Mining immune-related genes with prognostic value in the tumor microenvironment of breast invasive ductal carcinoma.

ABSTRACT: The tumor microenvironment (TME) plays an important role in the development of breast cancer. Due to limitations in experimental conditions, the molecular mechanism of TME in breast cancer has not yet been elucidated. With the development of bioinformatics, the study of TME has become convenient and reliable.Gene expression and clinical feature data were downloaded from The Cancer Genome Atlas database and the Molecular Taxonomy of Breast Cancer International Consortium database. Immune scores and stromal scores were calculated using the Estimation of Stromal and Immune Cells in Malignant Tumor Tissues Using Expression Data algorithm. The interaction of genes was examined with protein-protein interaction and co-expression analysis. The function of genes was analyzed by gene ontology enrichment analysis, Kyoto Encyclopedia of Genes and Genomes analysis and gene set enrichment analysis. The clinical significance of genes was assessed with Kaplan-Meier analysis and univariate/multivariate Cox regression analysis.Our results showed that the immune scores and stromal scores of breast invasive ductal carcinoma (IDC) were significantly lower than those of invasive lobular carcinoma. The immune scores were significantly related to overall survival of breast IDC patients and both the immune and stromal scores were significantly related to clinical features of these patients. According to the level of immune/stromal scores, 179 common differentially expressed genes and 5 hub genes with prognostic value were identified. In addition, the clinical significance of the hub genes was validated with data from the molecular taxonomy of breast cancer international consortium database, and gene set enrichment analysis analysis showed that these hub genes were mainly enriched in signaling pathways of the immune system and breast cancer.We identified five immune-related hub genes with prognostic value in the TME of breast IDC, which may partly determine the prognosis of breast cancer and provide some direction for development of targeted treatments in the future.

Prognostic Significance of Tumor-infiltrating Lymphocytes on Survival Outcomes of Patients With Resected Pancreatic Ductal Adenocarcinoma: A Systematic Review and Meta-Analysis.

Tumor-infiltrating lymphocytes (TILs) play an important role in mediating treatment response in pancreatic cancer. This meta-analysis investigated the prognostic significance of TIL subsets on overall survival (OS) and disease-free survival (DFS) of patients with pancreatic cancer. Studies were gathered via search of PubMed, Google Scholar, and Cochrane Library databases up to August 1, 2019. Using Review Manager version 5.3.5, pooled hazard ratios and 95% confidence intervals (CIs) were calculated using random or fixed-effects models, depending on the heterogeneity of studies. A total of 11 studies comprising 1760 patients were included in the meta-analysis. Pooled analysis revealed that high CD8 TILs were associated with improved OS [hazard ratio (HR)=0.59, 95% CI=0.51-0.69, P<0.00001] and DFS (HR=0.60, 95% CI=0.50-0.73, P<0.00001). Similarly, high CD3 TILs correlated with better OS (HR=0.64, 95% CI=0.54-0.75, P<0.00001) and DFS (HR=0.53, 95% CI=0.31-0.92, P<0.0001). In contrast, high FoxP3 TILs were associated with worse OS (HR=1.39, 95% CI=1.03-1.88, P=0.03). Finally, high CD4 TILs showed significant improvement in OS (HR=0.74, 95% CI=0.63-0.86, P=0.0001). TILs are a promising prognostic biomarker in pancreatic cancer. Prospective studies evaluating TILs are recommended as well as the establishment of standards in the assessment of TILs.

High EZH2 expression in ductal carcinoma in situ diagnosed on breast core needle biopsy is an independent predictive factor for upgrade on surgical excision.

