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. 2017 Aug 4:45:19.
doi: 10.1186/s41182-017-0059-4. eCollection 2017.

Tumor infiltrating leukocyte density is independent of tumor grade and molecular subtype in aggressive breast cancer of Western Kenya

Rispah T Sawe  1   2   3   4 , Simeon K Mining  1 , Ayub V Ofulla  2 , Kirtika Patel  1 , Bernard Guyah  2 , David Chumba  1 , Jenifer R Prosperi  3   4   5   6 , Maggie Kerper  3   4 , Zonggao Shi  3   4 , Mayra Sandoval-Cooper  3   4 , Katherine Taylor  3   7 , Sunil Badve  4   5 , M Sharon Stack  3   4 , Laurie E Littlepage  3   4
Affiliations

Tumor infiltrating leukocyte density is independent of tumor grade and molecular subtype in aggressive breast cancer of Western Kenya

Rispah T Sawe et al. Trop Med Health. .

Abstract

Background: Tumors commonly are infiltrated by leukocytes, or tumor infiltrating leukocytes (TILs). It remains unclear, however, if the density and type of individual TILs has a direct or simply correlative role in promoting poor prognosis in breast cancer patients. Breast cancer in Kenyan women is aggressive with presentation at a young age, with advanced grade (grade III), large tumor size (>2.0 cm), and poor prognosis. We previously observed that the tumors were predominantly estrogen receptor positive (ER+) but also included both a high percentage of triple negative tumors and also increased immune cell infiltration within the tumors. We used breast tumor tissues from each patient to make tissue microarrays that were then stained for leukocyte and myeloid markers including CD4, CD8, CD20, CD25, CD68, and CD163 using immunohistochemical techniques. The immune cell infiltration into the cancer tissue included increased numbers of macrophages (CD68+), helper T cells (CD4+), and CD25+ lymphocytes compared to benign tissue.

Results: This study characterized the grade, molecular subtypes, and proliferation index of these tumors and determined if TIL density was enriched across any of these factors. We analyzed 49 malignant patient tissue samples for this study. The patient population had a mean age of 51.9 years. The tumors analyzed were heterogeneous by grade: grade I (6%), grade II (47%), and grade III (39%). Most patients presented with large tumors (>2.0 cm) (69%). We classified the tumors into molecular subtypes based on clinical marker expression. Based on this analysis, the molecular subtype distribution was heterogeneous with luminal B (41%), basal/triple negative (TN) (37%), luminal A (14%) and HER2 (8%) breast cancer subtypes. While the basal/TN subtype had a much higher proliferative index (Ki-67+) than did the other molecular subtypes, we did not see a significant correlation between TIL density and either subtype or tumor grade. Therefore, TIL density is independent of molecular subtype and grade.

Conclusion: This study identified a Kenyan patient cohort that develops large, high-grade tumors primarily of the luminal B and basal molecular subtypes. After analyzing the TILs within these tumors, we found that immune cell infiltration of these tumors correlated with increased proliferation but not grade or molecular subtype. Future research is required to determine if the aberrant recruitment of TILs to tumors contributes to cancer progression and response to cancer treatments.

Keywords: Advanced; African; Aggressive breast cancer; Breast cancer subtypes; Kenyan; Tumor infiltrating leukocytes (TILs).

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Conflict of interest statement

Ethics approval and consent to participate

This study was approved by the Institutional Ethics and Review Committee of Moi Teaching and Referral Hospital (MTRH) and the University of Notre Dame Institutional Review Board and was conducted according to the principles in the Declaration of Helsinki (Deutsch 1989).

Patients with histologically diagnosed breast cancer at the MTRH were included in the study after giving their informed consent and have been described previously [12].

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Ki-67 status in Kenyan breast tumors by molecular subtype and grade. Breast cancer tissue samples were stained for Ki-67 and scored for percent Ki-67-positive cells. Ki-67 status then was compared across molecular subtype (a) by Kruskal-Wallis (P = 0.0009) followed by Dunn’s multiple comparison tests, ER status (b) by Mann-Whitney (P = 0.006), and grade (c) by Mann-Whitney (P = 0.1241) (the P value is indicated by * and ** for P < 0.05 and P<0.01, respectively)
Fig. 2
Fig. 2
Ki-67 and TIL density correlate in breast tumors from Kenya. Breast cancer tissue samples were stained for TILs, including CD68, CD163, CD4, CD8, CD20, and CD25. Each sample was scored for percentage of positively stained area for the indicated TIL. The Ki-67 positively stained areas/nuclei then were compared across TIL expression levels of CD68 a, CD163 b, CD4 c, CD8 d, CD20 e, and CD25 f by Spearman correlation (P = 0.02, 0.003, 0.057, 0.16, 0.52, and 0.0096 with r = 0.3419, 0.4023, 0.2741, 0.2140, 0.09378, and 0.3704, respectively)
Fig. 3
Fig. 3
The density of TILs does not differ between tumor grades in breast tumors from Kenya. Breast cancer tissue samples were stained for TILs, including CD68, CD163, CD4, CD8, CD20, and CD25. Each sample was scored for percentage of positively stained area for the indicated TIL. The positively stained areas then were compared across tumor grades 1 & 2 and 3 by Mann-Whitney CD68 a, CD163 b, CD4 c, CD8 d, CD20 e, and CD25 f (P = 0.98, 0.56, 0.73, 0.29, 0.68, and 0.47, respectively). The stained area did not significantly vary by grade
Fig. 4
Fig. 4
The density of TILs does not differ between molecular subtypes in breast tumors from Kenya. Breast cancer tissue samples were stained for TILs, including CD68, CD163, CD4, CD8, CD20, and CD25. Each sample was scored for percentage of positively stained area for the indicated TIL. The positively stained area then was compared across molecular subtypes basal/TN, HER2, luminal A, and luminal B. The positively stained areas then were compared across molecular subtypes basal/TN, HER2, luminal A, and luminal B by Kruskal-Wallis followed by Dunn’s multiple comparison tests CD68 a, CD163 b, CD4 c, CD8 d, CD20 e, and CD25 f (P = 0.1187, 0.3502, 0.0898, 0.6775, 0.1376, and 0.3128, respectively). The stained areas did not significantly vary by molecular subtype
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