Alcides Chaux

Immune Inhibitory Molecules LAG-3 and PD-1 Synergistically Regulate T-cell Function to Promote Tumoral Immune Escape

Inhibitory receptors on immune cells are pivotal regulators of immune escape in cancer. Among these inhibitory receptors, CTLA-4 (targeted clinically by ipilimumab) serves as a dominant off-switch while other receptors such as PD-1 and LAG-3 seem to serve more subtle rheostat functions. However, the extent of synergy and cooperative interactions between inhibitory pathways in cancer remain largely unexplored. Here, we reveal extensive coexpression of PD-1 and LAG-3 on tumor-infiltrating CD4(+) and CD8(+) T cells in three distinct transplantable tumors. Dual anti-LAG-3/anti-PD-1 antibody treatment cured most mice of established tumors that were largely resistant to single antibody treatment. Despite minimal immunopathologic sequelae in PD-1 and LAG-3 single knockout mice, dual knockout mice abrogated self-tolerance with resultant autoimmune infiltrates in multiple organs, leading to eventual lethality. However, Lag3(-/-)Pdcd1(-/-) mice showed markedly increased survival from and clearance of multiple transplantable tumors. Together, these results define a strong synergy between the PD-1 and LAG-3 inhibitory pathways in tolerance to both self and tumor antigens. In addition, they argue strongly that dual blockade of these molecules represents a promising combinatorial strategy for cancer.

ERG gene rearrangements are common in prostatic small cell carcinomas

Small cell carcinoma of the prostate is a rare subtype with an aggressive clinical course. Despite the frequent occurrence of ERG gene rearrangements in acinar carcinoma, the incidence of these rearrangements in prostatic small cell carcinoma is unclear. In addition, molecular markers to distinguish prostatic small cell carcinomas from lung and bladder small cell carcinomas may be clinically useful. We examined the occurrence of ERG gene rearrangements by fluorescence in situ hybridization in prostatic, bladder and lung small cell carcinomas. We also examined the expression of ERG, androgen receptor (AR) and NKX3-1 by immunohistochemistry in prostatic cases. Overall, 45% (10/22) of prostatic small cell carcinoma cases harbored ERG rearrangements, whereas no cases of bladder or lung small cell carcinomas showed ERG rearrangement (0/12 and 0/13, respectively). Of prostatic small cell carcinoma cases, 80% (8/10) showed ERG deletion and 20% (2/10) showed ERG translocation. In 83% (5/6) of prostatic small cell carcinoma cases in which a concurrent conventional prostatic acinar carcinoma component was available for analysis, there was concordance for the presence/absence of ERG gene rearrangement between the different subtypes. ERG, AR and NKX3-1 protein expression was detected in a minority of prostatic small cell carcinoma cases (23, 27 and 18%, respectively), while these markers were positive in the majority of concurrent acinar carcinoma cases (66, 83 and 83%, respectively). The presence of ERG rearrangements in nearly half of the prostatic small cell carcinomas is a similar rate of rearrangement to that found in prostatic acinar carcinomas. Furthermore, the high concordance rate of ERG rearrangement between the small cell and acinar components in a given patient supports a common origin for these two subtypes of prostate cancer. Finally, the absence of ERG rearrangement in bladder or lung small cell carcinomas highlights the utility of detecting ERG rearrangement in small cell carcinomas of unknown primary for establishing prostatic origin.

Immunohistochemistry for ERG Expression as a Surrogate for TMPRSS2-ERG Fusion Detection in Prostatic Adenocarcinomas