PURPOSE: Approximately 25 % of DCIS diagnosed on breast core needle biopsy (CNB) is upgraded to invasive carcinoma on surgical excision. Risk factors to predict the upgrade on excision are not well established, leading many patients to be over or under-treated. EZH2 was shown to be associated with aggressive behavior of cancer from many sites, including breast cancer. We aimed to analyze EZH2 expression and tumor infiltrating lymphocytes (TILs) in DCIS as predictive factors for an upgrade on excision. METHODS: We assessed EZH2 expression in 34 DCIS cases diagnosed on CNB and upgraded to invasive carcinoma on excision. Then, we compared these cases with 60 control cases that were not upgraded on excision. A staining score for DCIS (0-12) was obtained by multiplying the staining intensity (0-3) and the percentage of positive cells (1-4). The nuclear staining score ≥6 was considered as 'high' expression. RESULTS: 46 of 94 (49 %) DCIS on CNB showed high EZH2 expression. EZH2 expression was directly correlated with TILs density, nuclear grade, HER2 expression, Ki-67 index and negative ER status. On univariate analysis, upgrade on excision was associated with high EZH2 expression, high TILs density, negative ER status and high Ki-67 index. Multivariate analysis revealed the high EZH2 expression as the only independent predictive factor for upgrade on excision. CONCLUSIONS: Our study revealed the high EZH2 expression as the only independent predictive factor for an upgrade on excision. Future studies should focus on the evaluation of EZH2 expression in tumor-microenvironment interaction in terms of diagnostic, treatment and prognostic purposes.

Heat Shock Protein 90 Family Isoforms as Prognostic Biomarkers and Their Correlations with Immune Infiltration in Breast Cancer.

BACKGROUND: The heat shock protein 90 (HSP90s) family is composed of molecular chaperones composed of four isoforms in humans, which has been widely reported as unregulated in various kinds of cancers. Nevertheless, the role of each HSP90s isoform in prognosis and immune infiltration in distinct subtypes of breast cancer (BRAC) remains unclear. METHODS: Public online databases including the Oncomine, UALCAN, Kaplan-Meier Plotter, Tumor IMmune Estimation Resource (TIMER), Gene Expression Profiling Interactive Analysis (GEPIA), GeneMANIA, and Database for Annotation, Visualization, and Integrated Discovery (DAVID) were integrated to perform bioinformatic analyses and to explore the possible associations among HSP90s gene expression, prognosis, and immune infiltration in BRAC. RESULTS: The mRNA expression of all HSP90s members was elevated in distinct clinical stages and subtypes of BRAC, compared with the normal breast tissue (P < 0.05). Overexpressed HSP90AA1 was associated with poor prognosis, particularly, both short overall survival (OS) and release-free survival (RFS) in Basal-like BRAC patients; overexpressed HSP90AB1 and HSP90B1 were both associated with poor RFS in Luminal A BRAC patients, while overexpressed TRAP1 was associated with favorable RFS in Luminal A BRAC patients. Moreover, HSP90s gene expression in BRAC showed correlations with the infiltration of CD8+ T cells, neutrophils, macrophages, and dendritic cells (DCs), as well as the activation of tumor-associated macrophages (TAMs), DCs, and CD4+ helper T (Th) cells. The underlying mechanisms of HSP90s modulating tumor-infiltrating immune cells (TIICs) might be related with their functions in antigen processing and presentation, major histocompatibility complex (MHC) binding, and assisting client proteins. CONCLUSION: This study demonstrated that HSP90s family genes were overexpressed and might be serve as prognostic biomarkers in subtypes of BRAC. It might be a novel breakthrough point of BRAC treatment to regulate immune infiltration in BRAC microenvironment for more effective anticancer immunity through pharmacological intervention of HSP90s.

Regulation and modulation of antitumor immunity in pancreatic cancer.

Pancreatic ductal adenocarcinoma carries a dismal prognosis, and outcomes have improved little with modern therapeutics. Checkpoint-based immunotherapy has failed to elicit responses in the vast majority of patients with pancreatic cancer. Alongside tumor cell-intrinsic mechanisms associated with oncogenic KRAS-induced inflammation, the tolerogenic myeloid cell infiltrate has emerged as a critical impediment to adaptive antitumor immune responses. Furthermore, the discovery of an intratumoral microbiome and the elucidation of host-microbe interactions that curtail antitumor immunity also present opportunities for intervention. Here we review the mechanisms of immunotherapy resistance in pancreatic ductal adenocarcinoma and discuss strategies to directly augment T cell responses in parallel with myeloid cell- and microbiome-targeted approaches that may enable immune-mediated control of this malignancy.

Increased ERBB2 Gene Copy Numbers Reveal a Subset of Salivary Duct Carcinomas with High Densities of Tumor Infiltrating Lymphocytes and PD-L1 Expression.

