Project description:BACKGROUND:We sought to investigate associations between dynamic contrast-enhanced (DCE) magnetic resonance imaging (MRI) features and tumor-infiltrating lymphocytes (TILs) in breast cancer, as well as to study if MRI features are complementary to molecular markers of TILs. METHODS:In this retrospective study, we extracted 17 computational DCE-MRI features to characterize tumor and parenchyma in The Cancer Genome Atlas cohort (n?=?126). The percentage of stromal TILs was evaluated on H&E-stained histological whole-tumor sections. We first evaluated associations between individual imaging features and TILs. Multiple-hypothesis testing was corrected by the Benjamini-Hochberg method using false discovery rate (FDR). Second, we implemented LASSO (least absolute shrinkage and selection operator) and linear regression nested with tenfold cross-validation to develop an imaging signature for TILs. Next, we built a composite prediction model for TILs by combining imaging signature with molecular features. Finally, we tested the prognostic significance of the TIL model in an independent cohort (I-SPY 1; n?=?106). RESULTS:Four imaging features were significantly associated with TILs (P?<?0.05 and FDR?<?0.2), including tumor volume, cluster shade of signal enhancement ratio (SER), mean SER of tumor-surrounding background parenchymal enhancement (BPE), and proportion of BPE. Among molecular and clinicopathological factors, only cytolytic score was correlated with TILs (??=?0.51; 95% CI, 0.36-0.63; P?=?1.6E-9). An imaging signature that linearly combines five features showed correlation with TILs (??=?0.40; 95% CI, 0.24-0.54; P?=?4.2E-6). A composite model combining the imaging signature and cytolytic score improved correlation with TILs (??=?0.62; 95% CI, 0.50-0.72; P?=?9.7E-15). The composite model successfully distinguished low vs high, intermediate vs high, and low vs intermediate TIL groups, with AUCs of 0.94, 0.76, and 0.79, respectively. During validation (I-SPY 1), the predicted TILs from the imaging signature separated patients into two groups with distinct recurrence-free survival (RFS), with log-rank P?=?0.042 among triple-negative breast cancer (TNBC). The composite model further improved stratification of patients with distinct RFS (log-rank P?=?0.0008), where TNBC with no/minimal TILs had a worse prognosis. CONCLUSIONS:Specific MRI features of tumor and parenchyma are associated with TILs in breast cancer, and imaging may play an important role in the evaluation of TILs by providing key complementary information in equivocal cases or situations that are prone to sampling bias.

Project description:Metastatic disease remains one of the most urgent clinical challenges accounting for over 90% of cancer-related deaths. Yet, the identification of novel therapeutic targets to fight or prevent metastatic disease has been hampered by the limited availability of clinically relevant mouse models of metastasis formation. To address this caveat, we developed a novel preclinical mouse model of spontaneous metastatic breast cancer that recapitulates the key biological events of the metastatic cascade and mimics the clinical course of metastatic disease in humans. Exploiting the conditional K14cre;CdhF/F;Trp53F/F mouse model of de novo mammary tumor formation, we orthotopically transplanted K14cre;CdhF/F;Trp53F/F derived mouse invasive lobular carcinoma (mILC) fragments into mammary glands of wild-type syngeneic hosts. Once recipient mammary tumors were established, we mimicked the clinical setting and performed a mastectomy. Following surgery, recipient mice eventually succumbed to wide-spread clinically overt metastatic disease in lymph nodes, lungs and gastrointestinal tract. Using aCGH analyses, we explored the relationship between the genomic profiles of mammary donor tumors and paired recipient outgrowths and observed a strong correlation, indicating that the genomic profile of the parental K14cre;CdhF/F;Trp53F/F mILC is highly conserved in recipient mammary tumors. To investigate the genomic relationship between recipient mammary tumors and their metastases, we examined the correlation structure of genomic profiles derived from paired sets of primary tumors and metastases. Genomic profiles of clonally-related recipient mammary tumors were highly conserved in local and distant metastases, indicating that few genomic alterations occur during transition from a primary tumor to a distant site. To more thoroughly examine potential site-specific genomic alterations, we constructed so-called ‘delta-profiles’ by calculating the difference between the genomic profile of a recipient mammary tumor and its paired lymph node- and lung metastasis. Site-specific recurrent alterations were not observed in lymph node nor lung metastases. Taken together, these data show that genomic profiles of metastases are highly similar to those of parental recipient tumors and that, if changes occurred, they did not recur in different independent samples. We performed aCGH analyses on DNA isolated from K14cre;Cdh-/-;Trp53-/- derived donor mILCs (n=3) and their recipient mammary tumor outgrowths (n=10). Furthermore, we also analyzed genomic profiles derived from lung (n=10), tumor-draining (n=7) and distant lymph node metastases (n=5) isolated from the same recipient mice. DNA from each of these samples was hybridized against related donor splenic DNA.

