Gene Expression In Lymph Nodes Research Articles

Abstract 5169: Sentinel lymph node gene expression signature predicts survival outcome in cutaneous melanoma

Abstract Background: Sentinel lymph node status is an important prognostic factor for melanoma progression. We sought to develop a sentinel lymph node gene expression signature score predictive of disease progression in patients with cutaneous melanoma. Methods: Cases were retrieved from University of Pittsburgh protocol “Sentinel Node and Non-Sentinel Lymph Nodes (SLN and non-SLN) Procurement from Melanoma Subjects for Molecular Profiling.” Gene expression profiling was performed on SLN biopsies using U133A 2.0 Affymetrix gene chips. The top 25 genes associated with progression-free survival (PFS) were selected and a penalized regression function was used to select genes with a non-zero coefficient. A risk score was calculated from a 12 gene signature. A proportional hazards regression model was used to evaluate the association between clinical covariates, gene signature score, and PFS. Results: In total, 45 patients were included [female=18 (40%), median age (range)= 56 (16-81) years]. The median Breslow thickness (IQR) was 4.1 (3.3-6.0) mm, ulceration was present in 29 (67%) cases, and 25 (56%) lesions had nodular histology. Among patients, 23 (51%) had a positive SLN via H&E. Twenty-one (46.7%) patients developed disease recurrence/progression with a median follow-up of 8.3 years. The median time to progression was 9.6 years. Among the top 25 genes, 12 non-zero penalized coefficients were estimated (CLGN, C1QTNF3, ADORA3, ARHGAP8, ZNF223, PDE6G, CXCL3, CHRFAM7A, HLA-DRB-decreased risk, DCTN1, ASPSCR1, HEXIM1-increased risk). A 12-gene signature score was significantly associated with PFS (p<0.0001) and produced a bootstrap cross-validated C index of 0.888. Stratifying by SLN status the gene signature risk score remained highly significant (p<0.0001). In univariate analysis, Breslow thickness (HR=1.41), presence of ulceration (HR=3.61), SLN positivity (HR=2.50), and acral-lentiginous histology compared to nodular (HR=4.07) were each significantly associated with PFS (all p<0.05). After simultaneously adjusting for all of these factors plus the gene signature, the 12-gene score remained a significant predictor for PFS (p<0.0001). Conclusion: A SLN 12-gene signature risk score is associated with melanoma progression regardless of SLN status and can be used as a prognostic factor for PFS of patients. External validation of the gene signature is warranted. Citation Format: Lilit Karapetyan, William Gooding, Xi Yang, Andrew Knight, Aofei Li, Cindy Sander, Monica Panelli, Walter J. Storkus, Ahmad A. Tarhini, John M. Kirkwood. Sentinel lymph node gene expression signature predicts survival outcome in cutaneous melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5169.

Novel gene markers of immunosuppressive chemicals in mouse lymph node assay

The murine local lymph node assay (LLNA) is an immunologically based test of the sensitizing potential of immunotoxicants, but also evaluates immunosuppressive potential with good sensitivity and specificity. We conducted the LLNA with calcineurin inhibitors (tacrolimus and cyclosporin A), antimetabolites (methotrexate and azathioprine), steroids (dexamethasone and prednisolone), and an alkylator (cyclophosphamide). We performed a comprehensive analysis of results of gene expression in lymph nodes obtained in the LLNA using a highly sensitive DNA chip, 3D-Gene™, and the quantitative reverse transcription-polymerase chain reaction (qPCR). Zfp459 expression increased in all animals treated with immunosuppressive chemicals. Ltf, Cbll1 and Lias expression changed specifically in response to the calcineurin inhibitors, Fmo2 and 9630033F20Rik in response to the antimetabolites, Krt8, Gjb1, Hmha1 and Sfrs7 in response to the steroids, and Gbp1 and Mup5 in response to the alkylator. Therefore, these genes were considered novel markers for immunosuppression and their expression could be used to evaluate the mechanism of action of immunosuppressive chemicals.