Salivary duct carcinoma (SDC) commonly expresses androgen receptor (AR) and HER2, giving rise to treatment implications. SDC may also express programmed-death-ligand-1 (PD-L1), a predictive marker of response to checkpoint inhibitors. PD-L1 can be associated with genomic instability and high density of tumor infiltrating lymphocytes (TILs). Evaluation of HER2 immunohistochemistry (IHC) in SDC is not standardized, and relationships between ERBB2 copy numbers, PD-L1 expression and TILs in SDC are unknown. We evaluated 32 SDCs for HER2, AR and PD-L1 expression (IHC), ERBB2 status (FISH) and TILs (slide review). HER2 was scored with three different systems (breast, gastric, proposed salivary gland). PD-L1 was evaluated with the combined positive score. Most patients were older men, presenting at advanced clinical stage with nodal or distant metastases. During follow-up (mean 5 years, range 6 months to 21 years), 25 of the 32 patients (78%) died of SDC. We propose a HER2 IHC scoring system which accurately predicts underlying ERBB2 amplification or increased copy numbers in SDC. Most tumors had increased ERBB2 copy numbers (19/32 amplification, 6/32 aneusomy), a finding associated with higher TIL densities (p = 0.045) and PD-L1 expression (p = 0.025). Patients with TILs ≥ 40% had better prognoses (Log-Rank p = 0.013), with TILs being favorable prognosticators in univariate analysis (Hazard ratio: 0.18, p = 0.024). A subset of SDCs with increased ERBB2 copy numbers have higher TILs and PD-L1 expression. TILs ≥ 40% are associated with better prognosis.

The molecular landscape and microenvironment of salivary duct carcinoma reveal new therapeutic opportunities.

Purpose: Salivary duct carcinoma (SDC) is a rare and aggressive salivary gland cancer subtype with poor prognosis. The mutational landscape of SDC has already been the object of several studies, however little is known regarding the functional genomics and the tumor microenvironment despite their importance in oncology. Our investigation aimed at describing both the functional genomics of SDC and the SDC microenvironment, along with their clinical relevance. Methods: RNA-sequencing (24 tumors), proteomics (17 tumors), immunohistochemistry (22 tumors), and multiplexed immunofluorescence (3 tumors) data were obtained from three different patient cohorts and analyzed by digital imaging and bioinformatics. Adjacent non-tumoral tissue from patients in two cohorts were used in transcriptomic and proteomic analyses. Results: Transcriptomic and proteomic data revealed the importance of Notch, TGF-ß, and interferon-γ signaling for all SDCs. We confirmed an overall strong desmoplastic reaction by measuring α-SMA abundance, the level of which was associated with recurrence-free survival (RFS). Two distinct immune phenotypes were observed: immune-poor SDCs (36%) and immune-infiltrated SDCs (64%). Advanced bioinformatics analysis of the transcriptomic data suggested 72 ligand-receptor interactions occurred in the microenvironment and correlated with the immune phenotype. Among these interactions, three immune checkpoints were validated by immunofluorescence, including CTLA-4/DC86 and TIM-3/galectin-9 interactions, previously unidentified in SDC. Immunofluorescence analysis also confirmed an important immunosuppressive role of macrophages and NK cells, also supported by the transcriptomic data. Conclusions: Together our data significantly increase the understanding of SDC biology and open new perspectives for SDC tumor treatment. Before applying immunotherapy, patient stratification according to the immune infiltrate should be taken into account. Immune-infiltrated SDC could benefit from immune checkpoint-targeting therapy, with novel options such as anti-CTLA-4. Macrophages or NK cells could also be targeted. The dense stroma, i.e., fibroblasts or hyaluronic acid, may also be the focus for immune-poor SDC therapies, e.g. in combination with Notch or TGF-ß inhibitors, or molecules targeting SDC mutations.

CD8-positive memory T cells in tumor-draining lymph nodes of patients with breast cancer.