Project description:It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There is little knowledge about in situ, in vivo, tumor hypoxia during intracranial development of malignant brain tumors because of lack of efficient means to monitor it in these deep-seated orthotopic tumors. Bioluminescence imaging (BLI), based on the detection of light emitted by living cells expressing a luciferase gene, has been rapidly adopted for cancer research, in particular, to evaluate tumor growth or tumor size changes in response to treatment in preclinical animal studies. Moreover, by expressing a reporter gene under the control of a promoter sequence, the specific gene expression can be monitored non-invasively by BLI. Under hypoxic stress, signaling responses are mediated mainly via the hypoxia inducible factor-1? (HIF-1?) to drive transcription of various genes. Therefore, we have used a HIF-1? reporter construct, 5HRE-ODD-luc, stably transfected into human breast cancer MDA-MB231 cells (MDA-MB231/5HRE-ODD-luc). In vitro HIF-1? bioluminescence assay is performed by incubating the transfected cells in a hypoxic chamber (0.1% O?) for 24 hr before BLI, while the cells in normoxia (21% O?) serve as a control. Significantly higher photon flux observed for the cells under hypoxia suggests an increased HIF-1? binding to its promoter (HRE elements), as compared to those in normoxia. Cells are injected directly into the mouse brain to establish a breast cancer brain metastasis model. In vivo bioluminescence imaging of tumor hypoxia dynamics is initiated 2 wks after implantation and repeated once a week. BLI reveals increasing light signals from the brain as the tumor progresses, indicating increased intracranial tumor hypoxia. Histological and immunohistochemical studies are used to confirm the in vivo imaging results. Here, we will introduce approaches of in vitro HIF-1? bioluminescence assay, surgical establishment of a breast cancer brain metastasis in a nude mouse and application of in vivo bioluminescence imaging to monitor intracranial tumor hypoxia.

Project description:Metastatic disease remains one of the most urgent clinical challenges accounting for over 90% of cancer-related deaths. Yet, the identification of novel therapeutic targets to fight or prevent metastatic disease has been hampered by the limited availability of clinically relevant mouse models of metastasis formation. To address this caveat, we developed a novel preclinical mouse model of spontaneous metastatic breast cancer that recapitulates the key biological events of the metastatic cascade and mimics the clinical course of metastatic disease in humans. Exploiting the conditional K14cre;CdhF/F;Trp53F/F mouse model of de novo mammary tumor formation, we orthotopically transplanted K14cre;CdhF/F;Trp53F/F derived mouse invasive lobular carcinoma (mILC) fragments into mammary glands of wild-type syngeneic hosts. Once recipient mammary tumors were established, we mimicked the clinical setting and performed a mastectomy. Following surgery, recipient mice eventually succumbed to wide-spread clinically overt metastatic disease in lymph nodes, lungs and gastrointestinal tract. Using aCGH analyses, we explored the relationship between the genomic profiles of mammary donor tumors and paired recipient outgrowths and observed a strong correlation, indicating that the genomic profile of the parental K14cre;CdhF/F;Trp53F/F mILC is highly conserved in recipient mammary tumors. To investigate the genomic relationship between recipient mammary tumors and their metastases, we examined the correlation structure of genomic profiles derived from paired sets of primary tumors and metastases. Genomic profiles of clonally-related recipient mammary tumors were highly conserved in local and distant metastases, indicating that few genomic alterations occur during transition from a primary tumor to a distant site. To more thoroughly examine potential site-specific genomic alterations, we constructed so-called ‘delta-profiles’ by calculating the difference between the genomic profile of a recipient mammary tumor and its paired lymph node- and lung metastasis. Site-specific recurrent alterations were not observed in lymph node nor lung metastases. Taken together, these data show that genomic profiles of metastases are highly similar to those of parental recipient tumors and that, if changes occurred, they did not recur in different independent samples.

Project description: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.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.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.

Dataset Information

Molecular imaging of tumor-infiltrating macrophages in a preclinical mouse model of breast cancer.

Publications

Molecular imaging of tumor-infiltrating macrophages in a preclinical mouse model of breast cancer.

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