Maturation of lymph node fibroblastic reticular cells from myofibroblastic precursors is critical for antiviral immunity.

The stromal scaffold of the lymph node (LN) paracortex is built by fibroblastic reticular cells (FRCs). Conditional ablation of lymphotoxin-β receptor (LTβR) expression in LN FRCs and their mesenchymal progenitors in developing LNs revealed that LTβR-signaling in these cells was not essential for the formation of LNs. Although T cell zone reticular cells had lost podoplanin expression, they still formed a functional conduit system and showed enhanced expression of myofibroblastic markers. However, essential immune functions of FRCs, including homeostatic chemokine and interleukin-7 expression, were impaired. These changes in T cell zone reticular cell function were associated with increased susceptibility to viral infection. Thus, myofibroblasic FRC precursors are able to generate the basic T cell zone infrastructure, whereas LTβR-dependent maturation of FRCs guarantees full immunocompetence and hence optimal LN function during infection.

Endothelial cell-specific lymphotoxin-β receptor signaling is critical for lymph node and high endothelial venule formation.

The development of lymph nodes (LNs) and formation of LN stromal cell microenvironments is dependent on lymphotoxin-β receptor (LTβR) signaling. In particular, the LTβR-dependent crosstalk between mesenchymal lymphoid tissue organizer and hematopoietic lymphoid tissue inducer cells has been regarded as critical for these processes. Here, we assessed whether endothelial cell (EC)-restricted LTβR signaling impacts on LN development and the vascular LN microenvironment. Using EC-specific ablation of LTβR in mice, we found that conditionally LTβR-deficient animals failed to develop a significant proportion of their peripheral LNs. However, remnant LNs showed impaired formation of high endothelial venules (HEVs). Venules had lost their cuboidal shape, showed reduced segment length and branching points, and reduced adhesion molecule and constitutive chemokine expression. Due to the altered EC-lymphocyte interaction, homing of lymphocytes to peripheral LNs was significantly impaired. Thus, this study identifies ECs as an important LTβR-dependent lymphoid tissue organizer cell population and indicates that continuous triggering of the LTβR on LN ECs is critical for lymphocyte homeostasis.

Intratumoral budding (ITB) in preoperative biopsies predicts the presence of lymph node and distant metastasis in colon and rectal cancer patients

Schnüriger2, D. Inderbitzin2, I. Zlobec1, A. Lugli1,3

Prevalence and prognostic significance of TMPRSS2-ERG gene fusion in lymph node positive prostate cancers.

BACKGROUND TMPRSS2-ERG gene fusion is the most frequent genetic alteration in prostate cancer. However, information about its distribution in lymph node positive prostate cancers and the prognostic significance in these advanced tumors is unknown. METHODS Gene fusion status was determined by fluorescence in situ hybridization on a tissue-microarray constructed from 119 hormone-naïve nodal positive, surgically treated prostate cancers containing samples from the primary tumors and corresponding lymph node metastases. Data were correlated with various tumor features (Gleason score, stage, cancer volume, nodal tumor burden) and biochemical recurrence-free, disease-specific, and overall survival. RESULTS TMPRSS2-ERG fusion was detected in 43.5% of the primary tumors. Conversely, only 29.9% of the metastasizing components showed the fusion. Concordance in TMPRSS2-ERG status between primary tumors and metastases was 70.9% (Kappa 0.39); 20.9% and 8.1% of the patients showed the mutation solely in their primary tumors and metastases, respectively. TMPRSS2-ERG fusion was not correlated with specific histopathological tumor features but predicted favorable biochemical recurrence-free, disease-specific and overall survival independently when present in the primary tumor (P < 0.05 each). CONCLUSION TMPRSS2-ERG fusion is more frequent in primary prostate cancer than in corresponding metastases suggesting no selection of fusion-positive cells in the metastatic process. The gene fusion in primary tumors independently predicts favorable outcome.