BACKGROUND: Human immunological memory is a hallmark of the adaptive immune system and plays an important role in the development of effective immune responses against tumors. In the present study, we aimed to determine the frequencies of CD8+ memory T cell subsets including T stem cell memory (TSCM) in tumor-draining lymph nodes of patients with breast cancer (BC). METHODS: Mononuclear cells were obtained from axillary lymph nodes of 52 untreated patients with BC and stained for CD8, CCR7, CD45RO, CD95 markers to detect different subtypes of memory cells in the CD8+ lymphocyte population. Data were acquired on four-color flow cytometer and analyzed with CellQuest Pro software. RESULTS: We observed that 47.65 ± 2.66% of CD8+ lymphocytes expressed the CD45RO, a marker for memory T cells. Statistical analysis showed that the total frequency of central memory T cells (TCM) and their subset with low CD45RO expression was significantly higher in tumor-involved nodes compared to tumor-free ones (P = 0.024 and P = 0.017, respectively). The level of CD95 expression (based on mean fluorescence intensity) on the surface of TCM, their CD45ROhi and CD45ROlow subsets, and TSCM was higher in patients with stage II compared to those in stage I (P < 0.05). In addition, the percentage of naive CD8+ T cells was significantly lower in tumor-involved lymph nodes compared to tumor-free ones (P = 0.025). CONCLUSIONS: Our data collectively indicate no significant differences in the frequencies of CD8+ lymphocytes or their memory subsets in tumor-draining lymph nodes of patients with BC. However, the frequency of CD45low TCM was higher in tumor-involved nodes. Along with a decrease in the frequency of naive T cells, the higher frequency of CD45low TCM suggests that despite the immune reaction to provide a pool of effective memory cells, it is blocked in early-stage of memory cells' differentiation (CD45ROlow), probably by tumor-derived suppressive factors. Identifying the molecular and cellular mechanisms behind this suppression can provide invaluable tools for adoptive T cell therapies in cancer.

[Cytokine production by blood immune cells, tumor and its microenvironment, characteristic of extracellular matrix in patients with invasive ductal carcinoma of no special type]. / Produktsiia tsitokinov immunokompetentnymi kletkami krovi, opukhol'iu i ee mikrookruzheniem, osobennosti sostoianiia vnekletochnogo matriksa u bol'nykh invazivnoi kartsinomoi molochnoi zhelezy nespetsificheskogo tipa.

The aim of this research was to study cytokine production by blood immune cells, tumor, and its microenvironment, and characterize extracellular matrix of patients with invasive ductal carcinoma of no special type and lymphatic metastases. Spontaneous and polyclonal activators stimulated production of cytokines by blood immune cells, tumor and its microenvironment were studied in 95 patients with invasive ductal carcinoma of no special type. The concentration of IL-2, IL-4, IL-6, IL-8, IL-10, IL-17, IL-18, IL-1ß, IL-1Ra, TNF-α, IFN-γ, G-CSF, GM-CSF, VEGF and MCP-1 was determined by the solid-phase enzyme-linked immunosorbent assay. The condition of fibrous component and presence of neutral glycoproteins and sulfated glycosaminoglycans were evaluated during the research of extracellular matrix. Regional lymphatic metastases were detected in 35 of 95 patients. It was shown that in the presence or absence of lymphatic metastases index of polyclonal activators influence on the production of cytokines by blood immune cells was different for IL-6, IL-8, and IL-1ß; while in the case of cytokine production by tumor and its microenvironment the index of influence was different for IL-2 and IL-17. The presence of lymphatic metastases corresponded with the rise of cytokines spontaneous production, while the absence of lymphatic metastases corresponded with the rise of cytokines production stimulated by polyclonal activators. The value of indices of polyclonal activators influence on the production of cytokines by blood immune cells pointed to the highly stimulating effect of polyclonal activators while the value of indices of polyclonal activators influence on cytokines production by tumor and its microenvironments pointed to the low and sometimes even absent effect of polyclonal activators. Basing on these data we propose a ratio of indices of polyclonal activators influence for the better evaluation of the probability of lymphatic metastases during preoperative period. After characterizing extracellular matrix we found out a point threshold, which, in 100% of cases, predicted the presence of lymphatic metastases basing on the condition of extracellular matrix. Using the data acquired, we are proposing a risk group for metastasis among women with no lymphatic metastases in the moment of check-up.

Novel therapeutic targets in salivary duct carcinoma uncovered by comprehensive molecular profiling.

