Randomized, open-label, phase II, biomarker study of immune-mediated mechanism of action of neoadjuvant subcutaneous trastuzumab in patients with locally advanced, inflammatory, or early HER2-positive breast cancer-Immun-HER trial (GOIRC-01-2016).

BACKGROUND: It is possible to induce immunomodulation in HER2-positive breast cancer (BC) by modifying the route of administration of trastuzumab. METHODS: In this multicenter randomized phase II trial, all enrolled patients (pts) with T2-T4d HER2-positive BC received 3 cycles of neoadjuvant treatment (NAT) with fluorouracil, epirubicin and cyclophosphamide every 3 weeks (q21), followed by docetaxel/pertuzumab plus intravenous trastuzumab (arm A) or, docetaxel/pertuzumab plus subcutaneous (SC) trastuzumab (arm B) q21x4 cycles. After surgical operation, each pt was treated with trastuzumab q21x14 cycles using the same SC or intravenous formulation of NAT. Primary endpoint was the proportion of subjects with high stromal tumor-infiltrating lymphocytes (sTILs) in postneoadjuvant residual disease (RD). RESULTS: Sixty-three pts (31 (arm A) and 32 (arm B)) were enrolled. Pathological complete response was obtained by 20/31 pts (64.5%; 95% CI 45.4% to 80.1%) in arm A and 19/32 pts (59.4%; 95% CI 40.1% to 76.3%) in arm B. High sTILs were observed in 27% and 46% of postneoadjuvant residual tumors in arms A and B, respectively. CD8+ T cells increased significantly in RDs of both arms (p=0.014 and 0.002 for arm A and B, respectively), whereas a significant decline in the level of CD4+ FoxP3+ regulatory T cells was observed only in arm B (p=0.016). A significant upregulation of PD-1 on sTILs was found in RD of pts enrolled in arm B (p=0.012), while programmed death-ligand 1 (PD-L1) was significantly overexpressed in residual tumors of arm A (p=0.02). A strong negative correlation was reported in arm B between expression of PD-L1 on pretreatment sTILs and CD3 expression on sTILs in RD (τ: -0.73). Grade≥3 AE incidence rates were similar between the two arms. CONCLUSIONS: SC trastuzumab induced relevant sTILs enrichment, with favorable variations of immune parameters in HER2-positive BC pts with RD after NAT. Novel immunotherapy strategies should be tested to achieve SC-specific, antitumor immune response. TRIAL REGISTRATION NUMBER: NCT03144947, and EudraCT number: 2016-000435-41.

High Prognostic Value of 68Ga-PSMA PET/CT in Renal Cell Carcinoma and Association with PSMA Expression Assessed by Immunohistochemistry.

In oligo-metastatic renal cell carcinoma (RCC), neither computed tomography (CT) nor bone scan is sensitive enough to detect small tumor deposits hampering early treatment and potential cure. Prostate-specific membrane antigen (PSMA) is a transmembrane glycoprotein expressed in the neo-vasculature of numerous malignant neoplasms, including RCC, that can be targeted by positron emission tomography (PET) using PSMA-targeting radioligands. Our aim was to investigate whether PSMA-expression patterns of renal cancer in the primary tumor or metastatic lesions on immunohistochemistry (IHC) are associated with PET/CT findings using [68Ga]-PSMA-HBED-CC (PSMA-PET/CT). We then analyzed the predictive and prognostic role of the PSMA-PET/CT signal. In this retrospective single-center study we included patients with renal cancer submitted to PSMA-PET/CT for staging or restaging, with tumor specimens available for PSMA-IHC. Clinical information (age, tumor type, and grade) and IHC results from the primary tumor or metastases were collected. The intensity of PSMA expression at IHC was scored into four categories: 0: none; 1: weak; 2: moderate; 3: strong. PSMA expression was also graded according to the proportion of vessels involved (PSMA%) into four categories: 0: none; 1: 1-25%; 2: 25-50%; 3: >50%. The intensity of PSMA expression and PSMA% were combined in a three-grade score: 0-2 absent or mildly positive, 3-4 moderately positive, and 5-6 strongly positive. PSMA scores were used for correlation with PSMA-PET/CT results. Results: IHC and PET scans were available for the analysis in 26 patients (22 ccRCC, 2 papillary RCC, 1 chromophobe, 1 "not otherwise specified" RCC). PSMA-PET/CT was positive in 17 (65%) and negative in 9 patients (35%). The mean and median SUVmax in the target lesion were 34.1 and 24.9, respectively. Reporter agreement was very high for both distant metastasis location and local recurrence (kappa 1, 100%). PSMA-PET detected more lesions than conventional imaging and revealed unknown metastases in 4 patients. Bone involvement, extension, and lesion number were greater than in the CT scan (median lesion number on PET/CT 3.5). The IHC PSMA score was concordant in primary tumors and metastases. All positive PSMA-PET/CT results (15/22 ccRCC, 1 papillary cancer type II, and 1 chromofobe type) were revealed in tumors with strong or moderate PSMA combined scores (3-4 and 5-6). In ccRCC tissue samples, PSMA expression was strong to moderate in 20/22 cases. The SUVmax values correlated to the intensity of PSMA expression which were assessed using IHC (p = 0.01), especially in the ccRCC subgroup (p = 0.009). Median survival was significantly higher in patients with negative PSMA-PET/CT (48 months) compared to patients with a positive scan (24 months, p= 0.001). SUVmax ≥ 7.4 provides discrimination of patients with a poor prognosis. Results of PSMA-PET/CT changed treatment planning. Conclusions: in renal cancer, positive PSMA-PET/CT is strongly correlated to the intensity of PSMA expression on immunohistochemistry in both ccRCC and chromophobe cancer. PSMA-PET/CT signal predicts a poor prognosis confirming its potential as an aggressiveness biomarker and providing paramount additional information influencing patient management.