Bcl-2 predicts response to neoadjuvant chemotherapy and is overexpressed in lymph node metastases of urothelial cancer of the bladder

PURPOSE To assess whether Bcl-2, an inhibitor of the apoptotic cascade, can predict response to neoadjuvant chemotherapy in patients with urothelial cancer of the bladder (UCB). METHODS Bcl-2 expression was analyzed in 2 different tissue microarrays (TMAs). One TMA was constructed of primary tumors and their corresponding lymph node (LN) metastases from 152 patients with chemotherapy-naive UCB treated by cystectomy and pelvic lymphadenectomy (chemotherapy-naive TMA cohort). The other TMA was constructed of tumor samples obtained from 55 patients with UCB before neoadjuvant chemotherapy (transurethral resection of the bladder cancer) and after cystectomy with pelvic lymphadenectomy (residual primary tumor [ypT+], n = 38); residual LN metastases [ypN+], n = 24) (prechemotherapy/postchemotherapy TMA cohort). Bcl-2 overexpression was defined as 10% or more cancer cells showing cytoplasmic immunoreactivity. RESULTS In both TMA cohorts, Bcl-2 overexpression was significantly (P<0.05) more frequent in LN metastases than in primary tumors (chemotherapy-naive TMA group: 18/148 [12%] in primary tumors vs. 39/143 [27%] in metastases; postchemotherapy TMA: ypT+7/35 [20%] vs. ypN+11/19 [58%]). In the neoadjuvant setting, patients with Bcl-2 overexpression in transurethral resection of the bladder cancer specimens showed significantly (P = 0.04) higher ypT stages and less regression in their cystectomy specimens than did the control group, and only one-eighth (13%) had complete tumor regression (ypT0 ypN0). In survival analyses, only histopathological parameters added significant prognostic information. CONCLUSIONS Bcl-2 overexpression in chemotherapy-naive primary bladder cancer is related to poor chemotherapy response and might help to select likely nonresponders.

FGFR3 expression in primary invasive bladder cancers and matched lymph node metastases

PURPOSE FGFR3 is considered a good therapeutic target for bladder cancer. However, to our knowledge it is unknown whether the FGFR3 status of primary tumors is a surrogate for related metastases, which must be targeted by FGFR targeted systemic therapies. We assessed FGFR3 protein expression in primary bladder tumors and matched nodal metastases. MATERIALS AND METHODS We examined matched primary tumor and nodal metastases from 150 patients with bladder cancer clinically staged as N0M0. Four samples per patient were incorporated into a tissue microarray and FGFR3 expression was assessed by immunohistochemistry. FGFR3 expression was tested for an association with categorical clinical data using the Fisher exact test, and with overall and recurrence-free survival by Kaplan-Meier analysis. RESULTS Duplicate spots from primary tumors and lymph node metastases were highly concordant (OR 8.6 and 16.7, respectively, each p <0.001). Overall FGFR protein expression levels did not differ between primary and metastatic lesions (p = 0.78). Up-regulated expression was recorded in 53 of 106 evaluable primary tumor spots and 56 matched metastases. Concordance of FGFR3 expression levels in 79 matched primary tumor and metastasis specimens was high (OR 8.45, p <0.001). In 15 and 12 patients expression was up-regulated in only metastasis and in only the primary tumor, respectively. Overall and recurrence-free survival was not related to FGFR3 expression. CONCLUSIONS FGFR3 expression in matched primary and metastasized bladder cancer specimens showed good but not absolute concordance. Thus, in most patients primary tumor FGFR3 status can guide the selection of FGFR targeted therapy.

Metastases in normal-sized pelvic lymph nodes: detection with diffusion-weighted MR imaging