Salivary duct carcinoma (SDC) is a rare, aggressive salivary gland malignancy, which often presents at an advanced stage. A proportion of SDC are characterized by HER2 amplification and/or overexpression of androgen receptor (AR), which could be targeted in a subset of patients, but the presence of AR splice variant-7 (AR-V7) in some SDC cases could result in resistance to anti-androgen therapy. We evaluated a cohort of 28 cases of SDC for potentially targetable biomarkers and pathways using immunohistochemistry (IHC) and next-generation sequencing (DNA and RNA) assays. Pathogenic genetic aberrations were found in all but 1 case and affected TP53 (n = 19), HRAS (n = 7), PIK3CA, ERBB2 (HER2), and NF1 (n = 5 each); KMT2C (MLL3) and PTEN (n = 3 each); BRAF (p.V600E), KDM5C and NOTCH1 (n = 2 each). Androgen receptor was expressed in all cases and 13 of 27 harbored the AR-V7 splice variant (including a case without any other detectable genetic alteration). HER2 IHC was expressed in 11 of 28 cases. The majority of SDC cases had no biomarkers predictive of immunotherapy response: 5 cases exhibited low (1%-8%) programmed death ligand 1 (PD-L1) expression in tumor cells, 2 cases exhibited elevated TMB, and no samples exhibited microsatellite instability. Notably, the pre-treatment biopsies from 2 patients with metastatic disease, who demonstrated clinical responses to anti-androgen therapy, showed AR expression and no AR splice variants. We conclude that comprehensive molecular profiling of SDCs can guide the selection of patients for targeted therapies involving AR, HER2, PD-L1, mitogen-activated protein kinase, and PIK3CA pathways.

Granzyme B production by activated B cells derived from breast cancer-draining lymph nodes.

B lymphocytes with regulatory or effector functions synthesize granzyme B (GZMB). We investigated the frequency and phenotype of GZMB-producing B cells in breast tumor-draining lymph nodes (TDLNs). Mononuclear cells were isolated from 48 axillary lymph nodes and were stimulated with anti-BCR (B cell receptor), recombinant interleukin (IL)-21 and CD40 L alone or in combination. Flow cytometry was used to evaluate the expression of GZMB in B cells, and in 4 samples the phenotype of GZMB+ B cells was determined. B cells produced GZMB only when stimulated with a combination of IL-21 and anti-BCR for at least 16 h. Adding CD40 L to IL-21 and anti-BCR stimuli resulted in lower GZMB production in B cells. A small fraction of B cells was able to produce perforin in all stimulation conditions, and the majority of GZMB+ B cells were perforin-negative. Both naïve (CD24lowCD27-) and active/memory (CD24hiCD27+) B cells expressed GZMB. In patients with invasive ductal carcinoma, the frequency of GZMB+ B cells was significantly lower in metastatic compared to non-metastatic lymph nodes. The frequency of GZMB+ B cells did not significantly correlate with prognostic factors such as stage, tumor size or Her2 expression. In summary, a subpopulation of both naïve and memory B cells expressed GZMB in breast TDLNs. Our findings underscore the need to investigate the function of GZMB+ B cells in breast tumor immunity.

The immune microenvironment and expression of PD-L1, PD-1, PRAME and MHC I in salivary duct carcinoma.

AIMS: Salivary duct carcinoma (SDC) is an aggressive salivary malignancy that results in high mortality rates and is often resistant to chemotherapy. Anti-programmed death-1 (PD-1)/programmed death ligand-1 (PD-L1) checkpoint inhibitors have led to dramatic improvements in patients with various cancers. Other immunotherapeutic approaches, e.g. cancer vaccines, have shown promising results. Cancer testis antigens, e.g. preferentially expressed antigen in melanoma (PRAME), are regarded as promising vaccine targets because of their tumour-specific expression pattern. METHODS AND RESULTS: We analysed the immunoexpression of PD-L1, PD-1, major histocompatibility complex class I (MHC I) and PRAME in 53 SDCs. The immunoexpression levels of PD-L1 in tumour cells (TCs) and immune cells (ICs), PD-1 in ICs, PRAME in TCs and MHC I in TCs were analysed, and were correlated with outcome. PRAME expression was seen in 83% of SDCs. No PRAME staining was present in normal salivary gland tissue. With the three established diagnostic algorithms proposed for head and neck squamous cell carcinoma, the criteria being a combined positive score of ≥1, TC% ≥1%, and TC% ≥25%, 35 (66%), 17 (32%) and three cases (6%), respectively, were deemed to be positive for PD-L1. PD-1-positive ICs were seen in 35 (66%) cases. MHC I down-regulation was seen in 82% of SDCs. There was a significant correlation among PD-L1 expression in ICs, PD-1 expression in ICs, and PRAME expression in TCs. PD-L1 expression in TCs and lack of PD-1 expression in ICs were associated with decreased disease-specific survival in SDC patients. CONCLUSIONS: Alterations of the tumour immune microenvironment are common in SDCs, including expression of PD-1/PD-L1 and PRAME, which opens the way to potential novel immune therapies, such as cancer vaccination and PD-1/PD-L1 blockade, in these tumours.

