Myeloid cells in circulation and tumor microenvironment of breast cancer patients.

Pathological conditions including cancers lead to accumulation of a morphological mixture of highly immunosuppressive cells termed as myeloid-derived suppressor cells (MDSC). The lack of conclusive markers to identify human MDSC, due to their heterogeneous nature and close phenotypical and functional proximity with other cell subsets, made it challenging to identify these cells. Nevertheless, expansion of MDSC has been reported in periphery and tumor microenvironment of various cancers. The majority of studies on breast cancers were performed on murine models and hence limited literature is available on the relation of MDSC accumulation with clinical settings in breast cancer patients. The aim of this study was to investigate levels and phenotypes of myeloid cells in peripheral blood (n = 23) and tumor microenvironment of primary breast cancer patients (n = 7), compared with blood from healthy donors (n = 21) and paired non-tumor normal breast tissues from the same patients (n = 7). Using multicolor flow cytometric assays, we found that breast cancer patients had significantly higher levels of tumor-infiltrating myeloid cells, which comprised of granulocytes (P = 0.022) and immature cells that lack the expression of markers for fully differentiated monocytes or granulocytes (P = 0.016). Importantly, this expansion was not reflected in the peripheral blood. The immunosuppressive potential of these cells was confirmed by expression of Arginase 1 (ARG1), which is pivotal for T-cell suppression. These findings are important for developing therapeutic modalities to target mechanisms employed by immunosuppressive cells that generate an immune-permissive environment for the progression of cancer.

DNA methylation and repressive H3K9 and H3K27 trimethylation in the promoter regions of PD-1, CTLA-4, TIM-3, LAG-3, TIGIT, and PD-L1 genes in human primary breast cancer.

Background: High expression of immune checkpoints in tumor microenvironment plays significant roles in inhibiting anti-tumor immunity, which is associated with poor prognosis and cancer progression. Major epigenetic modifications in both DNA and histone could be involved in upregulation of immune checkpoints in cancer. Methods: Expressions of different immune checkpoint genes and PD-L1 were assessed using qRT-PCR, and the underlying epigenetic modifications including CpG methylation and repressive histone abundance were determined using bisulfite sequencing, and histone 3 lysine 9 trimethylation (H3K9me3) and histone 3 lysine 27 trimethylation (H3K27me3) chromatin immunoprecipitation assays (ChIP), respectively. Results: We first assessed the expression level of six immune checkpoints/ligands and found that PD-1, CTLA-4, TIM-3, and LAG-3 were significantly upregulated in breast tumor tissues (TT), compared with breast normal tissues (NT). We investigated the epigenetic modifications beyond this upregulation in immune checkpoint genes. Interestingly, we found that CpG islands in the promoter regions of PD-1, CTLA-4, and TIM-3 were significantly hypomethylated in tumor compared with normal tissues. Additionally, CpG islands of PD-L1 promoter were completely demethylated (100%), LAG-3 were highly hypomethylated (80-90%), and TIGIT were poorly hypomethylated (20-30%), in both NT and TT. These demethylation findings are in accordance with the relative expression data that, out of all these genes, PD-L1 was highly expressed and completely demethylated and TIGIT was poorly expressed and hypermethylated in both NT and TT. Moreover, bindings of H3K9me3 and H3K27me3 were found to be reduced in the promoter loci of PD-1, CTLA-4, TIM-3, and LAG-3 in tumor tissues. Conclusion: Our data demonstrate that both DNA and histone modifications are involved in upregulation of PD-1, CTLA-4, TIM-3, and LAG-3 in breast tumor tissue and these epigenetic modifications could be useful as diagnostic/prognostic biomarkers and/or therapeutic targets in breast cancer.

Intratumoral FoxP3+Helios+ Regulatory T Cells Upregulating Immunosuppressive Molecules Are Expanded in Human Colorectal Cancer.

Regulatory T cells (Tregs) can be antitumorigenic or pro-tumorigenic in colorectal cancer (CRC) depending on the presence of different Treg subsets with various immunosuppressive molecules. Some studies reported the phenotypic characteristics of tumor-infiltrating immune cells in CRC, but limited studies have focused on the co-expression of suppressive molecules on immune cells. The aim of this study was to characterize immune cells in the tumor microenvironment (TME), compared to paired adjacent non-tumor colon tissue of CRC patients. Additionally, we investigated co-expression of immunosuppressive molecules on different Treg subsets in the TME, normal colon tissue, and peripheral blood of CRC patients and healthy donors. In this preliminary study, we report that the majority of CD3+ T cells in the TME are CD4+ T cells with high co-expression of programmed death 1 (PD-1)/cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and PD-1/CD39 molecules. Levels of CD4+FoxP3+Helios+ Tregs were significantly increased in the TME. Furthermore, we observed increased levels of PD-1/CTLA-4 and PD-1/CD39 co-expressing cells within FoxP3+Helios+ and FoxP3+Helios- Treg subsets, indicative of their potent immunosuppressive potential. These results suggest synergistic associations between PD-1/CTLA-4 and PD-1/CD39 in dampening T-cell activation and function along with suppressing tumor-specific immune responses, suggesting that dual blockade of these molecules could be a more effective strategy for inducing antitumor immune responses in CRC.

Preferential accumulation of regulatory T cells with highly immunosuppressive characteristics in breast tumor microenvironment.

