SPARC in cancer-associated fibroblasts is an independent poor prognostic factor in non-metastatic triple-negative breast cancer and exhibits pro-tumor activity.

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype and lacks specific targeted therapeutic agents. The current mechanistic evidence from cell-based studies suggests that the matricellular protein SPARC has a tumor-promoting role in TNBC; however, data on the clinical relevance of SPARC expression/secretion by tumor and stromal cells in TNBC are limited. Here, we analyzed by immunohistochemistry the prognostic value of tumor and stromal cell SPARC expression in 148 patients with non-metastatic TNBC and long follow-up (median: 5.4 years). We also quantified PD-L1 and PD-1 expression. We detected SPARC expression in tumor cells (42.4%), cancer-associated fibroblasts (CAFs; 88.1%), tumor-associated macrophages (77.1%), endothelial cells (75.2%) and tumor-infiltrating lymphocytes (9.8%). Recurrence-free survival was significantly lower in patients with SPARC-expressing CAFs. Multivariate analysis showed that SPARC expression in CAFs was an independent prognostic factor. We also detected tumor and stromal cell SPARC expression in TNBC cytosols, and in patient-derived xenografts and cell lines. Furthermore, we analyzed publicly available single-cell mRNA sequencing data and found that in TNBC, SPARC is expressed by different CAF subpopulations, including myofibroblasts and inflammatory fibroblasts that are involved in tumor-related processes. We then showed that fibroblast-secreted SPARC had a tumor-promoting role by inhibiting TNBC cell adhesion and stimulating their motility and invasiveness. Overall, our study demonstrates that SPARC expression in CAFs is an independent prognostic marker of poor outcome in TNBC. Patients with SPARC-expressing CAFs could be eligible for anti-SPARC targeted therapy.

Phase II study evaluating the association of gemcitabine, trastuzumab and erlotinib as first-line treatment in patients with metastatic pancreatic adenocarcinoma (GATE 1).

In a previous phase II study (THERAPY), cetuximab and trastuzumab combination, as second-line after progression with gemcitabine, showed disease stabilization in 27% of 33 patients with pancreatic carcinoma. In the present phase II multicenter study, we assessed the efficacy and tolerance of gemcitabine, trastuzumab plus erlotinib as first-line treatment of metastatic pancreatic cancer. The primary endpoint was disease control rate (DCR, RECIST v.1); secondary endpoints were progression-free (PFS), overall (OS) survival and toxicity (NCI-CTCAE v3.0). Ancillary study addressed the predictive value of both EGFR/HER2 expression and KRAS mutational status. Sixty-three patients from four centers were included (62 evaluable for toxicity, 59 for efficacy), median age was 62 years (35-77), 59.7% men. The median treatment duration was 16.1 weeks (2.1-61). Eleven patients (19%) reported a partial tumor response, and 33 (56%) disease stabilization. DCR was 74.6% (95%CI: 61.8-85.0; 44/59 patients). After a median follow-up of 23.3 months (0.6-23.6), median PFS was 3.5 months (95%CI: 2.4-3.8) and median OS 7.9 months (95%CI: 5.1-10.2). PFS was significantly longer in patients with grade ≥ 2 cutaneous toxicities vs patients with grade 0-1 toxicities (HR = 0.55, 95%CI: 0.33-0.92, P = .020). Expression of EGFR and HER2 was correlated with PFS and OS in multivariate analysis; HER2 expression was correlated with the tumor response. Main severe toxicities were neutropenia (32%), cutaneous rash (37%) and thrombosis/embolisms (35.5%). This triplet combination is effective in terms of disease control, PFS and OS, and acceptable for safety. A larger study to investigate this combination compared to the standard regimen should be discussed.

Quantification of HER1, HER2 and HER3 by time-resolved Förster resonance energy transfer in FFPE triple-negative breast cancer samples.