Dynamic and significant changes of T-cell subgroups in breast cancer patients during surgery and chemotherapy.

OBJECTIVE: Breast cancer and its surgical treatment and chemotherapy have great impact on the immune system. This study aimed to monitor the various T cells in breast cancer patients and evaluate the immune functions. METHODS: Blood samples were collected from 249 breast cancer patients at the following time points: 1-3 days preoperative, postoperative (before the chemotherapy), after 3 chemotherapy cycles, and after 6 chemotherapy cycles. The percentages of the CD3+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells were measured using flow cytometry. Another 200 healthy women were used as control. RESULTS: Patients with stage II/III breast cancer had significantly lower percentages of CD3+, CD4+, CD8+, CD45RA+, and CD28+ T cells in comparison with normal control and those with stage I breast cancer (P < 0.05). The percentages of CD45RO+ T cells and CD4+CD25+ (Treg) cells were significantly higher in stage II/III malignancies versus stage I, and was significantly higher in stage I malignancies versus the normal control (P < 0.05). Breast cancer patients had significantly lower percentages of CD3+ and CD4+ T cells in comparison with the normal control (P < 0.05). The preoperative percentages of CD3+ and CD4+ T cells were significantly reduced after 3 cycles and after 6 cycles of chemotherapy (P < 0.05). In patients with stage II/III malignancies, there was a higher percentage of CD45RO+ T cells than CD45RA+ T cells, which was reversed after surgery. After 6 chemotherapy cycles, the percentage of Treg cells was significantly reduced in comparison with that before the chemotherapy in the patients with stage II/III malignancies. CONCLUSIONS: Patients with breast cancer had significantly suppressed immune functions. Surgical removal of the tumor may improve the immune functions. Chemotherapy significantly reduced the percentages of CD3+ and CD4+ T cells.

Multi-omics profiling of younger Asian breast cancers reveals distinctive molecular signatures.

Breast cancer (BC) in the Asia Pacific regions is enriched in younger patients and rapidly rising in incidence yet its molecular bases remain poorly characterized. Here we analyze the whole exomes and transcriptomes of 187 primary tumors from a Korean BC cohort (SMC) enriched in pre-menopausal patients and perform systematic comparison with a primarily Caucasian and post-menopausal BC cohort (TCGA). SMC harbors higher proportions of HER2+ and Luminal B subtypes, lower proportion of Luminal A with decreased ESR1 expression compared to TCGA. We also observe increased mutation prevalence affecting BRCA1, BRCA2, and TP53 in SMC with an enrichment of a mutation signature linked to homologous recombination repair deficiency in TNBC. Finally, virtual microdissection and multivariate analyses reveal that Korean BC status is independently associated with increased TIL and decreased TGF-ß signaling expression signatures, suggesting that younger Asian BCs harbor more immune-active microenvironment than western BCs.

Anti-nuclear antibodies in patients with breast cancer.

To study the prevalence of anti-nuclear antibodies (ANA) in breast cancer patients and its association with tumour characteristics. Ninety-one patients with breast mass detected by image studies and assigned to conduct diagnostic biopsy and eventual surgical treatment were studied for demographical, tumour data and presence of ANA. Serum of positive ANA patients was screened for the extractable nuclear antigen (ENA) profile. As comparison, 91 healthy individuals matched for age and from the same geographical area were included. In this sample 72 of 91 (79·1%) had malignant lesions (83% ductal infiltrative carcinoma). ANA was positive in 44·4% of patients with malignant tumour and in 15·7% of those with benign lesions (malignant versus benign with P = 0·03). Controls had ANA positivity in 5·4%, and when compared with tumour samples showed P < 0·0001. The most common immunofluorescence pattern was a fine dense speckled pattern. In the ANA-positive patients with malignant lesions, seven had positivity for ENA profile (three for anti-RNP and anti-Sm, one for just anti-RNP, two for anti-Ro and anti-La e two for just anti-La). It was not possible to associate ANA positivity with tumour histological characteristics or staging or with patient's age. A negative association of ANA with hormonal (oestrogen or oestrogen plus progesterone) receptor status was found (P = 0·01). In this sample, there was a high prevalence of ANA positivity in breast cancer patients with a negative association with the presence of hormonal receptors. More studies are needed to understand the real value of this finding.