Evaluation of Guanylyl Cyclase C Lymph Node Status for Colon Cancer Staging and Prognosis

The prognostic significance of guanylyl cyclase C (GCC) gene expression in lymph nodes (LNs) was evaluated in patients with stage II colon cancer who were not treated with adjuvant chemotherapy. We report a planned analysis performed on 241 patients. GCC mRNA was quantified by RT-qPCR using formalin-fixed LN tissues from patients with untreated stage II colon cancer who were diagnosed from 1999-2006 with at least ten LNs examined and blinded to clinical outcomes. Lymph node ratio (LNR) is the number of GCC-positive nodes divided by total number of informative LNs. Risk categories of low (0-0.1) and high (>0.1) for LNR were chosen by significance using Cox regression models. The data were tested for association with time to recurrence. Twenty-nine patients (12%) had a disease recurrence or cancer death. The LNR significantly predicted higher recurrence risk for 84 patients (34.9%) classified as high risk (hazard ratio (HR), 2.38; P=0.02). The estimated 5-year recurrence rates were 10% and 27% for the low- and high-risk groups, respectively. After adjusting for age, T stage, number of nodes assessed, and MMR status, a significant association remained (HR, 2.61; P=0.02). In a subset of patients (n=181) with T3 tumor, ≥12 nodes examined and negative margins, a significant association between the GCC LNR and recurrence risk also was observed (HR, 5.06; P=0.003). Our preliminary results suggest that detection of GCC mRNA in LNs is associated with risk of disease recurrence in patients with untreated stage II colon cancer. A larger validation study is ongoing.

Gene expression in formalin-fixed paraffin-embedded lymph nodes

Elucidation of molecular pathways involved in development of human lymphoma requires efficient methods for tackling gene expression in lymph nodes. Expression studies of transcription factors in these malignancies facilitate understanding the changes occurring in neoplastic transformation and lymphoma development. Excised lymph nodes are routinely fixed in formalin and embedded in paraffin for diagnosis and stored in many hospitals' pathology archives. These tissues represent a precious resource for research since they allow retrospective studies to cover a broad range of human lymphoma even the less frequent types. Reverse transcription polymerase chain reaction (RT-PCR) is a commonly used method for gene expression analysis and a reproducible protocol for RNA isolation from lymph nodes is an inevitable requirement for these studies. However, formalin fixation and paraffin-embedding interfere with the quality of RNA especially when isolated from lymph nodes being the most fragile lymphatic tissues. We present here a simple and fast method for RNA isolation from formalin-fixed paraffin-embedded lymph nodes that can be successfully applied for RT-PCR as well as for quantitative RT-PCR analysis. We tested diverse isolation reagents and combined a range of factors in order to get a high quality RNA for retrospective studies of gene expression in human lymphoma samples. Our modified method of RNA extraction from FFPE provides superior yields and purity based on qPCR data.

Expression of the high mobility group A1 (HMGA1) and A2 (HMGA2) genes in canine lymphoma: analysis of 23 cases and comparison to control cases

Overexpression of high mobility group A (HMGA) genes was described as a prognostic marker in different human malignancies, but its role in canine haematopoietic malignancies was unknown so far. The objective of this study was to analyse HMGA1 and HMGA2 gene expression in lymph nodes of canine lymphoma patients. The expression of HMGA1 and HMGA2 was analysed in lymph node samples of 23 dogs with lymphoma and three control dogs using relative quantitative real-time RT-PCR. Relative quantity of HMGA1 was significantly higher in dogs with lymphoma compared with reference samples. HMGA2 expression did not differ between lymphoma and control dogs. With the exception of immunophenotype, comparison of disease parameters did not display any differences in HMGA1 and HMGA2 expression. The present findings indicate a role of HMGA genes in canine lymphoma. This study represents the basis for future veterinary and comparative studies dealing with their diagnostic, prognostic and therapeutic values.

The Use of Gene-Expression Profiling to Predict Outcome of Follicular Lymphoma Patients Treated with Rituximab and Chemotherapy.

