Presence Of Metastatic Cells Research Articles

Insight into immune profile associated with vitiligo onset and anti-tumoral response in melanoma patients receiving anti-PD-1 immunotherapy.

Immunotherapy with checkpoint inhibitors is an efficient treatment for metastatic melanoma. Development of vitiligo upon immunotherapy represents a specific immune-related adverse event (irAE) diagnosed in 15% of patients and associated with a positive clinical response. Therefore, a detailed characterization of immune cells during vitiligo onset in melanoma patients would give insight into the immune mechanisms mediating both the irAE and the anti-tumor response. To better understand these aspects, we analyzed T cell subsets from peripheral blood of metastatic melanoma patients undergoing treatment with anti-programmed cell death protein (PD)-1 antibodies. To deeply characterize the antitumoral T cell response concomitant to vitiligo onset, we analyzed T cell content in skin biopsies collected from melanoma patients who developed vitiligo. Moreover, to further characterize T cells in vitiligo skin lesion of melanoma patients, we sequenced T cell receptor (TCR) of cells derived from biopsies of vitiligo and primary melanoma of the same patient. Stratification of patients for developing or not developing vitiligo during anti-PD-1 therapy revealed an association between blood reduction of CD8-mucosal associated invariant T (MAIT), T helper (h) 17, natural killer (NK) CD56bright, and T regulatory (T-reg) cells and vitiligo onset. Consistently with the observed blood reduction of Th17 cells in melanoma patients developing vitiligo during immunotherapy, we found high amount of IL-17A expressing cells in the vitiligo skin biopsy, suggesting a possible migration of Th17 cells from the blood into the autoimmune lesion. Interestingly, except for a few cases, we found different TCR sequences between vitiligo and primary melanoma lesions. In contrast, shared TCR sequences were identified between vitiligo and metastatic tissues of the same patient. These data indicate that T cell response against normal melanocytes, which is involved in vitiligo onset, is not typically mediated by reactivation of specific T cell clones infiltrating primary melanoma but may be elicited by T cell clones targeting metastatic tissues. Altogether, our data indicate that anti-PD-1 therapy induces a de novo immune response, stimulated by the presence of metastatic cells, and composed of different T cell subtypes, which may trigger the development of vitiligo and the response against metastatic tumor.

Investigation of white blood cell characteristics in cerebrospinal fluid samples at pediatric brain tumor diagnosis.

The role of white blood cells (WBC) in the pediatric central nervous system (CNS) tumor microenvironment is incompletely defined. We hypothesized that the WBC profile in cerebrospinal fluid (CSF) correlates with the presence of tumor cells and prognosis in pediatric CNS tumors, as well as other patient and disease characteristics, and differs by tumor type, thus giving insight into the tumor immune response. We conducted a retrospective analysis of CSF WBC profiles at CNS tumor diagnosis in 269 patients at our institution. We examined total nucleated cell count, absolute counts, and percentages by WBC subtype. We compared CSF WBC values by tumor cell presence, patient vital status, tumor location, and the most common tumor types. Patients who died of their tumor had a lower CSF lymphocyte percentage and a higher absolute monocyte count in CSF at diagnosis. The presence of tumor cells in CSF was associated with fewer lymphocytes and monocytes. Ventricular tumors had higher CSF lymphocyte, monocyte, macrophage, and total nucleated cell counts than extraventricular tumors. Germ cell tumors, low-grade glioma, high-grade glioma, and ependymoma had lower macrophage counts or percentages compared to other tumor types. WBC profile in CSF at pediatric CNS tumor diagnosis correlates with patient prognosis and presence of metastatic cells, along with tumor type and other tumor characteristics like relationship to the ventricles. Prospective CSF profiling and study may be useful to future immunotherapy and other pediatric CNS tumor clinical trials.

