Intratumoral heterogeneity affects tumor regression and Ki67 proliferation index in perioperatively treated gastric carcinoma

Abstract

Background: Intratumoral heterogeneity (ITH) is a major problem in gastric cancer (GC). We tested Ki67 and tumor regression for ITH after neoadjuvant/perioperative chemotherapy.

Methods: 429 paraffin blocks were obtained from 106 neoadjuvantly/perioperatively treated GCs (one to five blocks per case). Serial sections were stained with Masson's trichrome, antibodies directed against cytokeratin and Ki67, and finally digitalized. Tumor regression and three different Ki67 proliferation indices (PI), i.e., maximum PI (KiH), minimum PI (KiL), and the difference between KiH/KiL (KiD) were obtained per block. Statistics were performed in a block-wise (all blocks irrespective of their case-origin) and case-wise manner.

Results: Ki67 and tumor regression showed extensive ITH in our series (maximum ITH within a case: 31% to 85% for KiH; 4.5% to 95.6% for tumor regression). In addition, Ki67 was significantly associated with tumor regression (p < 0.001). Responders (<10% residual tumor, p = 0.016) exhibited prolonged survival. However, there was no significant survival benefit after cut-off values were increased ≥20% residual tumor mass. Ki67 remained without prognostic value.

Conclusions: Digital image analysis in tumor regression evaluation might help overcome inter- and intraobserver variability and validate classification systems. Ki67 may serve as a sensitivity predictor for chemotherapy and an indicator of ITH.

Fig. 1. Illustration of intratumoral heterogeneity of Ki67 expression and tumor regression in the examplary case (Supplemental Fig. 1).

Each row represents a seperate block of the same tumor. Tumor regression was evaluated as the ratio of residual (pan-cytokeratin) to former (Masson’s trichrome stain) tumor site. With increasing tumor regression, the hot spot Ki67 PI (KiH) decreased while the Ki67 PI at the lower proliferation end (KiL) increased (values in Supplemental Fig. 1). Ki67 images in 200-fold.

Fig. 2. Kaplan-Meier plots for the whole cohort depicting patient survival.

We performed three analyses setting the cut-off value for the median tumor bed ratio (mTBR) to 10% (a, b), to 20% (c, d) and additionally partitioned the cohort into quartiles (e, f). While patients with a mTBR below 10% exhibited prolonged survival (OS, p = 0.016), no prognostic benefit was shown at the cut-off value of 20%. Moreover, a finer grading into quartiles did not prove to be of prognostic relevance.

Fig. 3. Patient survival depicted using Kaplan-Meier plots.

Ki67 values were dichotomized at the median. No significant association with overall (a) or tumor-specific survival (b) was found for the maximum Ki67 hotspot proliferation index per case (maximum KiH).

Fig. 4. This schematic model illustrates the putative effect of neoadjuvant chemotherapy on Ki67 proliferation index (PI) and tumor regression.

Each pair of bars represents the Ki67 PI prior to neoadjuvant chemotherapy (light blue; expected value) and post-chemotherapy (dark blue; measured value). Ki67 high index (KiH), Ki67 low index (KiL), the difference (KiD) between KiH/KiL and tumor mass reduction were specified for each bar. Two main effects were observed (left). The decline in the KiH is interpreted as a result of the cytotoxic effect chemotherapy has on proliferating, chemo-sensitive subclones. The incline in the KiL can be attributed to selection pressure exerted by neoadjuvant chemotherapy on clonal diversity. Prior to neoadjuvant chemotherapy, chemo-resistant subclones were inhibited in their growth by better adapted (“fitter”), chemo-sensitive subclones. Following selection caused by neoadjuvant therapy, the surviving (chemo-resistant) subclones were able to exploit their growth advantage (increase in proliferation). Both effects were observed in tumor blocks with a large reduction of tumor mass (right; responder). Chemo-sensitive subclones perished with chemo-resistant subclones being selected through neoadjuvant chemotherapy. In contrast, in tumor blocks where chemo-resistant subclones accounted for a large proportion of the tumor mass (non-responder), only minor changes in KiH and KiL were observed.