromodeoxyuridine (incorporated into DNA during S phase) in replication complexes, and their order of appearances throughout the S phase are i d e n t i~a l~-~. This demonstrates that PCNA is tightly associated to the sites of DNA replication and probably must have a role in DNA synthesis8. This is supported by the use of antisense oligonucleotides both of which lower levels of PCNA protein and are associated with inhibition of DNA synthesis9. In the presence of PCNA and a multi-subunit complex called replication factor C, polymerase 6 catalyses elongation of Okazaki fragments to long DNA chains representing leading strand DNA synthesis10-12.PCNA is also involved in unscheduled DNA synthesis (i.e. nucleotide excision-repair) since tightly bound PCNA can be found associated with chromatin at all phases of the cell cycle after ultra violet (UV) irradiation in v i t r~'~. '~.The explanation for this lies in an understanding of the process of DNA excision repair. In the initial phase of DNA excision repair a multi subunit endonuclease forms at the site of damage and enzymatically removes the damaged strand. This is followed by DNA synthesis to repair the defect. Consequently there may be an overlap between the proteins involved in semi-conservative DNA replication and DNA repair. Direct experimental proof of this with demonstration of involvement of PCNA has recently come from anal...

Cutaneous melanoma is highly immunogenic, yet primary melanomas and metastases develop successfully in otherwise immunocompetent patients. To investigate the local immunosuppressive microenvironment, we examined the presence of suppressor T lymphocytes and tolerising dendritic cells (DCs), the expression of immunosuppressive cytokines (IL-10, TGFb1 and TGFb2) and the enzyme indoleamine 2,3-dioxygenase (IDO) using qRT -PCR and immunohistochemistry in primary skin melanomas, negative and positive sentinel lymph nodes (SLN), and lymph nodes with advanced metastases. Our results indicate that tolerogenic DCs and suppressor T lymphocytes are present in melanoma at all stages of disease progression. They express transforming growth factor b receptor 1 (TGFbR1), and are therefore susceptible to TGFb1 and TGFb2 specifically expressed by primary melanoma. We found that expression of IDO and interleukin 10 (IL-10) increased with melanoma progression, with the highest concentration in positive SLN. We suggest that negative SLN contain immunosuppressive cells and cytokines, due to preconditioning by tolerogenic DCs migrating from the primary melanoma site to the SLN. In primary melanoma, TGFb2 is likely to render peripheral DCs tolerogenic, while in lymph nodes IDO and TGFb1 may have a major effect. This mechanism of tumourassociated immunosuppression may inhibit the immune response to the tumour and may explain the discrepancy between the induction of systemic immunity by anti-melanoma vaccines and their poor performance in the clinic.

A number of different factors can profoundly influence the quantification of immunostained cells. Given the characteristics of immunohistological detection systems with non-linearity of signal and antigen concentration, we investigated the relationship of signal (number of stained cells) to the dilution of antibody employed. Three antibodies were studied which have been advocated as being effective in fixed material as markers of cell proliferation: PC10 (anti-proliferating cell nuclear antigen (PCNA)), Ki-S1 and MIB1 (a novel anti-Ki-67). Serial sections of tonsil were immunostained with a range of antibody dilutions using a fixed detection system and the number of stained cells quantified. Similar experiments were performed on tumour xenografts with known growth fraction and, in vitro, on human diploid fibroblasts in logarithmic growth phase. With both PC10 and Ki-S1 the number of stained cells increased with decreasing antibody dilution with no plateau being identified. In contrast, MIB1 showed a clear plateau. Immunocytological data indicate that PCNA and Ki-S1 antigen are present at low (but detectable) levels in at least some non-cycling cells and thus an artificial 'cut-off' has to be employed in assessing the number of proliferating cells with these antibodies. The superiority of MIB1 probably reflects the rapidity of catabolism of the Ki-67 antigen at the end of M phase. Taken together, these data point to the importance of carefully considering fundamental immunochemical properties such as antibody concentration (as well as antibody affinity and sensitivity of detection system) when employing immunological markers of cell proliferation in quantitative procedures.