BACKGROUND: Triple-negative breast cancer (TNBC) has a worse prognosis compared with other breast cancer subtypes, and biomarkers to identify patients at high risk of recurrence are needed. Here, we investigated the expression of human epidermal receptor (HER) family members in TNBC and evaluated their potential as biomarkers of recurrence. METHODS: We developed Time Resolved-Förster Resonance Energy Transfer (TR-FRET) assays to quantify HER1, HER2 and HER3 in formalin-fixed paraffin-embedded (FFPE) tumour tissues. After assessing the performance and precision of our assays, we quantified HER protein expression in 51 TNBC specimens, and investigated the association of their expression with relapse-free survival. RESULTS: The assays were quantitative, accurate, and robust. In TNBC specimens, HER1 levels ranged from ≈4000 to more than 2 million receptors per cell, whereas HER2 levels varied from ≈1000 to 60,000 receptors per cell. HER3 expression was very low (less than 5500 receptors per cell in all samples). Moderate HER2 expression was significantly associated with higher risk of recurrence (HR = 3.93; P = 0.003). CONCLUSIONS: Our TR-FRET assays accurately quantify HER1, HER2 and HER3 in FFPE breast tumour specimens. Moderate HER2 expression may represent a novel prognostic marker in patients with TNBC.

Clinical Correlations of Programmed Cell Death Ligand 1 Status in Liquid and Standard Biopsies in Breast Cancer.

BACKGROUND: Data regarding the prognostic value of programmed cell death ligand 1 (PD-L1) expression on circulating tumor cells (CTCs) are lacking. However, CTCs could represent an alternative approach to serial biopsies, allowing real-time monitoring of cancer phenotype. METHODS: We evaluated, in a dedicated prospective clinical trial, the clinicopathological correlations and prognostic value of PD-L1(+)-CTCs in 72 patients with metastatic breast cancer (MBC). RESULTS: Eighteen of 56 patients with available archival tissue presented at least one positive (≥1%) PD-L1 tumor sample. Baseline CTCs and PD-L1(+)-CTCs were detected in 57 (79.2%) and 26 (36.1%) patients. No significant correlation was found between PD-L1 tumors and CTC expression. In univariate analysis, triple negative (TN) phenotype, number of metastatic treatments, >2 metastatic sites, ≥5 CTCs and PD-L1(+)-CTCs were significantly associated with progression-free survival, while tissue PD-L1 expression was not. In multivariate analysis, TN phenotype, number of metastatic treatments and of metastatic sites were the only 3 variables independently associated with progression-free survival. Progesterone receptor negativity, TN phenotype, >2 metastatic sites and ≥5 CTCs were significantly associated with overall survival in univariate analysis. In multivariable analysis, TN phenotype and >2 metastatic sites were the only 2 independent variables. CONCLUSIONS: Unlike PD-L1(+)-tumor, PD-L1(+)-CTCs correlate to survival in MBC. Reappraisal of the role of PD-L1 expression by tumor tissue and by CTCs under anti-PD-1/PD-L1 treatment is necessary to evaluate its predictive value and potential role as a stratifying factor in strategies and trials for MBC patients with MBC. CLINICAL TRIAL REGISTRATION: NCT02866149.

Reproductive Decision-Making in MMR Mutation Carriers After Results Disclosure: Impact of Psychological Status in Childbearing Options.

Reproductive techniques such as prenatal diagnosis (PND) or preimplantation genetic diagnosis (PGD), although debated, are legally forbidden in France in case of Lynch syndrome. The preference of mutation carriers about their reproductive options is not systematically considered in France. We aimed to prospectively assess the reproductive preferences of mismatch repair mutation carriers consulting in our institution (2003-2010, n = 100). We also considered the short- and long-term post-disclosure psychological impact using the Impact of Events Scale-Revised questionnaire to measure the prevalence of posttraumatic stress disorder (PTSD) in those patients. Complete data were obtained for 34 respondents (17 males, 17 females, median age of 33.5 years [22-59]). Seventeen respondents (57 %) preferred spontaneous natural conception versus 28 % and 35 % choosing PND and PGD, respectively. At results disclosure, respondents mainly explained their distress by fear of premature death (43 %) and transmitting mutated genes (42 %). One year later, this last fear remained predominant in 55 % of subjects. None of the main socio-demographical, psychological or medical variables (including fear of transmitting mutations) was significantly associated with the reproductive preferences. Results disclosure had a real and time-decreasing psychological impact on mutation carriers. Reproductive techniques, expected to decrease the hereditary risk, were not significantly preferred to natural conception.

