Genetic and pharmacological targeting of CSF-1/CSF-1R inhibits tumor-associated macrophages and impairs BRAF-induced thyroid cancer progression.

Advanced human thyroid cancers are densely infiltrated with tumor-associated macrophages (TAMs) and this correlates with a poor prognosis. We used BRAF-induced papillary thyroid cancer (PTC) mouse models to examine the role of TAMs in PTC progression. Following conditional activation of BRAF(V600E) in murine thyroids there is an increased expression of the TAM chemoattractants Csf-1 and Ccl-2. This is followed by the development of PTCs that are densely infiltrated with TAMs that express Csf-1r and Ccr2. Targeting CCR2-expressing cells during BRAF-induction reduced TAM density and impaired PTC development. This strategy also induced smaller tumors, decreased proliferation and restored a thyroid follicular architecture in established PTCs. In PTCs from mice that lacked CSF-1 or that received a c-FMS/CSF-1R kinase inhibitor, TAM recruitment and PTC progression was impaired, recapitulating the effects of targeting CCR2-expressing cells. Our data demonstrate that TAMs are pro-tumorigenic in advanced PTCs and that they can be targeted pharmacologically, which may be potentially useful for patients with advanced thyroid cancers

TAMs are selectively depleted in advanced PTCs of <i>Tg-Braf/Ccr2-DTR</i> mice after treatment with DT.

<p>Representative IHC with Iba-1 in PTCs of control (A) and DT-treated (B) mice. C) Quantification of Iba-1 positively staining cells from thyroid sections of control and DT-treated mice using Metamorph software. D,E) FACS analysis with anti-Cd11b and anti-Gr-1 on cell suspensions isolated from thyroids of control (D) and DT-treated mice. In the absence of DT (D) there are abundant CD11b+ Gr1- TAMs (top left quadrant). Following one week of DT (E), TAMs are profoundly depleted with a small increase in CD11b+ Gr1+ neutrophils. F) Representative analysis from FACS data for the indicated markers of TAMs and neutrophils before and after DT treatment.</p

TAMs and CAFs densely infiltrate thyroid tissue during BRAF-induced PTC initiation.

<p>FACS analysis of thyroid tissue from T<i>g-rtTA/tetO-BRAF^V600E</i> mice, in the absence (A) or presence of dox (B–E). A, Wild type thyroids have a low abundance of resident tissue Mø as measured by anti-F4/80. Seven days after dox, ∼50% of cells are Cd45⁺ leukocytes (B), that are F4/80+ and Cd11b+ (C–D). Most TAMs co-express F4/80 and Cd11b (E). FACS analyses were done on pooled wild type (no dox: n = 6) and dox-induced (n = 4) thyroids. F) IHC stain of representative frozen section of dox-induced thyroid tissue with anti-Cd68⁺ (red stain) and DAPI (blue) showing dense staining of TAMs around thyroid follicular cell nests (asterix). G) IHC with anti-αSMA+ (brown stain) demonstrates strong staining (arrows) between nests of cancer cells (asterix). H) Immunofluorescent staining with vimentin (green) is similar to that of αSMA (G, I), consistent with a dense CAF stromal compartment. I) Dual label immunofluorescence with anti-Cd11b (red) and anti-αSMA (green) did not demonstrate co-localization, consistent with the presence of two different cell types (TAMs and CAFs, respectively). F, H) Sections were co-stained with DAPI (F,H: 20× magnification; G: 10× magnification).</p

The c-FMS/CSF-1R kinase inhibitor GW2580 decreases tumor macrophages and impairs Braf-induced thyroid cancer development.

<p>A–D) Representative thyroid sections for the indicated stains from 7 day dox-induced <i>Tg-rtTA/tetO-Braf</i> mice treated with either vehicle (n = 4) or GW2580-impregnated chow (n = 4): A) H/E; B) Iba-1; C) αSMA; D)Ki67. E) Thyroid weights of dox-induced, vehicle vs. GW2580-treated mice (*p = 0.02). F) Quantification for the indicated stains; * p<0.001, ** p<0.01.</p

PTC regression following treatment with DT in Tg-Braf/Ccr2-DTR mice.