Retrospective immunophenotypical evaluation of MET, PD-1/PD-L1, and mTOR pathways in primary tumors and pulmonary metastases of renal cell carcinoma: the RIVELATOR study addresses the issue of biomarkers heterogeneity.

Aim: In renal cell carcinoma (RCC), tumor heterogeneity generated challenges to biomarker development and therapeutic management, often becoming responsible for primary and acquired drug resistance. This study aimed to assess the inter-tumoral, intra-tumoral, and intra-lesional heterogeneity of known druggable targets in metastatic RCC (mRCC). Methods: The RIVELATOR study was a monocenter retrospective analysis of biological samples from 25 cases of primary RCC and their paired pulmonary metastases. The biomarkers analyzed included MET, mTOR, PD-1/PD-L1 pathways and the immune context. Results: High multi-level heterogeneity was demonstrated. MET was the most reliable biomarker, with the lowest intratumor heterogeneity: the positive mutual correlation between MET expression in primary tumors and their metastases had a significantly proportional intensity (P = 0.038). The intratumor heterogeneity grade was significantly higher for the mTOR pathway proteins. Combined immunophenotypical expression patterns and their correlations with the immune context were uncovered [i.e., mTOR expression in the metastases positively correlated with PD-L1 expression in tumor-infiltrating lymphocytes (TILs), P = 0.019; MET expression was related to PD-1 expression on TILs (P = 0.041, ρ = 0.41) and peritumoral lymphocytes (RILs; P = 0.013, ρ = 0.49)], suggesting the possibility of predicting drug response or resistance to tyrosine kinase, mTOR, or immune checkpoint inhibitors. Conclusions: In mRCC, multiple and multi-level assays of potentially predictive biomarkers are needed for their reliable translation into clinical practice. The easy-to-use immunohistochemical method of the present study allowed the identification of different combined expression patterns, providing cues for planning the management of systemic treatment combinations and sequences in an mRCC patient population. The quantitative heterogeneity of the investigated biomarkers suggests that multiple intralesional assays are needed to consider the assessment reliable for clinical considerations.

RAD51 as a biomarker for homologous recombination deficiency in high-grade serous ovarian carcinoma: robustness and interobserver variability of the RAD51 test.

The RAD51 test is emerging as a promising biomarker for the assessment of functional homologous recombination deficiency (HRD). Yet, the robustness and reproducibility of the immunofluorescence-based RAD51 test, in different academic laboratories, have not been systematically investigated. Therefore, we tested the performance of the RAD51 assay in formalin-fixed paraffin-embedded (FFPE) high-grade serous ovarian carcinoma (HGSOC) samples in four European laboratories. Here, we confirm that subtle differences in staining procedures result in low variability of RAD51 and γH2AX scores. However, substantial variability in RAD51 scoring was observed in some samples, likely due to complicating technical and biological features, such as high RAD51 signal-to-noise ratio and RAD51 heterogeneity. These results support the need to identify and perform additional quality control steps and/or automating image analysis. Altogether, resolving technical issues should be a priority, as identifying tumours with functional HRD is urgently needed to guide the individual treatment of HGSOC patients. Follow-up studies are needed to define the key tissue quality requirements to assess HRD by RAD51 in FFPE tumour samples, as this test could help in guiding the individual treatment of HGSOC patients.

