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Background: Colorectal carcinomas (CC) are one of the most commonly diagnosed malignancies. Tumor budding (the histologic process of dissociation that occurs at the invasive margin of colorectal cancer), has significant prognostic implications, in that higher tumor budding is associated with adverse histopathologic and clinical outcomes. Because of this prognostic significance, more research is needed to further understand the pathologic and immunohistochemical (IHC) associations pertaining to this important prognostic variable. In this study, we will further evaluate selective clinopathologic and IHC variables with possible association to tumor budding. Design: A total of 234 cases of CC diagnosed in our health system were retrospectively reviewed and routine hematoxylin and eosin–stained slides of these cases were collected. A representative slide for tumor budding was selected per case and selective IHC staining was performed. Clinicopathologic data were collected for each case and analyzed in relation to tumor budding scores. In exploratory analyses, tumor budding scores per individual investigator and consensus tumor budding scores were compared with selected IHC stains (MLH1, PMS2, and PHH3) as well as numerous clinicopathologic variables. Results: We found a paradoxical association between tumor budding and mitosis score using PHH3 immunostaining in univariate and multivariable analysis. Furthermore, patients with intact nuclear expression for MLH1 and/or PMS2 are more likely to have higher tumor budding compared with patients with lost expression. For multivariable analysis, the following covariates were significantly associated with higher tumor budding: the presence of lymphovascular invasion, higher pathologic tumor stage, and finally infiltrating border was more likely to be associated with higher tumor budding compared with cases with a pushing border. Regarding nonmucinous versus mucinous CC, nonmucinous adenocarcinoma (MCA) was more likely to be associated with higher tumor budding compared with MCA. Conclusion: Numerous clinicopathologic variables were found to be associated with tumor budding including lymphovascular invasion, tumor stage, infiltrating tumor border, non-MCA was more likely to be associated with higher tumor budding compared with MCA, possibly related to MUC-2 and MSI. Furthermore, regarding the paradoxical association between tumor budding and mitosis score using a PHH3 immunostaining (high tumor budding having lower mitosis), this is possibly related to the tumoral stomal microenvironment and cancer associated fibroblasts. An idea for a future study would be to look at the maturity of cancer-associated fibroblasts (immature vs. mature) and the tumoral stroma microenvironment, with regards to markers of tumor aggressiveness such as mitosis. In addition, we found that patients with intact nuclear expression for MLH1 and/or PMS2 were more likely to have higher tumor budding compared with patients with lost expression, possibly related to mismatch repair CC’s not being as reliant on tumor budding. Future research will hopefully concede further insight into the variables that affect tumor budding, especially regarding the tumoral microenvironment and variations between different patient populations, inclusive of patients lacking activity of the mismatch repair. Ultimately, this will allow for better prognostic information, and more precise treatment modalities.

We have developed a series of anti-cancer peptide drugs, PNC-27 and PNC-28 from the MDM2- binding domain of the p53 protein (AA12–26 and 17–26, respectively) and a newly designed membrane residency peptide (MRP) that allows the drug to insert itself into cellular plasma membrane. To date, we have treated in vitro cells from over 20 different human epithelial cancer cell lines, including pancreatic, colon, lung, breast and ovarian cancer with PNC-27 and PNC-28. In all cases both peptides induce tumor cell necrosis within minutes to hours. Recent biophysical and ultrastructural studies have shown that cancer cell necrosis is due to the formation of transmembrane pores by PNC-peptide inducing direct cell lysis. Furthermore, we have found that PNC-28 destroys a highly metatstatic pancreatic cancer (BMRPA1.TUC-3) xenotransplanted into Nu/Nu mice. Remarkably, the PNC-peptides have no effect on the growth or viability of normal cells in culture such as human fibroblasts, keratinocytes and normal pancreatic acinar cells. Most importantly, the PNC-peptides have shown no effect on human umbilical-cord derived human hematopoetic stem cells in vitro to differentiate into mature blood cells and in vivo on blood cell differentials from tumor bearing and PNC-peptide treated animals. These results strongly indicate that PNC-27 and PNC-28 are potent anti-cancer drugs. The results strongly suggest the drugs' considerable potential against different epithelial cancers without exerting the limiting side effects of suppressing a patient's bone marrow, hematopoesis, normal cell growth and differentiation. Clearly, the cytotoxic efficacy of the PNC-peptides against long-established cancer cell lines is not directly transferable to a primary cancer in a future patient. Thus, to study the effect of PNC-peptide on a primary human cancer as close as possible to the in vivo (the tumor) situation, we freshly established in culture, under an IRB-approved protocol, cancer cells from the tissues of two primary human ovarian cancers and human uterine cancers. The cells released from the tumor tissue placed into a tissue culture dish were collected and their derivation as a homogenous cell population and from their respective tumors confirmed by immune-cytopathological comparative analysis. The cells' cancerous properties were confirmed in vitro within the first four passages (p) of the newly established primary cells when the cells' genomic makeup is still virtually identical (>99%) to that present in the tumor tissue per se. Cell growth and cell death in cells from p1–p4 and treated with PNC-27 or a control peptide PNC-29 were measured using MTT cell proliferation and LDH cytotoxicity assays. The results clearly demonstrate that PNC-27 effectively kills these primary human ovarian and uterine cancer cells in a dose-dependant manner with an LD50 of 100 and 150μg/ml, respectively. Throughout these experiments the effect of PNC-27 on the cancer cells' morphology was recorded, demonstrating the cells' complete disruption during the course of the treatment whereas the cancer cells treated with PNC-29 (up to 500μg/ml) remained morphologically indistinguishable from untreated cancer cells. These findings demonstrate for the first time the potentials of PNC-27 anti-cancer peptide as an efficient drug against freshly established and thus primary cells from these rather frequent human gynecological malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr C44.