RIP140 regulates transcription factor HES1 oscillatory expression and mitogenic activity in colon cancer cells.

The transcription factor receptor-interacting protein 140 (RIP140) regulates intestinal homeostasis and tumorigenesis through Wnt signaling. In this study, we investigated its effect on the Notch/HES1 signaling pathway. In colorectal cancer (CRC) cell lines, RIP140 positively regulated HES1 gene expression at the transcriptional level via a recombining binding protein suppressor of hairless (RBPJ)/neurogenic locus notch homolog protein 1 (NICD)-mediated mechanism. In support of these in vitro data, RIP140 and HES1 expression significantly correlated in mouse intestine and in a cohort of CRC samples, thus supporting the positive regulation of HES1 gene expression by RIP140. Interestingly, when the Notch pathway is fully activated, RIP140 exerted a strong inhibition of HES1 gene transcription controlled by the level of HES1 itself. Moreover, RIP140 directly interacts with HES1 and reversed its mitogenic activity in human CRC cells. In line with this observation, HES1 levels were associated with a better patient survival only when tumors expressed high levels of RIP140. Our data identify RIP140 as a key regulator of the Notch/HES1 signaling pathway, with a dual effect on HES1 gene expression at the transcriptional level and a strong impact on colon cancer cell proliferation.

A novel Fc-engineered cathepsin D-targeting antibody enhances ADCC, triggers tumor-infiltrating NK cell recruitment, and improves treatment with paclitaxel and enzalutamide in triple-negative breast cancer.

INTRODUCTION: Triple-negative breast cancer (TNBC) prognosis is poor. Immunotherapies to enhance the antibody-induced natural killer (NK) cell antitumor activity are emerging for TNBC that is frequently immunogenic. The aspartic protease cathepsin D (cath-D), a tumor cell-associated extracellular protein with protumor activity and a poor prognosis marker in TNBC, is a prime target for antibody-based therapy to induce NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC). This study investigated whether Fc-engineered anti-cath-D antibodies trigger ADCC, their impact on antitumor efficacy and tumor-infiltrating NK cells, and their relevance for combinatory therapy in TNBC. METHODS: Cath-D expression and localization in TNBC samples were evaluated by western blotting, immunofluorescence, and immunohistochemistry. The binding of human anti-cath-D F1M1 and Fc-engineered antibody variants, which enhance (F1M1-Fc+) or prevent (F1M1-Fc-) affinity for CD16a, to secreted human and murine cath-D was analyzed by ELISA, and to CD16a by surface plasmon resonance and flow cytometry. NK cell activation was investigated by flow cytometry, and ADCC by lactate dehydrogenase release. The antitumor efficacy of F1M1 Fc-variants was investigated using TNBC cell xenografts in nude mice. NK cell recruitment, activation, and cytotoxic activity were analyzed in MDA-MB-231 cell xenografts by immunophenotyping and RT-qPCR. NK cells were depleted using an anti-asialo GM1 antibody. F1M1-Fc+ antitumor effect was assessed in TNBC patient-derived xenografts (PDXs) and TNBC SUM159 cell xenografts, and in combination with paclitaxel or enzalutamide. RESULTS: Cath-D expression on the TNBC cell surface could be exploited to induce ADCC. F1M1 Fc-variants recognized human and mouse cath-D. F1M1-Fc+ activated NK cells in vitro and induced ADCC against TNBC cells and cancer-associated fibroblasts more efficiently than F1M1. F1M1-Fc- was ineffective. In the MDA-MB-231 cell xenograft model, F1M1-Fc+ displayed higher antitumor activity than F1M1, whereas F1M1-Fc- was less effective, reflecting the importance of Fc-dependent mechanisms in vivo. F1M1-Fc+ triggered tumor-infiltrating NK cell recruitment, activation and cytotoxic activity in MDA-MB-231 cell xenografts. NK cell depletion impaired F1M1-Fc+ antitumor activity, demonstrating their key role. F1M1-Fc+ inhibited growth of SUM159 cell xenografts and two TNBC PDXs. In combination therapy, F1M1-Fc+ improved paclitaxel and enzalutamide therapeutic efficacy without toxicity. CONCLUSIONS: F1M1-Fc+ is a promising immunotherapy for TNBC that could be combined with conventional regimens, including chemotherapy or antiandrogens.

