Uncovering the Origin of Skin Side Effects from EGFR-Targeted Therapies

Stained skin samples obtained from wild type mice and mice lacking EGFR in the skin (EGFR KO)  at different ages are shown. After 7 days of age, EGFR KO mice increasingly developed skin alterations similar to patients treated with EGFR inhibitors. Neutrophilic pustules (red arrows), keratin plugs (yellow arrows), sebaceous glands (green arrows) and pigment masses (black arrows) are highlighted. Objective 10X, scale bar 100 mm.

Stained skin samples obtained from wild type mice and mice lacking EGFR in the skin (EGFR KO)  at different ages are shown. After 7 days of age, EGFR KO mice increasingly developed skin alterations similar to patients treated with EGFR inhibitors. Neutrophilic pustules (red arrows), keratin plugs (yellow arrows), sebaceous glands (green arrows) and pigment masses (black arrows) are highlighted. Objective 10X, scale bar 100 mm.

The epidermal growth factor receptor (EGFR), a key regulator of cell proliferation, is often mutated or overexpressed in a variety of cancer types. EGFR-targeted therapies, including monoclonal antibodies and small molecule inhibitors, can effectively treat patients whose tumors depend on aberrant EGFR signaling. Within a few weeks of initiating therapy, however, patients develop a characteristic rash with leukocyte infiltration into the skin accompanied by pruritus (itching), scaling of the skin, hair loss, and even changes in skin cell differentiation. The side effects can become so severe that patients take reduced doses, which can limit efficacy, or stop treatment altogether. To understand how EGFR inhibitors cause these skin changes in the hopes of identifying a means of preventing them, Stuart Yuspa, M.D., of CCR’s Laboratory of Cancer Biology and Genetics, and his colleagues examined patient samples and generated a mouse model of EGFR loss in the skin.

The researchers first measured changes in inflammatory proteins and blood cells in plasma samples obtained before and one month into treatment with the EGFR inhibitor gefitinib in a clinical trial of patients with epithelial ovarian cancer. In general, patients had increases in inflammatory factors on treatment, and those who had the lowest pretreatment levels of inflammatory proteins had more severe rash and pruritus. An increase in the percentage of circulating granulocytes and a decrease in the percentage of lymphocytes on gefitinib treatment were associated with severe rash and pruritus. Increased numbers of platelets also were associated with pruritus. These results suggest that changes in circulating levels of inflammatory mediators and cells are related to anti-EGFR therapy, but whether they were due to an effect on the tumor or on the skin was unclear.

To gain further insight, the investigators created a transgenic mouse model lacking EGFR expression specifically in the epidermis, the outer layer of skin. The mice appeared normal at birth and five to six days afterwards. At a week old, however, the mice began developing skin lesions similar to those of patients taking EGFR inhibitors, complete with leukocyte infiltration, pruritus, scaly skin, and destroyed hair follicles. The scientists then measured the levels of inflammatory proteins in the plasma of the EGFR skin knockout mice and wild type mice at ages two, three, and 12 weeks. Similar to the results observed in patients, the researchers found increases in a number of chemokines and cytokines. Blood cell counts in the knockout mice also resembled those in patients with higher numbers of neutrophils and platelets and reductions in lymphocytes.

Knowing that their model recapitulated the effects observed in patients taking anti-EGFR therapies, the investigators examined the epidermal structure of the knockout mice. They found that the skin barrier formed normally and that keratinocytes from seven-day-old knockout mice retained normal differentiation markers. At the same time, the scientists observed increased leukocyte infiltration in the skin. Two weeks later, they found overexpression and altered distribution of the epidermal markers, demonstrating that leukocyte invasion preceded the increase in differentiation markers.

The researchers found that macrophages and mast cells invaded the skin of the knockout mice first when the skin still appeared to be normal followed by T cells, eosinophils, and, much later, neutrophils, which were associated with hair follicle destruction. Looking at early changes in cytokine and chemokine expression in the skin, they found elevated CCL5 and its receptors CCR5 and CCR1, which are expressed on macrophages. Cultured skin keratinocytes lacking EGFR could secrete CCL5, and supernatants from these cells attracted macrophages better than those of EGFR-expressing keratinocytes. The investigators also found a number of other inflammatory markers expressed early in the skin that remained elevated in the circulation of older knockout mice.

To ameliorate the effects of EGFR loss in the skin, the researchers crossed the knockout mice with mice deficient in a number of immune signaling pathways, including tumor necrosis factor α, MyD88, nitric oxide synthase 2, CCR2, and Rag1. However, none of the double mutants showed an improvement in skin lesions. The researchers then decided to target macrophages specifically by injecting the EGFR knockout mice with liposomes containing clodronate, a bisphosphonate. Treatment improved skin architecture near the injection site and reduced macrophage numbers. Likewise, the levels of some highly expressed inflammatory genes and epithelial differentiation markers were normalized or reduced in clodronate-treated skin.

Together these results suggest that EGFR inhibition in the skin leads to increased macrophage infiltration, setting off an inflammatory cascade that eventually leads to skin damage and hair loss. Bisphosphonates, which are already approved to treat bone loss in cancer patients, may be promising therapies for preventing or improving the severe skin side effects of EGFR inhibitors.

Summary Posted: 08/2013

Reference

Mascia F, Lam G, Keith C, Garber C, Steinberg SM, Kohn E, Yuspa SH. Genetic Ablation of Epidermal EGFR Reveals the Dynamic Origin of Adverse Effects of Anti-EGFR Therapy. Science Translational Medicine. August 2013 PubMed Link