PURPOSE To prospectively assess the diagnostic performance of diffusion-weighted (DW) magnetic resonance (MR) imaging in the detection of pelvic lymph node metastases in patients with prostate and/or bladder cancer staged as N0 with preoperative cross-sectional imaging. MATERIALS AND METHODS This study was approved by an independent ethics committee. Written informed consent was obtained from all patients. Patients with no enlarged lymph nodes on preoperative cross-sectional images who were scheduled for radical resection of the primary tumor and extended pelvic lymph node dissection were enrolled. All patients were examined with a 3-T MR unit, and examinations included conventional and DW MR imaging of the entire pelvis. Image analysis was performed by three independent readers blinded to any clinical information. Metastases were diagnosed on the basis of high signal intensity on high b value DW MR images and morphologic features (shape, border). Histopathologic examination served as the standard of reference. Sensitivity and specificity were calculated, and bias-corrected 95% confidence intervals (CIs) were obtained with the bootstrap method. The Fleiss and Cohen κ and median test were applied for statistical analyses. RESULTS A total of 4846 lymph nodes were resected in 120 patients. Eighty-eight lymph node metastases were found in 33 of 120 patients (27.5%). Short-axis diameter of these metastases was less than or equal to 3 mm in 68, more than 3 mm to 5 mm in 13, more than 5 mm to 8 mm in five; and more than 8 mm in two. On a per-patient level, the three readers correctly detected metastases in 26 (79%; 95% CI: 64%, 91%), 21 (64%; 95% CI: 45%, 79%), and 25 (76%; 95% CI: 60%, 90%) of the 33 patients with metastases, with respective specificities of 85% (95% CI: 78%, 92%), 79% (95% CI: 70%, 88%), and 84% (95% CI: 76%, 92%). Analyzed according to hemipelvis, lymph node metastases were detected with histopathologic examination in 44 of 240 pelvic sides (18%); the three readers correctly detected these on DW MR images in 26 (59%; 95% CI: 45%, 73%), 19 (43%; 95% CI: 27%, 57%), and 28 (64%; 95% CI: 47%, 78%) of the 44 cases. CONCLUSION DW MR imaging enables noninvasive detection of small lymph node metastases in normal-sized nodes in a substantial percentage of patients with prostate and bladder cancer diagnosed as N0 with conventional cross-sectional imaging techniques.

Lymph node stromal cells negatively regulate antigen-specific CD4+ T cell responses

Lymph node (LN) stromal cells (LNSCs) form the functional structure of LNs and play an important role in lymphocyte survival and the maintenance of immune tolerance. Despite their broad spectrum of function, little is known about LNSC responses during microbial infection. In this study, we demonstrate that LNSC subsets display distinct kinetics following vaccinia virus infection. In particular, compared with the expansion of other LNSC subsets and the total LN cell population, the expansion of fibroblastic reticular cells (FRCs) was delayed and sustained by noncirculating progenitor cells. Notably, newly generated FRCs were preferentially located in perivascular areas. Viral clearance in reactive LNs preceded the onset of FRC expansion, raising the possibility that viral infection in LNs may have a negative impact on the differentiation of FRCs. We also found that MHC class II expression was upregulated in all LNSC subsets until day 10 postinfection. Genetic ablation of radioresistant stromal cell-mediated Ag presentation resulted in slower contraction of Ag-specific CD4(+) T cells. We propose that activated LNSCs acquire enhanced Ag-presentation capacity, serving as an extrinsic brake system for CD4(+) T cell responses. Disrupted function and homeostasis of LNSCs may contribute to immune deregulation in the context of chronic viral infection, autoimmunity, and graft-versus-host disease.

Long-term results of a prospective randomised trial assessing the impact of readaptation of the dorsolateral peritoneal layer following extended pelvic lymph node dissection and cystectomy.

OBJECTIVE To evaluate the long term oncological and functional outcomes after readaptation of the dorsolateral peritoneal layer following pelvic lymph node dissection (PLND) and cystectomy . PATIENTS AND METHODS A randomised, single-center, single-blinded, two-arm trial was conducted on 200 consecutive cystectomy patients who underwent PLND and cystectomy for bladder cancer (

Implications of intraglandular lymph node metastases in primary carcinomas of the parotid gland