BackgroundTMPRSS2-ERG fusions have been identified in about one-half of all prostatic adenocarcinomas (PCas). Fluorescence in situ hybridization (FISH) and reverse transcription polymerase chain reaction have been the most commonly used techniques in this setting. The aim of this study was to evaluate the utility of ERG immunoexpression as a surrogate for TMPRSS2-ERG fusion in a large series of PCa cases. Materials and MethodsFour hundred twenty-seven radical retropubic prostatectomy tissue samples were used to construct 10 tissue microarrays (TMAs). FISH analysis was previously conducted using dual-color interphase break-apart probes for the 5′ and 3′ regions of the ERG gene. ERG expression was evaluated using a commercial rabbit anti-ERG monoclonal antibody (clone EPR3864; Epitomics, Burlingame, CA). Each TMA spot was independently assessed, and any nuclear staining positivity was considered as indicative of ERG expression. ResultsTMPRSS2-ERG fusions were detected by FISH in 195 (45.7%) of the PCa cases. ERG immunoexpression was found in 192 (45.0%) of the PCa cases and in none of the nontumoral tissue samples. Mean ERG H-scores were significantly higher in tumors harboring FISH-detected TMPRSS2-ERG fusions (P<0.00001), and there was a strong association between ERG immunohistochemical expression and the TMPRSS2-ERG status defined by FISH (P<0.00001), with a sensitivity of 86% (95% CI, 80%-90%) and a specificity of 89% (95% CI, 84%-93%). Receiver-operating characteristic curve analysis showed that ERG immunoexpression had a high accuracy for identifying TMPRSS2-ERG fusions detected by FISH, with an area under the curve of 0.87 (95% CI, 0.84%-0.91; P<0.00001). ConclusionsWe found that ERG immunohistochemical expression has a high accuracy for defining the TMPRSS-ERG fusion status. ERG immunohistochemistry may offer an accurate, simpler, and less costly alternative for evaluation of ERG fusion status in PCa than FISH.

Expression of androgen and oestrogen receptors and its prognostic significance in urothelial neoplasm of the urinary bladder

Whats known on the subject? and What does the study add?

Histologic grade and perineural invasion are more important than tumor thickness as predictor of nodal metastasis in penile squamous cell carcinoma invading 5 to 10 mm.

Penile squamous cell carcinomas (SCCs) invading to a depth inferior to 5 mm usually have very low risk for regional metastasis, whereas tumors thicker than 10 mm have a high metastatic potential. A significant number of squamous cell carcinomas, however, belong to an intermediate category (5 to 10 mm in thickness) in which the incidence of regional lymph node metastasis is very difficult to predict. Consequently, a frequent clinical dilemma is whether to perform or not inguinal dissection in this group of lesions. The objective of this study was to evaluate multiple risk factors for regional metastasis in tumors 5 to 10-mm thick. One hundred thirty-four partial penectomies with invasive carcinomas 5 to 10-mm thick, all of which with corresponding inguinal lymph node dissection, were evaluated. Factors evaluated were—patients age, anatomic site, histologic grade, tumor thickness, anatomic levels of invasion, and vascular and perineural invasion. Grades were classified as 1, well; 2, moderately; and 3, poorly differentiated. To evaluate independent significance of various prognostic factors, a logistic regression analysis was performed, and a nomogram was prepared to evaluate metastatic risk according to histologic grade and perineural invasion. Groin metastasis was found in 66 of 134 patients (49%). High histologic grade and perineural invasion were statistically significant pathologic factors associated with groin metastasis. Nodal metastases were found in 2 of 25 grade 1 (8%), 24 of 46 grade 2 (52%), and 40 of 63 grade 3 carcinomas (63%) (P value 0.0001). Of 48 patients with perineural invasion, metastasis was found in 33 cases (69%) (P value 0.001). The average tumor thickness, anatomic level of invasion, and presence of vascular invasion were not statistically significantly different in metastasizing and nonmetastasizing neoplasms. Fifty percent of tumors invading 5 to 10 mm were not associated with metastasis and may be spared a nodal dissection. In this subset of patients, high histologic grade and perineural invasion were significant and useful risk factors associated with regional metastasis. The probability of inguinal node metastasis in patients with grade 1 tumors without perineural invasion is almost nonexistent whereas for high-grade tumors associated with perineural invasion is near 80%.

The basaloid cell is the best tissue marker for human papillomavirus in invasive penile squamous cell carcinoma: a study of 202 cases from Paraguay.