Notch-Jagged1 signaling and response to bevacizumab therapy in advanced colorectal cancer: A glance to radiomics or back to physiopathology?

Introduction: The Notch intracellular domain (NICD) and its ligands Jagged-1(Jag1), Delta-like ligand (DLL-3) and DLL4 play an important role in neoangiogenesis. Previous studies suggest a correlation between the tissue levels of NICD and response to therapy with bevacizumab in colorectal cancer (CRC). Another marker that may predict outcome in CRC is radiomics of liver metastases. The aim of this study was to investigate the expression of NICD and its ligands and the role of radiomics in the selection of treatment-naive metastatic CRC patients receiving bevacizumab. Methods: Immunohistochemistry (IHC) for NICD, Jag1 and E-cadherin was performed on the tissue microarrays (TMAs) of 111 patients with metastatic CRC treated with bevacizumab and chemotherapy. Both the intensity and the percentage of stained cells were evaluated. The absolute number of CD4+ and CD8+ lymphocytes was counted in three different high-power fields and the mean values obtained were used to determine the CD4/CD8 ratio. The positivity of tumor cells to DLL3 and DLL4 was studied. The microvascular density (MVD) was assessed in fifteen cases by counting the microvessels at 20x magnification and expressed as MVD score. Abdominal CT scans were retrieved and imported into a dedicated workstation for radiomic analysis. Manually drawn regions of interest (ROI) allowed the extraction of radiomic features (RFs) from the tumor. Results: A positive association was found between NICD and Jag1 expression (p < 0.001). Median PFS was significantly shorter in patients whose tumors expressed high NICD and Jag1 (6.43 months vs 11.53 months for negative cases; p = 0.001). Those with an MVD score ≥5 (CD31-high, NICD/Jag1 positive) experienced significantly poorer survival. The radiomic model developed to predict short and long-term survival and PFS yielded a ROC-AUC of 0.709; when integrated with clinical and histopathological data, the integrated model improved the predictive score (ROC-AUC of 0.823). Discussion: These results show that high NICD and Jag1 expression are associated with progressive disease and early disease progression to anti VEGF-based therapy; the preliminary radiomic analyses show that the integration of quantitative information with clinical and histological data display the highest performance in predicting the outcome of CRC patients.

COVID-19 in pregnancy: placental pathological patterns and effect on perinatal outcome in five cases.

INTRODUCTION: COVID-19, the disease caused by the novel coronavirus SARS-CoV-2, is a severe systemic thrombotic syndrome that emerged in 2019, with an ensuing pandemic. To evaluate the impact of this disease on placental tissue and perinatal outcome, histological, immunohistochemical and ultrastructural analyses of placental tissue were performed for five cases of pregnant women with COVID-19. CASE REPORTS: All five pregnant women in this series developed COVID-19 in late pregnancy. Two patients experienced respiratory distress, and computed tomography revealed signs of pneumonia, with bilateral involvement, multiple lobular and subsegmental areas of consolidation and ground-glass opacities. Histological studies of placental tissue revealed the presence of slight signs of maternal vascular underperfusion (MVUs) or foetal vascular underperfusion (FVUs) lesions and mild inflammatory lesions. CD15 immunoreactivity in the placental tissue was low in all cases, demonstrating that in these cases there was not severe foetal hypoxia/asphyxia risk for newborns or distal vascular immaturity. In all cases examined, ultrastructural analyses showed spherical-like coronavirus particles with an electron intermediate-density core as well as projections from the surface as spike-like structures in the syncytiotrophoblasts. At term, all of the women delivered newborns who were negative for SARS-CoV-2 by nasopharyngeal testing in their first day of life. All newborns were exclusively breastfed and were discharged on the 3rd day of life. CONCLUSIONS: In conclusion, placental patterns in pregnancy due to COVID-19 in the late stage of gestation indicate no evidence of vertical trans-placental SARS-CoV-2 transmission or a significant impact on the perinatal outcome of newborns, in both mild and more severe cases.