OBJECTIVES/HYPOTHESIS Assess the diagnostic and prognostic relevance of intraglandular lymph node (IGLN) metastases in primary parotid gland carcinomas (PGCs). STUDY DESIGN Retrospective study at a tertiary referral university hospital. METHODS We reviewed the records of 95 patients with primary PGCs, treated at least surgically, between 1997 and 2010. We assessed the clinicopathological associations of IGLN metastases, their prognostic significance, and predictive value in the diagnosis of occult neck lymph node metastases RESULTS Twenty-four (25.26%) patients had IGLN metastases. This feature was significantly more prevalent in patients with advanced pT status (P = .01), pN status (P < .01), and overall stage (P < .001); high-risk carcinomas (P = .01); as well as in patients with treatment failures (P < .01). IGLN involvement was significantly associated with decreased univariate disease-free survival (P < .001). Positive and negative predictive values and accuracy for IGLN involvement in the detection of occult neck lymph node metastases were 63.64%, 90.48%, and 84.91%, respectively. The diagnostic values were generally higher in patients with low-risk subtype of PGCs. CONCLUSIONS IGLN involvement provides prognostic information and is associated with advanced tumoral stage and higher risk of recurrence. This feature could be used as a potential readout to determine whether a neck dissection in clinically negative neck lymph nodes is needed or not. LEVEL OF EVIDENCE 4.

Sentinel lymph node biopsy in thick malignant melanoma: A 16-year single unit experience.

BACKGROUND The role of sentinel lymph node biopsy (SLNB) and its benefits in patients with thick melanoma is still controversial. OBJECTIVES We evaluated the clinical effect of SLNB in patients with thick melanoma. METHODS We performed a retrospective cohort review (1996-2012) of thick melanomas. Collected data included the patient and tumour characteristics. Locoregional recurrence, distant metastases, disease free and overall survival were compared between the patients with positive and negative SLNB. RESULTS 126 thick melanomas with a mean age of 64.09 years were included in the study. Positive SLNB were found in 47 (37.3%) patients. Significantly more locoregional recurrence (P = 0.002) and distant metastases (P = 0.030) were detected in the patients with positive SLNB. Furthermore, the patients with negative SLNB showed significantly better disease free survival (P = 0.021). CONCLUSIONS Positive SLNB might be prognostic factor in thick melanoma and aggravates the outcome of thick melanomas.

Laparoscopic Indocyanine Green Sentinel Lymph Node Mapping in Pregnant Cervical Cancer Patients.

We present cases of 2 pregnant patients with early-stage cervical cancer who have undergone indocyanine green (ICG) sentinel lymph node (SLN) mapping followed by laparoscopic SLN biopsy, pelvic lymphadenectomy, and cervical conization. Eight milliliters of ICG were injected in the 4 quadrants of the cervix after having obtained an adequate pneumoperitoneum and having inspected the abdominal cavity. SLNs were identified in both hemipelvises in both patients. In the final pathologic analysis, both SLNs and non-SLNs were negative for metastatic disease. No adverse events from ICG injection were recorded. ICG SLN mapping seems to be feasible in pregnant cervical cancer patients.

A Comparison of Radiocolloid and Indocyanine Green Fluorescence Imaging, Sentinel Lymph Node Mapping in Patients with Cervical Cancer Undergoing Laparoscopic Surgery.

BACKGROUND AND PURPOSE (99)TC combined with blue-dye mapping is considered the best sentinel lymph node (SLN) mapping technique in cervical cancer. Indocyanine green (ICG) with near infrared fluorescence imaging has been introduced as a new methodology for SLN mapping. The aim of this study was to compare these two techniques in the laparoscopic treatment of cervical cancer. METHODS Medical records of patients undergoing laparoscopic SLN mapping for cervical cancer with either (99)Tc and patent blue dye (Group 1) or ICG (Group 2) from April 2008 until August 2012 were reviewed. Sensitivity, specificity, and overall and bilateral detection rates were calculated and compared. RESULTS Fifty-eight patients were included in the study-36 patients in Group 1 and 22 patients in Group 2. Median tumor diameter was 25 and 29 mm, and mean SLN count was 2.1 and 3.7, for Groups 1 and 2, respectively. Mean non-SLN (NSLN) count was 39 for both groups. SLNs were ninefold more likely to be affected by metastatic disease compared with NSLNs (p < 0.005). Sensitivity and specificity were both 100 %. Overall detection rates were 83 and 95.5 % (p = nonsignificant), and bilateral detection rates were 61 and 95.5 % (p < 0.005), for Groups 1 and 2, respectively. In 75 % of cases, SLNs were located along the external or internal iliac nodal basins. CONCLUSIONS ICG SLN mapping in cervical cancer provides high overall and bilateral detection rates that compare favorably with the current standard of care.