Human papillomavirus (HPV) has been reported in 12-82% of penile squamous cell carcinomas (SCC). There is an association of the virus with basaloid and warty carcinomas but the reported prevalence is variable. The causes of these variations are not clear. They may be owing to geographic differences, the use of different techniques to detect HPV, the status of the original paraffin blocks, or to variable criteria in tumor classification. The aims of the study were to determine the prevalence of HPV in penile SCC and subtypes using a sensitive technique, to investigate genotypes involved, and to search for other morphologic features associated with the virus from a series of cases from Paraguay. HPV detection was done by SPF-10 polymerase chain reaction followed by DNA enzyme-immunoassay and genotyping by LIPA 25 (version 1). Samples were tested at Catalan Institute of Oncology, Barcelona, and cross testing was carried out at the Delft Diagnostic Laboratories in The Netherlands. HPV was detected in 64 of 202 cases (32%). Thirteen tumors had multiple HPV genotypes. Most prevalent genotypes were HPV-16 (46 cases), HPV-6 (6 cases), and HPV-18 (4 cases), either in single or in multiple infections. HPV was preferentially associated with warty-basaloid (82%), basaloid (76%), and warty (39%) carcinomas and not detected in verrucous, mixed verrucous-papillary, pseudohyperplastic, and pseudoglandular SCCs. There was a strong association between HPV and higher histologic grade. Basaloid cells were more frequently found in HPV positive tumors (72%) and this association was statistically significant in univariate and multivariate analyses. Cells with koilocytotic features and keratinizing squamous cells were also present but to a much lesser degree (47% and 19%, respectively). In summary, HPV was found in a third of the cases and the most common genotype was HPV-16. Low-risk genotypes were rarely found in single infections, representing 4 cases among all analyzed (2%). There was an association between HPV presence and higher histologic grade and with basaloid, warty-basaloid, and warty carcinomas. Our results also suggest that, in penile SCC, the basaloid cell is the best tissue marker for oncogenic HPV infection.

Loss of PTEN expression is associated with increased risk of recurrence after prostatectomy for clinically localized prostate cancer.

PTEN (phosphatase and tensin homolog on chromosome 10) is one of the most frequently lost tumor suppressor genes in human cancers and it has been described in more than two-thirds of patients with advanced/aggressive prostate cancer. Previous studies suggest that, in prostate cancer, genomic PTEN loss is associated with tumor progression and poor prognosis. Thus, we evaluated whether immunohistochemical PTEN expression in prostate cancer glands was associated with higher risk of recurrence, using a nested case–control study that included 451 men who recurred and 451 men who did not recur with clinically localized prostate cancer treated by radical prostatectomy. Recurrence was defined as biochemical recurrence (serum prostate-specific antigen >0.2 ng/ml) or clinical recurrence (local recurrence, systemic metastases, or prostate cancer-related death). Cases and controls were matched on pathological T stage, Gleason score, race/ethnicity, and age at surgery. Odds ratios of recurrence and 95% confidence intervals were estimated using conditional logistic regression to account for the matching factors and to adjust for year of surgery, preoperative prostate-specific antigen concentrations, and status of surgical margins. Men who recurred had a higher proportion of PTEN negative expression (16 vs 11%, P=0.05) and PTEN loss (40 vs 31%, P=0.02) than controls. Men with markedly decreased PTEN staining had a higher risk of recurrence (odds ratio=1.67; 95% confidence intervals 1.09, 2.57; P=0.02) when compared with all other men. In summary, in patients with clinically localized prostate cancer treated by prostatectomy, decreased PTEN expression was associated with an increased risk of recurrence, independent of known clinicopathological factors.

Penile Squamous Cell Carcinoma Clinicopathological Features, Nodal Metastasis and Outcome in 333 Cases

PURPOSE We evaluated clinicopathological features and outcomes in patients with penile squamous cell carcinoma. MATERIALS AND METHODS We studied 333 patients with homogeneous surgical treatment, including circumcision in 4, local excision in 2, partial penectomy in 194 and total penectomy in 133. Of the patients 153 also underwent bilateral groin dissection. Followup was 8 to 453 months (average 100). RESULTS The usual type of squamous cell carcinoma was noted in 65% of cases. Higher histological grade, deeper anatomical infiltration, and vascular and perineural invasion were common findings in sarcomatoid, basaloid and adenosquamous carcinoma cases, correlating with a higher rate of nodal metastasis and mortality. These features were unusual in verrucous, papillary and warty carcinoma cases. Recurrence in 22% of cases was common for the sarcomatoid, basaloid and adenosquamous types but was not noted for verrucous carcinoma. Locoregional relapse was more common in cases of usual, mixed, papillary and warty carcinoma, and systemic relapse was typical in sarcomatoid and basaloid carcinoma cases. The overall metastasis rate was 24% and the 10-year survival rate was 82%. The highest mortality rate was observed within the first 3 years of followup. High grade tumors were more common in penectomy cases and carcinoma exclusive of the foreskin had a better prognosis. The nodal metastasis risk groups were low--verrucous, papillary and warty, intermediate--usual and mixed, and high risk--sarcomatoid, basaloid and adenosquamous. Mortality risk groups were low--mixed, papillary and warty, intermediate--usual and basaloid, and high risk--sarcomatoid. CONCLUSIONS These data should help clinicians to design therapeutic strategies and followup protocols.