The anti-CD20 monoclonal antibody rituximab has been extensively evaluated for treatment of follicular lymphoma (FL) and is now an integral component of many treatment strategies. However, it is not known when and how to use this combination modality best in the overall treatment course of FL patients. Here we have analyzed the pattern of gene expression in lymph nodes from 28 FL patients who received rituximab in combination with chemotherapy. We demonstrate a novel feature selection method, in which genes whose expression correlates with treatment outcome are devided into groups based on similar expression patterns. By selecting representatives from these groups we built a three-gene predictor (for example RRAD, MGC5254, MARCO) which turned out to be successful in classifying all 28 samples correctly with respect to the responders versus nonresponders distinction. A similar four-gene predictor (for example USP9Y, CUL-4B, NSFS/REST, MADH) correctly classified the responding patients into two subgroups with very different event-free survival rates at the median follow up of 21 months (median not reached vs. 15 months, p<0.0001). The latter predictor also efficiently delineated patients within specific FLIPI risk groups. Our data demonstrate the ability of these microarray-based predictors to classify FL patients treated with rituximab and chemotherapy to subgroups with significantly distinct outcomes.

IκB Kinase Complex α Kinase Activity Controls Chemokine and High Endothelial Venule Gene Expression in Lymph Nodes and Nasal-Associated Lymphoid Tissue

The lymphotoxin (LT) beta receptor plays a critical role in secondary lymphoid organogenesis and the classical and alternative NF-kappaB pathways have been implicated in this process. IKKalpha is a key molecule for the activation of the alternative NF-kappaB pathway. However, its precise role and target genes in secondary lymphoid organogenesis remain unknown, particularly with regard to high endothelial venules (HEV). In this study, we show that IKKalpha(AA) mutant mice, who lack inducible kinase activity, have hypocellular lymph nodes (LN) and nasal-associated lymphoid (NALT) tissue characterized by marked defects in microarchitecture and HEV. In addition, IKKalpha(AA) LNs showed reduced lymphoid chemokine CCL19, CCL21, and CXCL13 expression. IKKalpha(AA) LN- and NALT-HEV were abnormal in appearance with reduced expression of peripheral node addressin (PNAd) explained by a severe reduction in the HEV-associated proteins, glycosylation-dependent cell adhesion molecule 1 (GlyCAM-1), and high endothelial cell sulfotransferase, a PNAd-generating enzyme that is a target of LTalphabeta. In this study, analysis of LTbeta(-/-) mice identifies GlyCAM-1 as another LTbeta-dependent gene. In contrast, TNFRI(-/-) mice, which lose classical NF-kappaB pathway activity but retain alternative NF-kappaB pathway activity, showed relatively normal GlyCAM-1 and HEC-6ST expression in LN-HEV. In addition, in this communication, it is demonstrated that LTbetaR is prominently expressed on LN- and NALT-HEV. Thus, these data reveal a critical role for IKKalpha in LN and NALT development, identify GlyCAM-1 and high endothelial cell sulfotransferase as new IKKalpha-dependent target genes, and suggest that LTbetaR signaling on HEV can regulate HEV-specific gene expression.

Lactic dehydrogenase virus infection reduces the expulsion of the nematode Nippostrongylus brasiliensis.

The interleukin (IL)-13-mediated goblet cell response is the major host effector system involved in the expulsion of Nippostrongylus brasiliensis. Lactic dehydrogenase virus (LDV) induced higher levels of N. brasiliensis egg production compared with controls, but the effect of LDV infection on worm expulsion of, and goblet cell and IL-13 responses to, N. brasiliensis have not been studied. In this study, the effects of LDV infection on these host responses against N. brasiliensis were examined. Mice with chronic LDV infection showed significantly lower worm expulsion rates than non-LDV-infected mice after N. brasiliensis infection, and there were no significant differences in the ratio of female versus male adult worms between control and LDV-infected mice. The number of goblet cells in LDV-infected mice was significantly lower than that in controls. In addition, the levels of IL-13 gene expression in lymph nodes were significantly lower in LDV-infected mice compared with controls. These results suggest that LDV infection reduces the protective immune responses against N. brasiliensis infection by the suppression of IL-13 production.