<p>A) Weight of thyroids of control and DT-treated <i>Tg-Braf/Ccr2-DTR</i> mice after 10 day treatment with intraperitoneal DT on alternate days. * p<0.01. B–C) Representative sections with the indicated stains. F) Quantification of Ki67 and TUNEL positive staining using Metamorph data analysis. Ki67 p<0.04; TUNEL p<0.01.</p

BRAF-induced PTCs derived from <i>Tg-Braf/Csf-1^−/−</i> mice have an attenuated phenotype.

<p>A) Thyroid weight of <i>Tg-Braf/Csf-1^+/−</i> (control) and <i>Tg-Braf/Csf-1^−/−</i> (<i>Csf-1</i> null) mice at 6–12 weeks of age. B, C) Representative sections stained with anti-Mac-2 (B) and H/E (C) show TAM depletion and preservation of follicular architecture in PTCs of <i>Csf-1</i> null mice.</p

Ibrutinib as initial therapy for elderly patients with chronic lymphocytic leukaemia or small lymphocytic lymphoma: an open-label, multicentre, phase 1b/2 trial

BACKGROUND: Chemoimmunotherapy has led to improved numbers of patients achieving disease response, and longer overall survival in young patients with chronic lymphocytic leukaemia; however, its application in elderly patients has been restricted by substantial myelosuppression and infection. We aimed to assess safety and activity of ibrutinib, an orally administered covalent inhibitor of Bruton tyrosine kinase (BTK), in treatment-naive patients aged 65 years and older with chronic lymphocytic leukaemia. METHODS: In our open-label phase 1b/2 trial, we enrolled previously untreated patients at clinical sites in the USA. Eligible patients were aged at least 65 years, and had symptomatic chronic lymphocytic leukaemia or small lymphocytic lymphoma requiring therapy. Patients received 28 day cycles of once-daily ibrutinib 420 mg or ibrutinib 840 mg. The 840 mg dose was discontinued after enrolment had begun because comparable activity of the doses has been shown. The primary endpoint was the safety of the dose-fixed regimen in terms of frequency and severity of adverse events for all patients who received treatment. This study is registered with ClinicalTrials.gov, number NCT01105247. FINDINGS: Between May 20, 2010, and Dec 18, 2012, we enrolled 29 patients with chronic lymphocytic leukaemia and two patients with small lymphocytic lymphoma. Median age was 71 years (range 65–84), and 23 (74%) patients were at least 70 years old. Toxicity was mainly of mild-to-moderate severity (grade 1–2). 21 (68%) patients had diarrhoea (grade 1 in 14 [45%] patients, grade 2 in three [10%] patients, and grade 3 in four [13%] patients). 15 (48%) patients developed nausea (grade 1 in 12 [39%] patients and grade 2 in three [10%] patients). Ten (32%) patients developed fatigue (grade 1 in five [16%] patients, grade 2 in four [13%] patients, and grade 3 in one [3%] patient). Three (10%) patients developed grade 3 infections, although no grade 4 or 5 infections occurred. One patient developed grade 3 neutropenia, and one developed grade 4 thrombocytopenia. After a median follow-up of 22·1 months (IQR 18·4–23·2), 22 (71%) of 31 patients achieved an objective response (95% CI 52·0–85·8); four patients (13%) had a complete response, one patient (3%) had a nodular partial response, and 17 (55%) patients had a partial response. INTERPRETATION: The safety and activity of ibrutinib in elderly, previously untreated patients with symptomatic chronic lymphocytic leukaemia, or small lymphocytic lymphoma is encouraging, and merits further investigation in phase 3 trials. FUNDING: Pharmacyclics, Leukemia and Lymphoma Society, D Warren Brown Foundation, Mr and Mrs Michael Thomas, Harry Mangurian Foundation, P50 CA140158 to Prof J C Byrd MD