Prognostic Relevance of Tumor-Infiltrating Immune Cells in Cervix Squamous Cell Carcinoma.

There exists a variety of studies about tumor-infiltrating immune cells (TIICs) in cervical cancer, but their prognostic value in correlation with the histopathological subtype has never been investigated. Therefore, the aim of this study was to quantify TIICs in a panel of 238 sporadic cervical cancers and investigate the correlation with cervical cancer subtype and patient survival. TIICs levels were significantly increased in the subgroup of CSCC (191 samples) in comparison to CAC (47 samples). In CSCC, TIICs' infiltration showed a negative correlation with age, FIGO stage and with the histone protein modification H3K4me3. Moreover, in CAC, it was positively correlated with p16 and with the glucocorticoid receptor and inversely correlated with the MDM2 protein and with H3K4me3. Interestingly, immune infiltration was an independent positive prognosticator for disease-free survival (DFS) in patients with CSCC, those bearing tumors with the strongest TIICs infiltration showing the better DFS. Altogether, the present study provides a differentiated overview of the relations between TIIC levels and prognosis in patients with CSCC vs. patients with CAC.

Improving the response to oxaliplatin by targeting chemotherapy-induced CLDN1 in resistant metastatic colorectal cancer cells.

BACKGROUND: Tumor resistance is a frequent cause of therapy failure and remains a major challenge for the long-term management of colorectal cancer (CRC). The aim of this study was to determine the implication of the tight junctional protein claudin 1 (CLDN1) in the acquired resistance to chemotherapy. METHODS: Immunohistochemistry was used to determine CLDN1 expression in post-chemotherapy liver metastases from 58 CRC patients. The effects of oxaliplatin on membrane CLDN1 expression were evaluated by flow cytometry, immunofluorescence and western blotting experiments in vitro and in vivo. Phosphoproteome analyses, proximity ligation and luciferase reporter assays were used to unravel the mechanism of CLDN1 induction. RNAseq experiments were performed on oxaliplatin-resistant cell lines to investigate the role of CLDN1 in chemoresistance. The "one-two punch" sequential combination of oxaliplatin followed by an anti-CLDN1 antibody-drug conjugate (ADC) was tested in both CRC cell lines and murine models. RESULTS: We found a significant correlation between CLDN1 expression level and histologic response to chemotherapy, CLDN1 expression being the highest in resistant metastatic residual cells of patients showing minor responses. Moreover, in both murine xenograft model and CRC cell lines, CLDN1 expression was upregulated after exposure to conventional chemotherapies used in CRC treatment. CLDN1 overexpression was, at least in part, functionally related to the activation of the MAPKp38/GSK3ß/Wnt/ß-catenin pathway. Overexpression of CLDN1 was also observed in oxaliplatin-resistant CRC cell lines and was associated with resistance to apoptosis, suggesting an anti-apoptotic role for CLDN1. Finally, we demonstrated that the sequential treatment with oxaliplatin followed by an anti-CLDN1 ADC displayed a synergistic effect in vitro and in in vivo. CONCLUSION: Our study identifies CLDN1 as a new biomarker of acquired resistance to chemotherapy in CRC patients and suggests that a "one-two punch" approach targeting chemotherapy-induced CLDN1 expression may represent a therapeutic opportunity to circumvent resistance and to improve the outcome of patients with advanced CRC.

KRAS genotyping in rectal adenocarcinoma specimens with low tumor cellularity after neoadjuvant treatment.