Evaluating for Pseudoprogression in Colorectal and Pancreatic Tumors Treated With Immunotherapy.

Pseudoprogression has been observed in patients with various tumor types treated with immunotherapy. However, the frequency of pseudoprogression is unknown in gastrointestinal malignancies. Metastatic colorectal cancer (mCRC) and advanced pancreatic ductal adenocarcinoma (PDAC) patients who progressed on treatment with immunotherapy beyond RECIST version 1.1 criteria were analyzed. Degree of progression, tumor markers, time to progression, overall survival, Eastern Cooperative Oncology Group Performance Status (ECOG PS), and costs were analyzed for patients treated beyond progression (TBP) and not treated beyond progression. Fifty-nine of 159 (37%) patients with mCRC or PDAC were TBP (31 mCRC, 28 PDAC). Fifty-four of 59 (92%) patients were microsatellite stable. Zero of these 59 patients with initial treatment beyond progression demonstrated subsequent radiographic tumor shrinkage at a median 42 days from first scan documenting progression. A pseudoprogression rate of >6% could be excluded with 95% confidence. Compared with baseline, median growth on the first and second scan that showed progression was 29.8% and 43%, respectively. In those not treated beyond progression, median growth at first restaging was 31.2%. The trend in change in tumor size positively correlated with the trend in tumor markers in all patients TBP. Fifteen patients (25%) experienced grade 3/4 adverse events by continuing treatment beyond progression, whereas 19 (32%) experienced deterioration in ECOG PS. Pseudoprogression was not seen in microsatellite stable patients with mCRC or PDAC treated with immunotherapy. Changes in tumor markers correlated with changes in tumor volume. This data may help inform future treatment decisions and/or trial design in patients with mCRC or advanced PDAC treated with immunotherapy.

The effect of neoadjuvant chemotherapy on the correlation of tumor-associated macrophages with CD31 and LYVE-1.

Angiogenesis and lymphangiogenesis play a crucial role in tumor growth, invasion and metastasis. Tumor-associated macrophages (TAM) induce both angiogenesis and lymphangiogenesis in mouse breast cancer models and positively correlate with these processes in human breast cancer patients. Neoadjuvant chemotherapy (NAC) is a widely used therapeutic option for cancer treatment. However, the effect of NAC on the distribution of TAM within intratumoral compartments and their correlation with angiogenesis and lymphangiogenesis remained unknown. In the present study we analyzed the effect of NAC on the distribution of CD68+ and stabilin-1+ TAM in five functionally distinct areas of human breast cancer and their correlations with microvessel density (MVD) and lymphatic microvessel density (LMVD), identified by CD31 and LYVE1, respectively. We found that NAC enhances blood vessel density in soft fibrous stroma and in coarse fibrous stroma. Without NAC the amount of CD68+ TAM in gaps of ductal tumor structures positively correlate with CD31+ microvessel density in soft fibrous stroma. NAC had enhancing effect on the amount of CD68+ TAM but not stabilin-1+ TAM in soft fibrous stroma. However, no correlation between TAM and CD31+ microvessel density was identified after NAC. NAC did not enhance the lymphatic microvessel density. But after NAC stabilin-1 expressing subpopulation of TAM positively correlated with expression of LYVE-1. We hypothesized that CD68+ TAM can support tumor angiogenesis primarily before NAC, while stabilin-1+ TAM can contribute to the maintenance of lymphatic microvessel density after NAC.

Prognostic significance of infiltrating immune cell subtypes in invasive ductal carcinoma of the breast.