The breast cancer immune microenvironment is modified by neoadjuvant chemotherapy

Neoadjuvant chemotherapy (NAT) in breast cancer (BC) has been used to reduce tumor burden prior to surgery. However, the impact on prognosis depends on the establishment of Pathological Complete Response (pCR), which is influenced by tumor-infiltrating lymphocyte levels and the activation of the antitumor immune response. Nonetheless, NAT can affect immune infiltration and the quality of the response. Here, we showed that NAT induces dynamic changes in the tumor microenvironment (TME). After NAT, an increase of regulatory T cells and a decrease of CD8+ T cells was found in tumor, correlated with the presence of metastatic cells in lymph nodes. In addition, an increase of polymorphonuclear myeloid-derived suppressor like cells was found in luminal patients post-NAT. pCR patients showed a balance between the immune populations, while non-pCR patients presented an inverse relationship in the frequency of CD68+ versus CD3+, CD8+, and CD20+ cells. Moreover, activated T cells were found in peripheral blood, as well as an increase in T cell clonality with a lower diversity post-NAT. Overall, these results shown that NAT induces an activation of immune response, however, a balance in the TME seems to be related to a better antigenic presentation and therefore a better response to treatment.

The Amino-Terminal Oligomerization Domain of Angiopoietin-2 Affects Vascular Remodeling, Mammary Gland Tumor Growth, and Lung Metastasis in Mice.

Angiopoietin-2 (ANGPT2) is a context-dependent TIE2 agonistic or antagonistic ligand that induces diverse responses in cancer. Blocking ANGPT2 provides a promising strategy for inhibiting tumor growth and metastasis, yet variable effects of targeting ANGPT2 have complicated drug development. ANGPT2443 is a naturally occurring, lower oligomeric protein isoform whose expression is increased in cancer. Here, we use a knock-in mouse line (mice expressing Angpt2443), a genetic model for breast cancer and metastasis (MMTV-PyMT), a syngeneic melanoma lung colonization model (B16F10), and orthotopic injection of E0771 breast cancer cells to show that alternative forms increase the diversity of Angpt2 function. In a mouse retina model of angiogenesis, expression of Angpt2443 caused impaired venous development, suggesting enhanced function as a competitive antagonist for Tie2. In mammary gland tumor models, Angpt2443 differentially affected primary tumor growth and vascularization; these varying effects were associated with Angpt2 protein localization in the endothelium or in the stromal extracellular matrix as well as the frequency of Tie2-positive tumor blood vessels. In the presence of metastatic cells, Angpt2443 promoted destabilization of pulmonary vasculature and lung metastasis. In vitro, ANGPT2443 was susceptible to proteolytical cleavage, resulting in a monomeric ligand (ANGPT2DAP) that inhibited ANGPT1- or ANGPT4-induced TIE2 activation but did not bind to alternative ANGPT2 receptor α5β1 integrin. Collectively, these data reveal novel roles for the ANGPT2 N-terminal domain in blood vessel remodeling, tumor growth, metastasis, integrin binding, and proteolytic regulation. SIGNIFICANCE: This study identifies the role of the N-terminal oligomerization domain of angiopoietin-2 in vascular remodeling and lung metastasis and provides new insights into mechanisms underlying the versatile functions of angiopoietin-2 in cancer.See related commentary by Kamiyama and Augustin, p. 35.

Cytomorphological analysis of body fluids in tertiary care centre

Cytological study of body fluids has become integral part in both diagnostic and management of suspected malignant cases. The meticulous examination of body fluid for there cytomorphological properties throws light on the cause, presence of metastatic cells, typing of unknown cases, staging and prognosis of cancer.1) To study and evaluate current trends in cytological evaluation of body fluids for various pathologi­cal conditions in a tertiary care centre. 2) To analyse their frequency in relation to diagnosis.The present study is prospective type of analysis of 311 body fluids received in Department of pathology for duration of one year from Jan 2018 to Dec 2018 in our tertiary care centre. A total of 311 body fluid samples were analyzed in this study. Pleural fluid comprised of the major bulk of the study with 120 cases followed by peritoneal fluid with 100 cases. Non-malignant diagnosis was given in majority of the cases. Slight male preponderance was noted with male to female ration 1.37:1. Cytological analysis of body fluid is routine diagnostic investigation which offers definite aid to the treating physician. Preliminary body fluid analysis in resource limited setups still remains the most simple, convenient and cost effective technique in reaching to a particular diagnosis.