Alterations of intralesional and lymph node gene expression and cellular composition induced by IL‐12 administration during leishmaniasis

Changes in gene expression and cellular distribution in the lymph node and at the site of infection, the footpad, during Leishmania major infection and/or IL-12 administration were evaluated. Otherwise susceptible BALB/c mice given IL-12 are able to resolve infection. Interestingly, iNOS was not induced in the lymph node by IL-12, yet, nitric oxide is critical in the control of leishmaniasis. However, we observed an increase in iNOS at the lesion site in response to IL-12. These results reflect the importance of examining the primary site of infection. We observed no changes in inflammation at the lesion site; however, IL-12 promoted an early inflammatory response in the lymph nodes. IL-12 administration differentially affected both the local and systemic immune response to invading leishmanial parasites. IL-12 induced iNOS at the lesion site and an early granulomatous inflammation in the lymph node; therefore, we hypothesize that these are key events responsible for the resolution of disease in BALB/c mice treated with IL-12.

Early cytokine and chemokine gene expression in lymph nodes of macaques infected with SIV is predictive of disease outcome and vaccine efficacy

Early cytokine and chemokine gene expression in lymph nodes of macaques infected with SIV is predictive of disease outcome and vaccine efficacy

Early cytokine and chemokine gene expression in lymph nodes of macaques infected with SIV is predictive of disease outcome and vaccine efficacy

Early cytokine and chemokine gene expression in lymph nodes of macaques infected with SIV is predictive of disease outcome and vaccine efficacy

Early cytokine and chemokine gene expression in lymph nodes of macaques infected with simian immunodeficiency virus is predictive of disease outcome and vaccine efficacy.

Competitive PCR was used to evaluate the expression of cytokine, granzyme B, and chemokine genes in lymph nodes of macaques recently infected with the simian immunodeficiency virus (SIV) pathogenic molecular clone SIVmac239 (n = 16), the nonpathogenic vaccine strain SIVmac239 delta nef (n = 8), and the nonpathogenic molecular clone SIVmac1A11 (n = 8). For both SIVmac239 and its nef-deleted derivative, strong expression was observed as early as 7 days postinfection for interleukin 1beta (IL-1beta), IL-6, tumor necrosis factor alpha, gamma interferon, and IL-13. The levels of gene induction were equally intense for both viruses despite a lower viral load for SIVmac239 deltanef compared with that for SIVmac239. However, the nature of the cytokine network activation varied with the viral inocula. Primary infection with SIVmac239 was characterized by a higher level of IL-4, IL-10, MIP-1alpha, MIP-1beta, MCP-1, and RANTES gene expression and a lower level of IL-12 and granzyme B gene expression compared with infection with SIVmac239 delta nef. Thus, infection with nef-deleted SIV was associated with a preferential Th1 versus Th2 pattern of cytokine production. Infection with SIVmac1A11 was characterized by a delayed immune response for all markers tested. The unique patterns of cytokine and chemokine gene expression in lymph nodes correlated nicely with the pathogenic potential of the SIV strains used as well as with differences in their ability to serve as protective vaccines.

Variable region usage in B cell deficient mice in a model of experimental herpes simplex virus retinitis.

By using PCR, we have found previously that differences in T cell receptor alpha/beta chain variable region (TCR alpha/beta) gene expression in lymph nodes (LN) existed between susceptible and resistant mice following anterior chamber (AC) inoculation with herpes simplex virus (HSV). We now report and compare the immunoglobulin gene variable region (Ig VH) and the TCR V beta mRNA expression in normal congenic mice (BALB/c and C.B-17) and also in B cell modulated C.B-17 mice (B-). RNA prepared from spleen and LN of these mice before and after HSV-AC inoculation was analysed by PCR using oligoprimers specific for 11 VH gene families and 19 V beta gene families. Densitometry analysis revealed that VH family mRNA expression levels in spleen and lymph nodes did not correlate with HSV susceptibility or resistance patterns in B-, BALB/c and C.B-17 mice. Analysis of TCR V beta chain genes showed that spleen of resistant C.B-17 and B-Ab transferred mice showed a preference for V beta 11 gene expression not seen in susceptible mice. In contrast, LN of BALB/c and B-, both susceptible mice, made TCR V beta transcripts that were indistinguishable from those generated by resistant C.B-17 mice, following HSV-AC inoculation. Finally, TCR V beta gene family repertoire appears to be severely diminished in uninfected B- mice (50% in LN and 45% in spleen) by anti-mu treatment.