Small Cell Lung Cancer Transformation as a Resistance Mechanism to Osimertinib in Epidermal Growth Factor Receptor-Mutated Lung Adenocarcinoma: Case Report and Literature Review.

Introduction: Small cell lung cancer (SCLC) transformation represents a mechanism of resistance to osimertinib in EGFR-mutated lung adenocarcinoma, which dramatically impacts patients' prognosis due to high refractoriness to conventional treatments. Case Description: We present the case of a patient who developed a SCLC phenotypic transformation as resistance mechanism to second-line osimertinib for T790M-positive EGFR-mutated NSCLC. Our patient received platinum-etoposide doublet following SCLC switch and achieved a modest clinical benefit which lasted 4 months. NGS and IHC analyses for p53 and Rb were performed on subsequent liver biopsies, revealing baseline TP53 mutation and complete absence of p53 and Rb expression. Primary cell cultures were established following a liver biopsy at the time of SCLC transformation, and drug sensitivity assays showed meaningful cell growth inhibition when osimertinib was added to platinum-etoposide compared with control (p < 0.05). A review of the current literature regarding SCLC transformation after failure of osimertinib was performed. Conclusions: Based on retrospective data available to date, platinum-etoposide chemotherapy is the preferred treatment choice in the occurrence of SCLC transformation after osimertinib failure. The extension of osimertinib in combination with chemotherapy in the occurrence of SCLC transformation as resistance mechanism to osimertinib is a matter of debate. The combination of osimertinib and platinum-etoposide was effective in inhibiting cell growth in our primary cell cultures. Clinical studies are needed to further explore this combination in the occurrence of SCLC transformation as a resistance mechanism to osimertinib.

Case Report: Irreversible Watery Diarrhea, Severe Metabolic Acidosis, Hypokalemia and Achloridria Syndrome Related to Vasoactive Intestinal Peptide Secreting Malignant Pheochromocytoma.

Background: Pheochromocytoma (PHEO) clinical manifestations generally mirror excessive catecholamines secretion; rarely the clinical picture may reflect secretion of other hormones. Watery diarrhea, hypokalemia and achlorhydria (WDHA) is a rare syndrome related to excessive secretion of vasoactive intestinal peptide (VIP). Clinical Case: A 73-year-old hypotensive man affected by adrenal PHEO presented with weight loss and watery diarrhea associated with hypokalemia, hyperchloremic metabolic acidosis (anion gap 15 mmol/l) and a negative urinary anion gap. Abdominal computed tomography scan showed a right adrenal PHEO, 8.1 cm in maximum diameter, with tracer uptake on 68GaDOTA-octreotate positron emission tomography. Metastasis in lumbar region and lung were present. Both chromogranin A and VIP levels were high (more than10 times the normal value) with slightly elevated urine normetanephrine and metanephrine excretion. Right adrenalectomy was performed and a somatostatin analogue therapy with lanreotide started. Immunostaining showed chromogranin A and VIP co-expression, with weak somatostatin-receptor-2A positivity. In two months, patient clinical conditions deteriorated with severe WDHA and multiple liver and lung metastasis. Metabolic acidosis and hypokalemia worsened, leading to hemodynamic shock and exitus. Conclusions: A rare case of WDHA syndrome caused by malignant VIP-secreting PHEO was diagnosed. High levels of circulating VIP were responsible of the rapidly evolving clinical picture with massive dehydration and weight loss along with severe hyperchloremic metabolic acidosis and hypokalemia due to the profuse untreatable diarrhea. The rescue treatment with lanreotide was unsuccessful because of the paucity of somatostatin-receptor-2A on VIP-secreting PHEO chromaffin cells.

Neuroendocrine small cell carcinoma of the cervix: A case report.