Breast Cancer Metastasis to the Axillary Lymph Nodes: Are Changes to the Lymph Node “Soil” Localized or Systemic?

Metastasis is a multistep process that is not well understood. Colonization of a secondary organ requires specific molecular alterations of the host microenvironment. To determine the temporal and spatial changes associated with metastatic dissemination to the axillary lymph nodes, gene expression profiles were compared between histologically normal lymph nodes from node-positive patients and tumor-free nodes from node-negative patients. Using a stringent false discovery rate correction (<0.05) for multiple hypothesis testing, we did not detect any differentially expressed genes between the lymph node groups. Thus, the presence of metastatic cells within the lymphatic system does not elicit widespread changes in gene expression through the axillary basin; rather, lymph nodes independently respond to disseminated tumor cells.

Significance of Matrix Metalloproteinase 9 Expression as Supporting Marker to Cytokeratin 19 mRNA in Sentinel Lymph Nodes in Breast Cancer Patients.

One-step nucleic acid amplification (OSNA) detects and quantifies, with the use of a polymerase chain reaction, the presence of cytokeratin 19 mRNA in sentinel lymph nodes. The main advantage of the OSNA assay is the avoidance of second surgery in case of positive sentinel lymph node diagnosis. The objective of this study was to evaluate the significance of matrix metalloproteinase 9 expression by immunohistochemistry as supporting marker to cytokeratin 19 mRNA in sentinel lymph nodes in breast cancer patients and to relate this expression with clinicopathological data. This study was conducted on fresh sentinel lymph nodes obtained from 40 patients with tumors classified as carcinoma of no special type. The presence of metastatic cells in the slices of lymph nodes was evaluated by immunohistochemistry using antibodies for CK19 and MMP-9. Expression of CK19 and MMP-9 in lymph nodes was also confirmed by means of Western blot analysis. Results indicated that the strongest correlation with CK19 mRNA was displayed by MMP-9, CK19 (by immunohistochemistry, IHC), and nodal metastases (p < 0.001). Higher histological grading also positively correlated with CK19 mRNA, however that correlation was less significant. Since MMP-9 shows very strong correlation with CK19 mRNA in breast carcinoma of no special type metastases, expression of MMP-9 in sentinel lymph nodes should be considered as useful method whenever OSNA analysis is not available.

Abstract A29: Cooption of the reactive stroma by brain metastasis relies on protease inhibitors

Abstract Metastatic cancer cells are known to get benefit from the interaction with their microenvironment. The presence of metastatic cells in a target organ induces changes in the stroma. This reactive stroma is very evident in the brain, where cancer cells are ubiquitously associated with reactive glia. Since reactive astrocytes are known sources not only of trophic factors but also deleterious signals, we hypothesized that the ability of metastatic cells to develop in a target organ requires the ability to neutralize these deleterious signals. We detected reactive astrocytes in the proximity of brain metastasis producing plasminogen activators (tPA and uPA), which activate Plasmin, one of the main proteases in the brain. Plasmin activity in the brain acts as a deleterious factor to metastatic cells in this organ. Cancer cells selected in vivo for their ability to metastasize in the brain overexpress Serine Protease Inhibitors (SERPINs) that block Plasmin activation. Moreover, a significant cohort of human brain metastasis samples from both lung and breast adenocarcinoma scored positive for specific members of the SERPIN family. When the cancer cells were depleted of SERPINs their brain metastatic activity was significantly reduced in all experimental models, including breast and lung adenocarcinoma xenografts and syngeneic mouse models. Brain metastatic cells are sensitive to Plasmin activated killer cytokines (sFASL), and their ability to survive and to grow in the brain depends on genes whose products are inactivated by Plasmin cleavage (VEGFA and L1CAM). The cooption of SERPINs by brain metastatic cells is required to avoid the deleterious consequences of the reactive stroma and subsequently benefit from the interaction with the microenvironment. Citation Format: Manuel Valiente, Derek J. Lee, Anna Obenauf, Jason T. Huse, Jamie E. Chaft, Mark G. Kris, Edi Brogi, Joan Massagué. Cooption of the reactive stroma by brain metastasis relies on protease inhibitors. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A29.

