Neutral evolution of drug resistant colorectal cancer cell populations is independent of their KRAS status

Abstract

Emergence of tumor resistance to an anti-cancer therapy directed against a putative target raises several questions including: (1) do mutations in the target/pathway confer resistance? (2) Are these mutations pre-existing? (3) What is the relative fitness of cells with/without the mutation? We addressed these questions in patients with metastatic colorectal cancer (mCRC). We conducted an exhaustive review of published data to establish a median doubling time for CRCs and stained a cohort of CRCs to document mitotic indices. We analyzed published data and our own data to calculate rates of growth (g) and regression (d, decay) of tumors in patients with CRC correlating these results with the detection of circulating MT-KRAS DNA. Additionally we estimated mathematically the caloric burden of such tumors using data on mitotic and apoptotic indices. We conclude outgrowth of cells harboring intrinsic or acquired MT-KRAS cannot explain resistance to anti-EGFR (epidermal growth factor receptor) antibodies. Rates of tumor growth with panitumumab are unaffected by presence/absence of MT-KRAS. While MT-KRAS cells may be resistant to anti-EGFR antibodies, WT-KRAS cells also rapidly bypass this blockade suggesting inherent resistance mechanisms are responsible and a neutral evolution model is most appropriate. Using the above clinical data on tumor doubling times and mitotic and apoptotic indices we estimated the caloric intake required to support tumor growth and suggest it may explain in part cancer-associated cachexia.

Fig 1. Example of a case of CRC [40X magnification].

Stains include hematoxylin and eosin (H&E), Ki-67 (MIB-1) and phosphohistone immunohistochemistry. The Ki-67 protein is associated with cell proliferation and expression can be detected during all active phases of the cell cycle (G₁, S, G₂, and mitosis), but is absent in resting cells (G₀)¹⁸. The MIB-1 antibody was used to determine the Ki-67 labeling index. Phospho-histone H3^{(Ser 10)} is expressed in the nuclei of cells during M-phase (mitosis) allowing one to use phosphohistone staining as a means to identify cells in mitosis. H&E stain (A, x400) including atypical mitotic figures (B, x400). MIB-1/Ki-67 stain demonstrating a high proliferation index (C, x40). Phosphohistone stain demonstrating cells in mitosis [D, x400].

Fig 2. Evolution of tumors in patients is shown as graphs of RECIST measurements.

A: Nine patients with detectable MT KRAS DNA in serum. B: Nine patients without detectable MT KRAS DNA in serum. As can be seen the kinetics of tumor regression and growth of these tumors is indistinguishable. Detection of a mutant KRAS DNA is serum, interpreted as evidence of “acquiring” this genetic alteration, does not impact the growth of the tumor. The blue lines are the lines drawn by the model and this demonstrates how well the theoretical (blue line) fit the actual (red symbols). The fit of the actual data to the theoretical had p values less than 0.05 in all cases, and in most much less than that.

Fig 3. Comparison of growth rate (g) using tumor measurements from Diaz et al [17] and from an independent CRC tumor data set of patients treated in second line with conventional chemotherapy.

Top 2 panels: The growth (g, p = 0.244) and regression (d, p = 0.858) rates of tumors treated with panitumumab in which mutant KRAS DNA could be detected in serum (MT) do not differ from those of tumors without detectable mutant KRAS DNA in serum (WT). Lower 2 panels: Compared to tumors independently treated in second line with conventional chemotherapy the growth rates (g) of the panitumumab-treated tumors is similar for both the subgroup with detectable circulating mutant KRAS DNA (MT) in serum and those without detectable mutant KRAS DNA in serum (WT).

Fig 4

Estimates (A) the cumulative number of additional kilocalories and (B) the number of additional kilocalories per day required by a tumor for each successive 60-day doubling period required due to the tumor. The estimates are for two different mitotic and apoptotic indexes. The doubling time is 60 days and the tumor mass progresses through 11 doublings. The estimates include the number of calories to produce both the cell that survive, as well as those that undergoes apoptosis. (C) Estimates for the number of calories needed to support a tumor.

Fig 5

(A) Progression of tumor quantity from 1 gram to 2 kilograms with a doubling time of 60 days. The blue curve depicts a tumor that does not decrease at all during progression, while the red curve depicts an example where intermittent treatments lead to transient decreases in tumor size. Resistance then emerges and the tumor again beings to grow. Thus the blue model that we use provides estimates for the minimum amount of new tumor formed and the minimum caloric requirements. (B) Total amount of tumor that undergoes apoptosis as the net tumor quantity increases from 1 gram to 2 kilograms for two different mitotic (μ) and apoptotic (α) indexes. The doubling time is 60-days and the tumor mass progresses through 11 doublings.