KRAS status assessment is mandatory in patients with metastatic colorectal cancer before therapy with anti-epidermal growth factor receptor monoclonal antibodies, as KRAS mutations are associated with resistance to this treatment. However, KRAS genotyping may be very challenging in case of poor tumor cellularity, particularly when major tumor regression is achieved in locally advanced rectal adenocarcinomas after radiochemotherapy. We aimed at identifying the most reliable strategy to detect KRAS mutations in such samples. DNA was extracted from 31 surgical specimens with major tumor regression, following manual dissection, and from paired pre-treatment biopsies and analyzed by high-resolution melting. DNA samples displaying altered melting curve shapes were then sequenced. Samples with unmodified melting curves or wild-type sequence were further investigated by using an allele-specific PCR assay (TheraScreen) and laser microdissection (followed by high-resolution melting and sequencing analyses). In the 31 post-radiochemotherapy surgical specimens, seven KRAS mutations were identified by high-resolution melting analysis/sequencing. One additional mutation was detected by the TheraScreen assay and two mutations, including the one identified by the TheraScreen assay, were detected following laser microdissection. Altogether, 9/31 surgical specimens (29%) presented KRAS mutations. In the manually dissected pre-treatment biopsies, 12 mutations (39%) were identified by high-resolution melting analysis and sequencing. No additional mutations were found by using the TheraScreen assay or laser microdissection. These results indicate that, in the case of post-radiochemotherapy surgical specimens of colorectal cancer with low tumor cellularity, pre-treatment biopsies might represent the most cost-effective option for reliable KRAS genotyping. The use of more sensitive assays, such as allele-specific PCR or laser microdissection, can be envisaged but with higher costs and longer delays.

In situ hybridization for the assessment of urokinase plasminogen activator and plasminogen activator inhibitor type­1 in formalin­fixed paraffin­embedded breast cancer specimens.

Urokinase plasminogen activator (uPA) and its inhibitor, plasminogen activator inhibitor type 1 (PAI­1), have been reported as prognostic and predictive biomarkers in breast cancer, particularly in patients with node­negative tumors. uPA and PAI­1 expression levels classify patients into a poor­prognosis subgroup, requiring adjuvant chemotherapy and a favorable­prognosis subgroup, which can be considered for de­escalation. However, the clinical use of these two biomarkers remains limited, since fresh­frozen/fresh tumor samples are currently required for their quantification. The aim of the present study was to compare PLAU and SERPINE1 mRNA expression levels (corresponding to uPA and PAI­1 proteins, respectively), assessed using in situ hybridization in 83 formalin­fixed paraffin­embedded (FFPE) breast tumor samples, with uPA and PAI­1 protein expression assessed using immunometric assay with paired fresh­frozen breast cancer samples. The results from the two methods significantly correlated as regards uPA quantification; however, >30% of the samples were discordant, according to the clinically validated threshold. Concordance between the two analytical methods was less prominent for PAI­1 protein and SERPINE1 mRNA. Taken together, the results of the present study indicate that although PLAU and SERPINE1 mRNA may be reliably detected in FFPE samples using in situ hybridization, this technology cannot be used as a substitute for the replacement of the immunometric assay­derived quantification on fresh­frozen samples.

Correlation of the TIGIT-PVR immune checkpoint axis with clinicopathological features in triple-negative breast cancer.

