The French phase 3 trial (OS 2006) testing zoledronic acid, an osteoclast inhibitor, with chemotherapy and surgery did not improve the outcome of patients with osteosarcoma (OS). To understand this unexpected result, the presence of infiltrating immune cells was investigated in 124 pre-therapeutic biopsies of patients enrolled in the trial. The percentage of CD68/CD163 tumor-infiltrating macrophages (TAMs), CD8+ lymphocytes, osteoclasts, and the PD1/PDL-1 checkpoint were assessed by immunohistochemistry. M1/M2 macrophage polarization was characterized by pSTAT1/CMAF staining. The expression of these biomarkers was correlated with clinical outcome. No statistical correlations were found with response to chemotherapy. High CD163 levels (>50% of cells per core; 43.8% of patients) were associated with CMAF nuclear expression and significantly correlated with better overall survival (p = 0.0025) and longer metastasis progression-free survival (MPFS, p = 0.0315) independently of metastatic status (p = 0.002). Only a trend was observed for patients with high CD68-positive cells (p = 0.0582). CD8+ staining was positive in >50% of cases with a median staining of 1%. Lower CD8+ levels were associated with metastatic disease at diagnosis and the presence of CD8-positive cells significantly correlated with improved overall survival in zoledronate-treated patients (p = 0.0415). PD1/PDL-1 staining was negative in >80% of cases and was not correlated with outcome. Finally, CD163-positive TAMs and CD8 positive cells are crucial prognostic biomarkers in OS, whereas PD1/PDL-1 checkpoint plays a minor role. For the first time, we described a correlation between CD8 positive cells and survival in zoledronate-treated patients. The immunohistochemical analysis of the microenvironment in biopsies may represent a novel tool for therapeutic stratification.

Background: Some authors have suggested that the semimembranosus tendon is involved in the pathophysiology of ramp lesions. This led us to conduct a gross and microscopic analysis of the posterior horn of the medial meniscus and the structures inserted on it. Hypothesis: (1) The semimembranosus tendon has a tendinous branch inserting into the posterior horn of the medial meniscus, and (2) the meniscotibial ligament is inserted on the posteroinferior edge of the medial meniscus. Study Design: Descriptive laboratory study. Methods: In total, 14 fresh cadaveric knees were dissected. From each cadaveric donor, a stable anatomic specimen was harvested en bloc, including the medial femoral condyle, medial tibial plateau, whole medial meniscus, cruciate ligaments, joint capsule, and distal insertion of the semimembranosus tendon. The harvested blocks were cut along the sagittal plane to isolate the distal insertion of the semimembranosus tendon on the posterior joint capsule and the posterior horn of the medial meniscus in a single slice. Histological slides were made from these samples and analyzed under a microscope. Results: In all knees, gross examination revealed a direct branch of the semimembranosus and a tendinous capsular branch ending behind the posterior horn of the medial meniscus. This capsular branch protruded over the joint capsule, over the meniscotibial ligament below and the meniscocapsular ligament above, but never ended directly in the meniscal tissue. The capsular branch was 14.3 ± 4.4 mm long (mean ± SD). The direct tendon inserted 11 ± 2.8 mm below the articular surface of the tibial plateau. The meniscotibial ligament inserted on the posteroinferior edge of the medial meniscus, and the meniscocapsular ligament insertion was on its posterosuperior edge. Highly vascularized adipose tissue was found, delimited by the posterior horn of the medial meniscus, meniscotibial ligament, meniscocapsular ligament, and capsular branch of the semimembranosus tendon. Conclusion: In all knees, our study found a capsular branch of the semimembranosus tendon inserted behind the medial meniscus. The meniscotibial ligament was inserted on the posteroinferior edge of the medial meniscus. Histological analysis of this area revealed that this ligament inserted differently from the insertion previously described in the literature. Clinical Relevance: This laboratory study provides insight into the pathophysiology of ramp lesions frequently associated with anterior cruciate ligament injury. To restore anatomy, it is mandatory to reestablish meniscotibial ligament continuity in ramp repairs.

To date, all studies conducted on breast cancer diagnosis have focused on the expression of the full-length 66-kDa estrogen receptor alpha (ERα66). However, much less attention has been paid to a shorter 46-kDa isoform (ERα46), devoid of the N-terminal region containing the transactivation function AF-1. Here, we investigated the expression levels of ERα46 in breast tumors in relation to tumor grade and size, and examined the mechanism of its generation and its specificities of coregulatory binding and its functional activities. Using approaches combining immunohistochemistry, Western blotting, and proteomics, antibodies allowing ERα46 detection were identified and the expression levels of ERα46 were quantified in 116 ERα-positive human breast tumors. ERα46 expression upon cellular stress was studied, and coregulator bindings, transcriptional, and proliferative response were determined to both ERα isoforms. ERα46 was expressed in over 70% of breast tumors at variable levels which sometimes were more abundant than ERα66, especially in differentiated, lower-grade, and smaller-sized tumors. We also found that ERα46 can be generated via internal ribosome entry site-mediated translation in the context of endoplasmic reticulum stress. The binding affinities of both unliganded and fully-activated receptors towards co-regulator peptides revealed that the respective potencies of ERα46 and ERα66 differ significantly, contributing to the differential transcriptional activity of target genes to 17β estradiol (E2). Finally, increasing amounts of ERα46 decrease the proliferation rate of MCF7 tumor cells in response to E2. We found that, besides the full-length ERα66, the overlooked ERα46 isoform is also expressed in a majority of breast tumors. This finding highlights the importance of the choice of antibodies used for the diagnosis of breast cancer, which are able or not to detect the ERα46 isoform. In addition, since the function of both ERα isoforms differs, this work underlines the need to develop new technologies in order to discriminate ERα66 and ERα46 expression in breast cancer diagnosis which could have potential clinical relevance.