Merkel cell polyomavirus (MCPyV) has been found in patients with Merkel cell carcinoma and respiratory tract infections. Merkel cell carcinoma is a primary aggressive neuroendocrine carcinoma of the skin. It has been demonstrated that MCPyV can be transmitted during sexual activity and may be present in the oral and anogenital mucosa. The aim of the present study was to evaluate whether MCPyV coexisted with HPV in three cases of neuroendocrine small cell carcinoma of the cervix using PCR and immunohistochemical analysis Three cases of NSC of the cervix were identified in the pathology archives of Parma University (Italy). Of these, two cases were associated with an adenocarcinomatous component. A set of general primers from the L1 region (forward, L1C1 and reverse, L1C2 or L1C2M) was PCR amplified to detect the broad-spectrum DNA of genital HPV. The presence of MCPyV was investigated via immunohistochemistry using a mouse monoclonal antibody against the MCPyV LT antigen and through PCR analysis to separate viral DNA. HPV DNA was present in all three neuroendocrine carcinomas and in the adenocarcinoma component of the two mixed cases. None of the cases were immunoreactive to CM2B4 and did not contain viral DNA in either their neuroendocrine or adenocarcinomatous component. Whilst it is difficult to draw definitive conclusions from such a small sample size, these data suggested that MCPyV does not coexist with HPV in the cervix. However, in the present study, the absence of detectable MCPyV may have been due to the presence of a genotype that was not detected by the primers used in the PCR analysis or by the antibody used for the immunohistochemical study. MCPyV microRNA may also have been present, inhibiting LT expression. Additional studies with larger cohorts and more advanced molecular biology techniques are required to confirm the hypothesis of the current study.

Myeloma Cells Deplete Bone Marrow Glutamine and Inhibit Osteoblast Differentiation Limiting Asparagine Availability.

Multiple myeloma (MM) cells consume huge amounts of glutamine and, as a consequence, the amino acid concentration is lower-than-normal in the bone marrow (BM) of MM patients. Here we show that MM-dependent glutamine depletion induces glutamine synthetase in stromal cells, as demonstrated in BM biopsies of MM patients, and reproduced in vitro by co-culturing human mesenchymal stromal cells (MSCs) with MM cells. Moreover, glutamine depletion hinders osteoblast differentiation of MSCs, which is also severely blunted by the spent, low-glutamine medium of MM cells, and rescued by glutamine restitution. Glutaminase and the concentrative glutamine transporter SNAT2 are induced during osteoblastogenesis in vivo and in vitro, and both needed for MSCs differentiation, pointing to enhanced the requirement for the amino acid. Osteoblastogenesis also triggers the induction of glutamine-dependent asparagine synthetase (ASNS), and, among non-essential amino acids, asparagine rescues differentiation of glutamine-starved MSCs, by restoring the transcriptional profiles of differentiating MSCs altered by glutamine starvation. Thus, reduced asparagine availability provides a mechanistic link between MM-dependent Gln depletion in BM and impairment of osteoblast differentiation. Inhibition of Gln metabolism in MM cells and supplementation of asparagine to stromal cells may, therefore, constitute novel approaches to prevent osteolytic lesions in MM.

Bovine pestivirus is a new alternative virus for multiple myeloma oncolytic virotherapy.

BACKGROUND: The oncolytic viruses have shown promising results for the treatment of multiple myeloma. However, the use of human viruses is limited by the patients' antiviral immune response. In this study, we investigated an alternative oncolytic strategy using non-human pathogen viruses as the bovine viral diarrhea virus (BVDV) that were able to interact with CD46. METHODS: We treated several human myeloma cell lines and non-myeloma cell lines with BVDV to evaluate the expression of CD46 and to study the effect on cell viability by flow cytometry. The possible synergistic effect of bortezomib in combination with BVDV was also tested. Moreover, we infected the bone marrow mononuclear cells obtained from myeloma patients and we checked the BVDV effect on different cell populations, defined by CD138, CD14, CD3, CD19, and CD56 expression evaluated by flow cytometry. Finally, the in vivo BVDV effect was tested in NOD-SCID mice injected subcutaneously with myeloma cell lines. RESULTS: Human myeloma cells were selectively sensitive to BVDV treatment with an increase of cell death and, consequently, of apoptotic markers. Consistently, bone marrow mononuclear cells isolated from myeloma patients treated with BVDV, showed a significant selective decrease of the percentage of viable CD138+ cells. Interestingly, bortezomib pre-treatment significantly increased the cytotoxic effect of BVDV in myeloma cell lines with a synergistic effect. Finally, the in vitro data were confirmed in an in vivo myeloma mouse model showing that BVDV treatment significantly reduced the tumoral burden compared to the vehicle. CONCLUSIONS: Overall, our data indicate, for the first time, a direct oncolytic effect of the BVDV in human myeloma cells suggesting its possible use as novel alternative anti-myeloma virotherapy strategy.