Vibrational spectroscopic changes of B‐lymphocytes upon activation

The first study interpreting B-lymphocyte activation in normal lymph nodes using vibrational micro-spectral imaging is reported. Lymphocyte activation indicates the presence and response against a pathogen, regardless of the inciting pathogen's etiology, whether a benign, reactive or malignant process. Understanding the biochemical makeup of lymphocyte activation during early stages of disease and immune response may offer significant aid in determining a tumor's origin without the presence of malignant metastatic cells but within lymph nodes that are reactive and displaying regions of hyperplasia. Infrared and Raman data scrutinized via unsupervised multivariate methods may provide a physical and reproducible method to determine the biochemical components and variances therein of activated lymph nodes with distinguishing characteristics depending on the malignancy present in the region or elsewhere in the body. The results reported here provide a proof-of-concept study that reveal a potential to screen lymph nodes for disease without the presence of metastatic cells.

In-transit sentinel lymph nodes in malignant melanoma. What is their importance?

The sentinel lymph node (SLN) is the first node in a lymph node basin to receive direct drainage from the primary tumour. However, in some cases, lymphoscintigraphy images demonstrate the presence of lymph nodes located in the area between the primary tumour and the first regional lymph node basin. These nodes are called in-transit nodes and have to be considered SLNs as well. It was to determine the incidence and location of in-transit SLNs in patients with malignant melanoma and to evaluate whether it is necessary to harvest them. METHOD. Nine hundred patients with malignant melanoma were included. Lymphoscintigraphy was performed on the day before surgery following intradermal injection of 74-111 MBq of 99mTc-Nanocolloid in four doses around the primary lesion or the biopsy scar. The presence of in-transit SLNs was revealed in 80 patients. During surgery, in-transit SLNs were identified and excised in all but 3 patients (96.2 %). Metastatic cell deposits were identified in these in-transit SLNs in 15 patients (19.4 %), with 4 patients with no tumour involvement of the regional lymph node basin. Lymphoscintigraphy is mandatory in the location of in-transit SLNs. Moreover, although the incidence of these nodes is relatively low in malignant melanoma, the presence of metastatic cells in these in-transit SLNs reaches a significant percentage. Therefore, excision of in-transit SLNs is necessary in all cases.

Prognostic value of an extended tumor staging in a prospective study with detection of disseminated tumor cells in bone marrow, peritoneal cavity and lymph nodes in colorectal cancer