CRITICAL ROLE OF INTERLEUKIN 4 IN THE INDUCTION OF NEONATAL TRANSPLANTATION TOLERANCE

Neonatal injection of semiallogeneic cells is known to promote differentiation of donor-specific CD4+ T cells into TH2-like cells in the peripheral lymphoid organs. We reasoned that the propensity of neonatal T cells to synthesize high levels of IL-4 might be involved in this polarization of the alloreactive response and thereby in the development of neonatal transplantation tolerance. First, analysis of cytokine gene expression in lymph nodes after neonatal injection of 10(7) (A/J x BALB/c)F1 cells in BALB/c mice indicated that IL-4 but not IL-2 is rapidly produced by CD4+ cells after allogeneic challenge in vivo. To determine whether the early production of IL-4 was involved in the establishment of allotolerance, BALB/c mice neonatally injected with (A/J x BALB/c)F1 spleen cells received on days 1 and 3 after birth 1 mg of anti-IL-4 mAb (11B11) or the same amount of control mAb. When grafted with A/J skin at 4 weeks, 88% of mice treated with control mAb retained their graft for more than 50 days, whereas rejection occurred within 30 days in 93% of mice treated with anti-IL-4 mAb. Analysis of T cell functions after in vitro restimulation with A/J spleen cells indicated that early IL-4 neutralization did not prevent donor-specific CTL unresponsiveness but allowed the emergence of alloreactive T cells secreting increased levels of IL-2 and IFN-gamma. We conclude that early production of IL-4 is critical for the establishment of neonatal transplantation tolerance in this strain combination, which has disparities across the entire H-2 region.

Tumor necrosis factor/cachectin and lymphotoxin gene expression in lymph nodes from lymphoma patients

We investigated the mRNA content for tumor necrosis factor (TNF)/cachectin and lymphotoxin (LT) in tumoral tissues of a prospective series of 35 non-Hodgkin's (NHL) and 23 Hodgkin's (HL) lymphomas, to assess their postulated contribution to systemic symptoms. Total RNAs were extracted from diagnostic tissue specimens and submitted to Northern blot analysis, using specific TNF and LT cRNA probes. High amounts of TNF mRNA were found exclusively in NHL (12/35). The majority (9/12) of these were low grade B-cell NHL, which contained a uniform population of malignant cells. In contrast, abundant LT mRNA production was detected in most HL (21/23) and in 19 of 35 NHL. The highest LT mRNA levels were observed in high grade NHL and in lymphocytic predominant subtypes of HL specimens. A significant correlation was found between TNF/cachectin and LT gene expression in NHL and the presence of constitutional symptoms. The biologic and prognostic implications of these preliminary findings are presently unknown, but they demonstrate that lymphoma tissues sharing common histologic features are highly heterogeneous in their ability to synthesize cytokines susceptible to playing a role in the growth control of malignant cells. These results suggest that the evaluation of TNF/cachectin and LT production in lymphomas may help to elucidate the mechanisms of tumoral fever and cachexia.

Tumor Necrosis Factor/Cachectin and Lymphotoxin Gene Expression in Lymph Nodes From Lymphoma Patients

Abstract We investigated the mRNA content for tumor necrosis factor (TNF)/cachectin and lymphotoxin (LT) in tumoral tissues of a prospective series of 35 non-Hodgkin's (NHL) and 23 Hodgkin's (HL) lymphomas, to assess their postulated contribution to systemic symptoms. Total RNAs were extracted from diagnostic tissue specimens and submitted to Northern blot analysis, using specific TNF and LT cRNA probes. High amounts of TNF mRNA were found exclusively in NHL (12/35). The majority (9/12) of these were low grade B-cell NHL, which contained a uniform population of malignant cells. In contrast, abundant LT mRNA production was detected in most HL (21/23) and in 19 of 35 NHL. The highest LT mRNA levels were observed in high grade NHL and in lymphocytic predominant subtypes of HL specimens. A significant correlation was found between TNF/cachectin and LT gene expression in NHL and the presence of constitutional symptoms. The biologic and prognostic implications of these preliminary findings are presently unknown, but they demonstrate that lymphoma tissues sharing common histologic features are highly heterogeneous in their ability to synthesize cytokines susceptible to playing a role in the growth control of malignant cells. These results suggest that the evaluation of TNF/cachectin and LT production in lymphomas may help to elucidate the mechanisms of tumoral fever and cachexia.