Immunohistochemical expression of Napsin A in normal human fetal lungs at different gestational ages and in acquired and congenital pathological pulmonary conditions.

Surfactant protein B (SP-B) is a key component of pulmonary surfactant. SP-B is processed to a mature, surface-active protein from a pro-peptide by two distinct cleavage events in its N-terminal and C-terminal regions. Napsin A, a protease expressed in type II pneumocytes, is responsible for the N-terminal cleavage event. Here, for the first time, we have evaluated the expression of Napsin A in normal fetal lungs at different gestational ages and in lungs from fetuses and neonates with congenital and acquired pathological pulmonary conditions. Lung samples were collected from fetal and neonatal autopsies at the Department of Medicine and Surgery's Pathology Unit of Parma University (Italy). Immunohistochemical analysis was performed using a primary anti-Napsin A (clone IP64 clone) monoclonal antibody. A section of lung adenocarcinoma was used as an external positive control. Napsin A was expressed early in normal fetal lungs throughout the epithelium of the distal pseudoglandular tracts. In fetuses at 30 weeks of gestation and term newborns, Napsin A was already expressed only in isolated cells within the alveolar epithelium, similar to adult subjects. Furthermore, increased expression of Napsin A compared with a control group was observed in lung tissue from fetuses and a newborn with pathological conditions (inflammatory diseases and pulmonary hypoplasia). In conclusion, this study demonstrates that Napsin A is produced early in fetal life, and that its production is increased in many diseases, presumably in an effort to remedy functional pulmonary failure.

Optimizing PD-L1 evaluation on cytological samples from advanced non-small-cell lung cancer.

Aim: Programmed cell death-ligand 1 (PD-L1) predicts response to immune checkpoint inhibitors in non-small-cell lung cancer (NSCLC) patients. Most NSCLCs are diagnosed at an advanced stage and using minimally invasive diagnostic procedures that yield small biopsies or cytological samples. Methods: Cytological smears and paired histological samples from 52 advanced NSCLC patients were tested for PD-L1 expression by immunocyto/histochemistry (ICC/IHC) and for PD-L1 gene status by FISH. Results:PD-L1 was overexpressed in 9/52 (17%) cytological samples and in seven (13.5%) matched biopsies. The concordance between immunocytochemistry and IHC was 92.3% (48/52; p < 0.001). The concordance between PD-L1 gene status on cytology and histology was 69.2% (18/26; p < 0.001). No correlation between IHC and fluorescence in situ hybridization results was found. Conclusion: Our data support the feasibility and reliability of PD-L1 protein and PD-L1 gene assessment on direct cytological smears from NSCLC patients whenever histological sample are inadequate.

Immune context characterization and heterogeneity in primary tumors and pulmonary metastases from renal cell carcinoma.

AIM: The knowledge of the immune context of renal cell carcinoma (RCC) is useful to predict benefit from immunotherapy. We retrospectively characterized the immune context of RCC patients underwent primary nephrectomy and pulmonary metastasectomy. MATERIALS & METHODS: Intratumoral infiltrating lymphocytes and peritumoral renal infiltrating lymphocytes, lymphocyte subpopulations (CD4+, CD8+), PD-1, PD-L1 were explored in paired samples of primary RCC (T) and respective pulmonary metastases (M). RESULTS: The immune variables demonstrated intralesional and intratumoral heterogeneity. Intralesional lymphocyte heterogeneity reached 76% of cases in T, 28% in M. The heterogeneity rate for PD-L1 expression was from 44% (T) to 56% (M); it correlated with better survival. CONCLUSION: The immune context of RCC is highly variable both within a given tumor and among primary and metastases.

Correlations between tumor-infiltrating and circulating lymphocyte subpopulations in advanced renal cancer patients treated with nivolumab.