Background: T cell immunoreceptor with Ig and ITIM domains (TIGIT) interacts with poliovirus receptor (PVR) to contribute to cancer immune escape. Recently, TIGIT and PVR have been identified as promising immunotherapy targets. Their gene expression is upregulated in many solid tumors, but their protein expression level is not well documented, particularly in triple negative breast cancer (TNBC), the breast cancer subtype that most benefit from immunotherapy. Methods: TIGIT and PVR expression levels were assessed by immunohistochemistry in 243 surgically resected localized TNBC and then their relationship with clinical-pathological features and clinical outcome was analyzed. Results: TIGIT expression was observed in immune cells from the tumor microenvironment, whereas PVR was mainly expressed by tumor cells. High TIGIT expression was significantly associated with age (p=0.010), histological grade (p=0.014), non-lobular histology (p=0.024), adjuvant chemotherapy (p=0.006), and various immune cell populations (tumor infiltrating lymphocytes (TILs), CD3+, CD8+, PD-1+ cells; all p<0.0001), PD-L1+ tumor cells (p<0.0001), and PD-L1+ stromal cells (p=0.003). Infiltration by TIGIT+ cells tended to be higher in non-molecular apocrine tumors (p=0.088). PVR was significantly associated with histological grade (p<0.0001), the basal-like (p=0.003) and non-molecular apocrine phenotypes (p=0.039), high TILs infiltration (p=0.011), CD3+ (p=0.002), CD8+ (p=0.024) T cells, and PD-L1 expression in tumor (p=0.003) and stromal cells (p=0.001). In univariate analysis, only known prognostic factors (age, tumor size, lymph node status, adjuvant chemotherapy, TILs and CD3+ T-cell infiltrate) were significantly associated with relapse-free survival (RFS) and overall survival. High TIGIT and PVR expression levels tended to be associated with longer RFS (p=0.079 and 0.045, respectively). The analysis that included only non-molecular apocrine TNBC revealed longer RFS for tumors that strongly expressed TIGIT or PVR (p=0.025 for TIGIT and 0.032 for PVR). Conclusions: These results indicated that in TNBC, TIGIT+ cells can easily interact with PVR to exert their inhibitory effects. Their wide expression in TNBC and their association with other immune checkpoint components suggest the therapeutic interest of the TIGIT-PVR axis.

Association of CD206 Protein Expression with Immune Infiltration and Prognosis in Patients with Triple-Negative Breast Cancer.

Background: Triple-negative breast cancers (TNBCs) have a worse prognosis, but might respond to immunotherapies. Macrophages are plastic cells that can adopt various phenotypes and functions. Although they are a major immune population in TNBCs, the relationship between tumor-associated macrophages (TAMs) and TNBC progression has been rarely explored, with controversial results. Methods: We evaluated the prognostic impact of TAMs, quantified by immunohistochemistry with anti-CD68, -IRF8, -CD163, and -CD206 antibodies, in a well-described cohort of 285 patients with non-metastatic TNBC. Results: CD68 (p = 0.008), IRF8 (p = 0.001), and CD163 (p < 0.001) expression positively correlated with higher tumor grade, while CD206 was associated with smaller tumor size (p < 0.001). All macrophage markers were associated with higher tumor-infiltrating lymphocyte numbers and PD-L1 expression. Univariate survival analyses reported a significant positive correlation between CD163+ or CD206+ TAMs and relapse-free survival (respectively: HR = 0.52 [0.28−0.97], p = 0.027, and HR = 0.51 [0.31−0.82], p = 0.005), and between CD206+ TAMs and overall survival (HR = 0.54 [0.35−0.83], p = 0.005). In multivariate analysis, there was a trend for an association between CD206+ TAMs and relapse-free survival (HR = 0.63 [0.33−1.04], p = 0.073). Conclusions: These data suggest that CD206 expression defines a TAM subpopulation potentially associated with favorable outcomes in patients with TNBC. CD206 expression might identify an immune TNBC subgroup with specific therapeutic options.

Prognostic Relevance of Nuclear Receptors in Relation to Peritumoral Inflammation and Tumor Infiltration by Lymphocytes in Breast Cancer.

The prognostic impact of tumor-infiltrating lymphocytes (TILs) is intensively investigated in breast cancer (BC). It is already known that triple-negative breast cancer (TNBC), the most aggressive type of BC, has the highest percentage of TILs. In addition, there is an influence of steroid hormone receptor expression (type I nuclear receptors) on TIL subpopulations in breast cancer tissue. The link between type II nuclear receptors and the level of TILs is unclear. Therefore, the aim of this study was to quantify TILs in a panel of 264 sporadic breast cancers and investigate the correlation of TIL levels with type I and II nuclear receptors expression. TIL levels were significantly increased in the subgroup of TNBC. By contrast, they decreased in estrogen (ER)- or progesterone receptor (PR)-positive cases. Moreover, TIL levels were correlated with type II nuclear receptors, including PPARγ, with a significant inverse correlation of the nuclear form (r = −0.727, p < 0.001) and a weak positive correlation of the cytoplasmic form (r = 0.202, p < 0.002). Surprisingly, BC cases with a TIL Salgado score of >15% showed a significantly decreased overall survival. In addition, peritumoral inflammation was also quantified in BC tissue samples. In our cohort, although the level of peritumoral inflammation was not correlated with OS, it determined the prognostic value of ER, PR, and PPARγ in BC. Altogether, the present study provides a differentiated overview of the relations between nuclear receptor expression, TIL levels, peritumoral inflammation, and prognosis in BC.