Immunosuppressive cells such as regulatory T cells (Tregs) have an ambiguous role in breast cancer prognosis, with studies reporting both positive and negative correlations between Treg infiltration and prognosis. This discrepancy could be due to the different immunosuppressive molecules present in these cells. In the present study, we phenotypically characterize different Treg subsets infiltrating the tumor microenvironment (TME), compared to adjacent normal tissue and peripheral blood of primary breast cancer (PBC) patients. We report that the majority of tumor-infiltrating CD4+ and CD8+ T cells have terminally exhaustive phenotype as assessed by CD39 and PD-1 expressions. We also show that Tregs are accumulated in breast TME compared to normal tissue. Further characterization of Tregs showed that these are mainly FoxP3+Helios+ and express high levels of CTLA-4 and PD-1. This preferential accumulation of FoxP3+Helios+ Treg subset with co-expression of different immune inhibitory molecules might have a negative effect on breast cancer prognosis. Taken together, our results suggest that breast tumor cells might utilize Tregs, and different suppressive pathways involving CD39, PD-1 and CTLA-4 molecules in creating an immune-subversive environment for them to survive, and a dual blockade of these immunosuppressive molecules might be considered as an effective method in breast cancer treatment.

Increased Levels of Circulating and Tumor-Infiltrating Granulocytic Myeloid Cells in Colorectal Cancer Patients.

Increased levels of myeloid cells, especially myeloid-derived suppressor cells (MDSCs), have been reported to correlate with bad prognosis and reduced survival in cancer patients. However, limited data are available on their conclusive phenotypes and their correlation with clinical settings. The aim of this study was to investigate levels and phenotype of myeloid cells in peripheral blood and tumor microenvironment (TME) of colorectal cancer (CRC) patients, compared to blood from healthy donors (HDs) and paired, adjacent non-tumor colon tissue. Flow cytometric analysis was performed to examine the expression of different myeloid markers in fresh peripheral blood samples from CRC patients and HDs, and tissue-infiltrating immune cells from CRC patients. We found significantly higher levels of cells expressing myeloid markers and lacking the expression of major histocompatibility complex class II molecule HLA-DR in blood and tumor of CRC patients. Further analysis revealed that these cells were granulocytic and expressed Arginase 1 indicative of their suppressive phenotype. These expanded cells could be neutrophils or granulocytic MDSCs, and we refer to them as granulocytic myeloid cells (GMCs) due to the phenotypical and functional overlap between these cell subsets. Interestingly, the expansion of peripheral GMCs correlated with higher stage and histological grade of cancer, thereby suggesting their role in cancer progression. Furthermore, an increase in CD33+CD11b+HLA-DR-CD14-CD15- immature myeloid cells was also observed in CRC tumor tissue. Our work shows that GMCs are expanded in circulation and TME of CRC patients, which provides further insights for developing immunotherapeutic approaches targeting these cell subsets to enhance antitumor immune and clinical responses.

Deficiency of mitochondrial modulator MCJ promotes chemoresistance in breast cancer.

Despite major advances in early detection and prognosis, chemotherapy resistance is a major hurdle in the battle against breast cancer. Identifying predictive markers and understanding the mechanisms are key steps to overcoming chemoresistance. Methylation-controlled J protein (MCJ, also known as DNAJC15) is a negative regulator of mitochondrial respiration and has been associated with chemotherapeutic drug sensitivity in cancer cell lines. Here we show, in a retrospective study of a large cohort of breast cancer patients, that low MCJ expression in breast tumors predicts high risk of relapse in patients treated with chemotherapy; however, MCJ expression does not correlate with response to endocrine therapy. In a prospective study in breast cancer patients undergoing neoadjuvant therapy, low MCJ expression also correlates with poor clinical response to chemotherapy and decreased disease-free survival. Using MCJ-deficient mice, we demonstrate that lack of MCJ is sufficient to induce mammary tumor chemoresistance in vivo. Thus, loss of expression of this endogenous mitochondrial modulator in breast cancer promotes the development of chemoresistance.

Uncommon metastasis of laryngeal cancer to small bowel causing intestinal obstruction treated by laparoscopic approach.

Metastatic laryngeal cancer to the small bowel is extremely rare. Management of small bowel obstruction used to constitute a relative contraindication for the use of laparoscopic modality. We are reporting a case of an elderly man known to have laryngeal cancer who presented with small bowel obstruction due to metastatic deposit to the small bowel. The condition was successfully treated by laparoscopic assisted approach. A review of the natural history of advanced laryngeal cancer, common and uncommon sites of metastasis, and the rare presentation as small bowel obstruction is illustrated in this review.

Management of peri-anal giant condyloma acuminatum--a case report and literature review.

Giant condyloma acuminatum (GCA), originally described by Buschke and Loewenstein in 1925 as a lesion of the penis, is more rarely seen in the anorectum and is characterized by clinical malignancy in the face of histologic benignity; however, malignant transformation to frankly invasive squamous-cell carcinoma has been described in about one-third of patients. In addition, malignant transformation has been reported in patients with "ordinary" condylomata acuminata. Human papillomavirus, known to cause condylomata acuminata, is also known to induce these tumors and was found in 96% of 63 cases reviewed in the last 10 years. These lesions have a propensity for recurrence and a likelihood of malignant transformation, and lead to significant mortality. Therefore, early and radical R0 excision, along with vigilant follow-up, provides the hope for cure. Conservative and/or multimodal therapy has been reported in a few cases, but its effect is not yet proved. The authors report one case of GCA; in addition, they reviewed the literature over the last 10 years and compared with previous reviews.