PURPOSE: To explore the correlation between tumor-infiltrating immune cell subsets and breast cancer prognosis. MATERIALS AND METHODS: Specimens of 102 patients with invasive ductal carcinoma of the breast were analyzed for immune-related markers (CD8, CD20, FOXP3 and CD68). The number of positive cells in the 3 most highly stained intratumoral stroma areas of the primary tumor was counted. The mean number was calculated and used to divide patients into 2 groups for each marker (CD8-high/CD8-low, CD20-high/CD20-low, FOXP3-high/FOXP3-low, and CD68-high/CD68-low). RESULTS: Kaplan-Meier survival analysis showed (a) for all patients that high tumor-infiltrating CD8+ and CD20+ B lymphocytes, low tumor-infiltrating FOXP3+ regulatory T cells (Tregs), and CD68+ macrophages all increased OS and DFS (p<0.05); (b) for both the 35 ER-negative and 45 lymph-node-negative patients, high CD8+ cytotoxic T lymphocytes (CTLs) increased OS and DFS (p<0.05). Multivariate analysis of OS and DFS showed that for all patients high CD8+ CTLs and low FOXP3+ Tregs were related to good OS and DFS (p<0.05). CONCLUSION: High numbers of tumor-infiltrating CD8+ and low numbers of FOXP3+ T lymphocytes both could function as potential independent prognostic markers for invasive ductal breast carcinoma.

Polyclonal Antibody against Different Extracellular Subdomains of HER2 Induces Tumor Growth Inhibition in vitro.

BACKGROUND: Human epidermal growth factor receptor 2 (HER2) has a crucial role in several malignancies. The extracellular domain of HER2 (HER2-ECD) has been extensively employed as an important target in passive and active immunotherapy. Isolated recombinant prokaryotic HER2-ECD subdomains were previously found to be ineffective in inducing anti-tumor antibody response. OBJECTIVE: To employ recombinant eukaryotic HER2-ECD subdomains to raise anti-HER2 antibodies and determine their anti-tumor activity in vitro. METHODS: Two paired subdomains of HER2-ECD (DI+II and DIII+IV), representing Pertuzumab and Trastuzumab binding domains, respectively, along with the full extracellular domain of HER2 were generated in CHO-K1 cells. Polyclonal antibodies were raised against these subdomains and characterized using ELISA, flow cytometry, and immunoblot and their anti-tumor activity was assessed by XTT assay. The cross-reactivity of these antibodies was specified along with other members of the human HER family. RESULTS: Similar to Trastuzumab and anti-HER2-ECD antibody, anti-DI+II and DIII+IV polyclonal antibodies reacted with recombinant HER2-ECD and native HER2 expressed on tumor cells. These two polyclonal antibodies were able to inhibit the binding of Pertuzumab and Trastuzumab to HER2, respectively, and did not cross-react with other members of HER family. These antibodies were able to inhibit tumor cell growth in vitro, similar to Trastuzumab. CONCLUSION: The high immunogenicity of human HER2 DI+II and DIII+IV subdomains in rabbits and the tumor inhibitory activity of the purified specific antibodies imply that they might be suitable for active immunotherapy in formulation with appropriate adjuvants and in combination with other HER2 specific therapeutics.

Focused ultrasound for immuno-adjuvant treatment of pancreatic cancer: An emerging clinical paradigm in the era of personalized oncotherapy.

Current clinical treatment regimens, including many emergent immune strategies (e.g., checkpoint inhibitors) have done little to affect the devastating course of pancreatic ductal adenocarcinoma (PDA). Clinical trials for PDA often employ multi-modal treatment, and have started to incorporate stromal-targeted therapies, which have shown promising results in early reports. Focused ultrasound (FUS) is one such therapy that is uniquely equipped to address local and systemic limitations of conventional cancer therapies as well as emergent immune therapies for PDA. FUS methods can non-invasively generate mechanical and/or thermal effects that capitalize on the unique oncogenomic/proteomic signature of a tumor. Potential benefits of FUS therapy for PDA include: (1) emulsification of targeted tumor into undenatured antigens in situ, increasing dendritic cell maturation, and increasing intra-tumoral CD8+/ T regulatory cell ratio and CD8+ T cell activity; (2) reduction in intra-tumoral hypoxic stress; (3) modulation of tumor cell membrane protein localization to enhance immunogenicity; (4) modulation of the local cytokine milieu toward a Th1-type inflammatory profile; (5) up-regulation of local chemoattractants; (6) remodeling the tumor stroma; (7) localized delivery of exogenously packaged immune-stimulating antigens, genes and therapeutic drugs. While not all of these results have been studied in experimental PDA models to date, the principles garnered from other solid tumor and disease models have direct relevance to the design of optimal FUS protocols for PDA. In this review, we address the pertinent limitations in current and emergent immune therapies that can be improved with FUS therapy for PDA.