EGFR and HER3 mRNA expression levels predict distant metastases in locally advanced rectal cancer.

Aberrant activation of the HER signaling pathways plays a critical role in the invasive and metastatic potential of tumors. The aim of this study was to address whether, in rectal cancer, alterations of these pathways could have a value as prognostic factors to be used to identify patients who are at risk of distant metastases. Therefore, the mRNA expression of the four members of the HER family as well as the frequency of PTEN allelic loss and KRAS/BRAF mutations were determined in pretreatment biopsies from a series of 100 locally advanced rectal cancers and then their ability to predict distant metastases was evaluated. Over-expression of EGFR (p = 0.021), HER2 (p = 0.011) and HER3 (p = 0.020) was significantly associated with worse metastasis-free survival in univariate analysis. In multivariate analysis, both over-expression of EGFR (p = 0.028) and HER3 (p = 0.011) remained independent prognostic factors for distant metastasis. In conclusion, quantification of EGFR and HER3 mRNA expression in pretreatment biopsies may be useful to identify patients who are at risk of developing metastases.

[Locally advanced rectal cancer management: which role for the pathologist in 2011?]. / Prise en charge des cancers du rectum localement avancés : quel rôle pour le pathologiste en 2011 ?

Locally advanced rectal cancers mainly correspond to lieberkünhien adenocarcinomas and are defined by T3-T4 lesions with or without regional metastatic lymph nodes. Such tumors benefit from neoadjuvant treatment combining chemotherapy and radiotherapy, followed by surgery with total mesorectum excision. Such a strategy can decrease the rate of local relapse and lead to an easier complementary surgery. The pathologist plays an important role in the management of locally advanced rectal cancer. Indeed, he is involved in the gross examination of the mesorectum excision quality and in the exhaustive sampling of the most informative areas. He also has to perform a precise histopathological analysis, including the determination of the circumferential margin or clearance and the evaluation of tumor regression. All these parameters are major prognostic factors which have to be clearly included in the pathology report. Moreover, the next challenge for the pathologist will be to determine and validate new prognostic and predictive markers, notably by using pre-therapeutic biopsies. The goal of this mini-review is to emphasize the pathologist's role in the different steps of the management of locally advanced rectal cancers and to underline its implication in the determination of potential biomarkers of aggressiveness and response.

CXCR2 Levels Correlate with Immune Infiltration and a Better Prognosis of Triple-Negative Breast Cancers.

Chemokines and their receptors are key players in breast cancer progression and outcome. Previous studies have shown that the chemokine receptor CXCR2 was expressed at higher levels by cells of the tumor microenvironment in triple-negative breast cancers (TNBCs). The aim of this study was to focus our attention on a retrospective cohort of 290 TNBC cases and analyze the involvement of CXCR2, CD11b (a marker of granulocytes) and CD66b (a marker of neutrophils) and their link with immune infiltration and immune checkpoint markers. We report that high densities of CXCR2-, CD11b- and CD66b-positive cells were associated with high-grade tumors. Moreover, molecular apocrine TNBCs, defined here as tumors that express both AR and FOXA1 biomarkers, exhibited low levels of CXCR2 and CD11b. High CXCR2 and CD11b levels were correlated with elevated density of tumor-infiltrating lymphocytes (TILs), CD8+ cytotoxic lymphocytes, expression of PD-L1 by tumor and stromal cells and of PD-1 by stromal cells. On the other hand, CD66b levels were associated only with CD8+, stromal PD-L1 and PD-1 expression. In univariate analysis, low levels of CXCR2 were correlated with poor OS and RFS. In multivariate analysis, low levels of CXCR2 were associated with poor OS. Finally, in TNBC treated with adjuvant chemotherapy, CXCR2 density was associated with longer RFS. Overall, our data highlight the potential beneficial association of high levels of CXCR2 with a subgroup of TNBC patients characterized by a better prognosis.