3539 Background: The traditional definition of localized and surgical resectable colorectal cancer (CRC) is challenged by the discovery of disseminated tumor cells in various body compartments. But the influence of minimal residual disease (MRD) on survival remains unclear. Methods: MRD was determined prospectively in 222 patients operated for colorectal tumors using immunohistochemistry with anti-pan-cytokeratin and Ber-EP4 antibodies. This is the first study with a simultaneous detection of cell dissemination in all possible compartments in a large patient cohort. Prognostic significance was determined in relation to 140 clinical and pathological data. In a median follow-up time of 44,6 months the main endpoint was disease free survival (DFS). Results: Out of 205 patients having undergone surgery for CRC, 67 (32,7%) died of CRC-related causes, 22 patients (10,7%) developed local recurrence. Presence of epithelial cells was detected in bone marrow in 135 of 205 patients (65,9%) and was not associated with tumor stage nor with poorer DFS. Presence of metastatic cells in peritoneal cavity are detectable in 50 of 194 patients (25,8%). They are associated with lymph node involvement (p<0,001), UICC stage and tumor invasion (p=0,001), CEA-level (p=0,003), tumor stenosis (p=0,004) and, in univariate analysis, with reduced DFS (p=0.006). Lymph nodes micrometastases were found in 50 of 107 (46,7%) UICC stage I or II patients, correlated with UICC stage (p=0.03), tumor invasion (p=0,025) tumor shape (p=0,002) and macroscopic tumor type (p=0,044), but not with DFS. Conclusions: Tumor cells disseminate into various body compartments throughout all stages of CRC. In about two-third of patients, tumor cells are left in the body after curative surgery. We have to memorize this fact for future adjuvant therapies. However, only the peritoneal tumor cells have an influence, but not an independent prognostic significance, in relation to established parameters. Thus, other factors such as the presence of a cellular metastatic phenotype and/or ineffective immunological response must play an important role in diagnosis and prognosis of MRD. No significant financial relationships to disclose.

Sentinel lymphadenectomy in cutaneous melanoma.

In the last ten years validation of the sentinel lymph node (SLN) concept has led to modification of the surgical approach for patients with intermediate-risk cutaneous melanoma. Forty-eight patients affected by cutaneous melanoma with a Breslow thickness between 0.65 and 4 mm were enrolled in the study. Approximately 2 mCi of radiotracer and 1 mL of vital blue dye were injected in each patient around the site of the primary lesion. Lymphoscintigraphy was performed until the lymphatic basin and the respective SLN were localized. The whole surgical procedure consisted of enlargement of the surgical margins followed by localization and excision of the SLN(s) by using both radiotracer and vital dye. Whenever the SLN proved to be histologically positive for metastasis, complete regional lymphadenectomy was performed. Within 15 minutes of radiotracer administration the lymphatic basin was localized in all 4 patients by lymphoscintigraphy. Vital dye and radiotracer successfully allowed SLN localization and excision in 46 of 48 patients (97%); in one case the SLN was detected by radiotracer alone. The SLN proved to be metastatic in six (13%) of 46 evaluable patients; interestingly, in three of them the presence of metastatic cells was revealed only by immunohistochemistry. All patients with tumor-positive SLNs had primary lesions with a Breslow thickness = 2 mm. Sentinel lymphadenectomy is able to identify lymph node involvement in patients with cutaneous melanoma with a Breslow thickness > 1 mm, thus avoiding the risks associated with radical regional lymphadenectomy. Lymphoscintigraphy proved to be an important tool to obtain correct preoperative localization of the drainage basin, especially for melanomas located on the face and trunk.

Early detection of peritoneal dissemination of gastrointestinal cancers by reverse-transcriptase polymerase chain reaction

We examined the usefulness of the reverse-transcriptase polymerase chain reaction (RT-PCR) analysis for E-cadherin mRNA in the early diagnosis of peritoneal dissemination of gastric and colorectal cancers. The RT-PCR analysis was performed on RNA samples extracted from cells which were collected preoperatively from ascites or intraperitoneally infused fluid. E-cadherin mRNA was detected in 2/2 cases (100%) graded into P-1 peritoneal dissemination, indicating the presence of metastatic cells. In the cases with P-2 and P-3 dissemination the gene expression was detected in 2/3 (67%) and 3/5 (60%), respectively. The RT-PCR analysis was more sensitive for P-1 dissemination than cytological examination. These results suggest that determination of E-cadherin gene expression is useful for the early detection of peritoneal dissemination in gastrointestinal cancer because of its high sensitivity and specificity.