An EGFR-ERK-SOX9 Signaling Cascade Links Urothelial Development and Regeneration to Cancer

Like many carcinomas, urothelial carcinoma (UroCa) is associated with chronic injury. A better understanding of this association could inform improved strategies for preventing and treating this disease. We investigated the expression, regulation, and function of the transcriptional regulator SRY-related high-mobility group box 9 (Sox9) in urothelial development, injury repair, and cancer. In mouse bladders, Sox9 levels were high during periods of prenatal urothelial development and diminished with maturation after birth. In adult urothelial cells, Sox9 was quiescent but was rapidly induced by a variety of injuries, including exposure to the carcinogen cyclophosphamide, culture with hydrogen peroxide, and osmotic stress. Activation of extracellular signal-regulated kinases 1/2 (ERK1/2) was required for Sox9 induction in urothelial injury and resulted from activation of the epidermal growth factor receptor (Egfr) by several Egfr ligands that were dramatically induced by injury. In UroCa cell lines, SOX9 expression was constitutively upregulated and could be suppressed by EGFR or ERK1/2 blockade. Gene knockdown showed a role for SOX9 in cell migration and invasion. Accordingly, SOX9 protein levels were preferentially induced in invasive human UroCa tissue samples (n = 84) compared with noninvasive cancers (n = 56) or benign adjacent urothelium (n = 49). These results identify a novel, potentially oncogenic signaling axis linking urothelial injury to UroCa. Inhibiting this axis is feasible through a variety of pharmacologic approaches and may have clinical utility.

Immunoexpression Status and Prognostic Value of mTOR and Hypoxia-induced Pathway Members in Primary and Metastatic Clear Cell Renal Cell Carcinomas

The need for effective targeted therapies for renal cell carcinomas (RCCs) has fueled the interest for understanding molecular pathways involved in the oncogenesis of kidney tumors. Aiming to analyze the expression status and prognostic significance of mTOR and hypoxia-induced pathway members in patients with clear cell RCC (ccRCC), tissue microarrays were constructed from 135 primary and 41 metastatic ccRCCs. Immunoexpression levels were compared and correlated with clinicopathologic parameters and outcome. PTEN levels were significantly lower in primary and metastatic ccRCCs compared with benign tissues (P<0.001). Levels of phos-AKT, phos-S6, and 4E-binding protein-1 (4EBP1) were higher in metastatic ccRCC (P⩽0.001). For phos-S6 and 4EBP1, levels were higher in primary ccRCC compared with benign tissues (P<0.001). c-MYC levels were higher in metastatic ccRCC (P<0.0001), and incremental p27 levels were observed in benign, primary ccRCC, and metastatic ccRCC (P<0.0001). HIF-1&agr; levels were significantly higher in primary and metastatic ccRCCs compared with benign tissues (P<0.0001). In primary ccRCC, levels of all mTOR and hypoxia-induced pathway members were significantly associated with pT stage (P⩽0.036), p27 levels with Fuhrman grade (P=0.031), and 4EBP1, p27, and HIF-1&agr; levels with tumor size (P⩽0.025). Tumor size, HIF-1&agr;, and phos-S6 levels were associated with disease-specific survival (DSS) (P⩽0.032) and tumor progression (P⩽0.043). In conclusion, both mTOR and hypoxia-induced pathways were activated in primary and metastatic ccRCC. PTEN loss seems to be an early event during tumorigenesis. Tumor size, HIF-1&agr;, and phos-S6 expression were found to be independent predictors of both DSS and tumor progression in primary ccRCC.