Prognostic Value of HER2-Low Expression in Non-Metastatic Triple-Negative Breast Cancer and Correlation with Other Biomarkers.

HER2-low breast cancer (i.e., HER 1+ or 2+, without gene amplification) is an emerging subtype for which very few data are available, especially within the triple-negative breast cancer (TNBC) group. Our aim was to evaluate HER2 expression and its prognostic value in a large retrospective series of patients with non-metastatic TNBC (median age: 57.7 years; range: 28.5-98.6). Among the 296 TNBC samples, 83.8% were HER2 0, 13.5% were HER2 1+, and 2.7% were HER2 2+ (HercepTestTM and 2018 ASCO/CAP guidelines for HER2 scoring). CK5/6 and/or EGFR-expressing androgen receptors and FOXA1-expressing tumors were classified as basal-like (63.8%) and molecular apocrine-like (MA, 40.2%), respectively. Compared with HER2 0 tumors, HER2 1+/2+ tumors exhibited a lower histological grade (1/2) (35.4% vs. 18.2%, p = 0.007) and MA profile (57.5% vs. 36.7%, p = 0.008). Moreover, patients with HER2 1+/2+ tumors were older (p = 0.047). After a median follow-up of 9.7 years, HER2 2+ tumors (compared with HER2 0/1+ tumors) were associated with worse relapse-free survival (RFS) (HR = 3.16, 95% CI [1.27; 7.85], p = 0.034) in a univariate analysis. Overall survival (OS) and RFS were not different in the HER2 0 and 1+/2+ groups. HER2 levels were not significantly associated with OS or RFS in a multivariate analysis.

Clinicopathological Correlates of γδ T Cell Infiltration in Triple-Negative Breast Cancer.

The prognostic impact of the different tumor-infiltrating lymphocyte (TIL) subpopulations in solid cancers is still debated. Here, we investigated the clinicopathological correlates and prognostic impact of TILs, particularly of γδ T cells, in 162 patients with triple-negative breast cancer (TNBC). A high γδ T cell density (>6.625 γδ T cells/mm2) was associated with younger age (p = 0.008), higher tumor histological grade (p = 0.002), adjuvant chemotherapy (p = 0.010), BRCA1 promoter methylation (p = 0.010), TIL density (p < 0.001), and PD-L1 (p < 0.001) and PD-1 expression (p = 0.040). In multivariate analyses, γδ T cell infiltration (cutoff = 6.625 γδ T cells/mm2) was an independent prognostic factor (5-year relapse-free survival: 63.3% vs. 89.8%, p = 0.027; 5-year overall survival: 73.8% vs. 89.9%, p = 0.031, for low vs. high infiltration). This prognostic impact varied according to the tumor PIK3CA mutational status. High γδ T cell infiltration was associated with better survival in patients with PIK3CA wild-type tumors, but the difference was not significant in the subgroup with PIK3CA-mutated tumors. Altogether, these data suggest that high γδ T cell infiltrate is correlated with immune infiltration and might represent a candidate prognostic tool in patients with TNBC.

MAGI1 inhibits the AMOTL2/p38 stress pathway and prevents luminal breast tumorigenesis.

Alterations to cell polarization or to intercellular junctions are often associated with epithelial cancer progression, including breast cancers (BCa). We show here that the loss of the junctional scaffold protein MAGI1 is associated with bad prognosis in luminal BCa, and promotes tumorigenesis. E-cadherin and the actin binding scaffold AMOTL2 accumulate in MAGI1 deficient cells which are subjected to increased stiffness. These alterations are associated with low YAP activity, the terminal Hippo-pathway effector, but with an elevated ROCK and p38 Stress Activated Protein Kinase activities. Blocking ROCK prevented p38 activation, suggesting that MAGI1 limits p38 activity in part through releasing actin strength. Importantly, the increased tumorigenicity of MAGI1 deficient cells is rescued in the absence of AMOTL2 or after inhibition of p38, demonstrating that MAGI1 acts as a tumor-suppressor in luminal BCa by inhibiting an AMOTL2/p38 stress pathway.