BACKGROUND: In clinical trials with immunotherapy, histological features such as tumor-infiltrating lymphocytes (TILs) are investigated as potential predictive biomarkers, with the limit of an outdated parameter for a typically dynamic element. METHODS: This explorative study compared, in metastatic renal cell carcinoma (mRCC) patients, basal pathological data about TILs on diagnostic histological specimens with circulating lymphocyte subpopulations measured before and during therapy with nivolumab. RESULTS: Of 11 mRCC patients, 5 had low presence of TILs (L-TILs), 3 moderate amount (M-TILs) and 3 high number (H-TILs). Overall, 8 patients had low intratumoral pathological CD4+/CD8+ ratio (LIPR) ≤1 and 3 cases high intratumoral pathological ratio (HIPR) ≥2. Of 8 patients with LIPR, only 2 matched with low circulating CD4+/CD8+ ratio (LCR) ≤1; 5 had high circulating ratio (HCR) ≥2. All 3 cases with HIPR (≥2) conversely had LCR (≤1). Circulating CD4+/CD8+ ratio remained unchanged during therapy (mean -0.12 in 8 weeks). The respective percentage values of CD4+ and CD8+ circulating T cells also remained stable (variation 0%); the absolute value of CD4+ was more likely to increase (mean +46.3/mm3); the level of CD8+ tended to slightly decrease (mean -6.5/mm3). No correlation of lymphocyte subpopulations with treatment outcome was found. Of note, we did not evidence correspondence between histopathological and circulating findings in terms of T-lymphocyte subpopulations, also suggesting the inconsistency of circulating data in terms of relative variations. CONCLUSIONS: Considering the likely high dynamism of TILs, rebiopsy before therapy might be proposed to assess the utility of TILs characterization for predictive purpose. (www.actabiomedica.it).

Loss of heterozygosity of key tumor suppressor genes in advanced renal cancer patients treated with nivolumab.

AIM: We studied the possible clinical significance of loss of heterozygosity (LOH) at key tumor suppressor genes loci in advanced renal cancer patients treated with nivolumab. METHODS: LOH study was performed on 3p14.2 (FHIT gene); 3p21.3-21.2; 9p21 (BDMF gene); 9p22 (SH3GL2 gene). RESULTS: Of 12 patients, 8 (67%) had LOH. The most affected gene was FHIT. All five patients with LOH at FHIT locus had good outcome, mean progression free survival of 6.8 months. The patients LOH negative at FHIT locus had mean progression free survival of 4 months, 67% were treatment refractory. Overall, 75% of patients with LOH of at least one gene had benefit; 75% of LOH negative cases were refractory. CONCLUSION: LOH at key tumor suppressor genes should be further investigated as predictive for immunotherapy.

Immunohistochemical Detection of Hematopoietic Cell-specific Protein-Tyrosine Phosphatase (Tyrosine Phosphatase SHP-1) in a Series of Endometrioid and Serous Endometrial Carcinoma.

In this study, we evaluated the expression of SHP-1 (PTPN6) in endometrioid (Ec) and serous (Sc) subtypes of endometrial carcinoma by immunohistochemical analysis. In total, 114 patients with Ec carcinoma and 48 patients with Sc carcinoma were enrolled in this study. The correlation between the type of histology, the grade of tumor, the stage of development, and immunoreactivity to SHP-1 was evaluated. Kaplan-Meier and multivariate survival analyses, using a Cox regression model, were performed to establish whether this marker has prognostic value in these malignancies, on the basis of follow-up and stratification of the patients according to their SHP-1 immunoreactivity. A significantly higher SHP-1 expression was observed in the Ec group compared with the Sc group (P=0.0005, Fisher exact test). In the Ec group, SHP-1 immunoreactivity was correlated with grading, demonstrating that more differentiated lesions expressed SHP-1 more frequently than less differentiated neoplasms (G1 vs. G2, P=0.0243, statistically significant value, Fisher exact test; G1 vs. G3, P=0.0088, extremely significant value, Fisher exact test). Instead, in the Sc group, SHP-1 expression was not correlated with grading, as Sc is now defined as a high-grade carcinoma. SHP-1 expression did not change with neoplastic progression in Ec and Sc groups. From both univariate and multivariate analysis in the Ec group, expression of SHP-1 remained a positive prognostic factor (P=0.004, log-rank test) [HR=0.32 (0.11 to 0.94), P=0.039]. In contrast, in the Sc group, no correlation between SHP-1 expression and survival was noted (P=0.77, log-rank test). In this study, we observed that the absence of SHP-1 in immunohistochemical analysis might serve as a marker of poor prognosis for a subset of high-grade endometrial cancer.

Pulmonary metastasectomy for colorectal cancer: analysis of prognostic factors affecting survival.

BACKGROUND: Pulmonary metastasectomy is considered a standard procedure in the treatment of metastatic colorectal cancer (CRC). Different prognostic factors including multiple metastatic nodules, the presence of extra-pulmonary metastases and BRAF mutation status have been associated with poor survival. The aim of this study was to evaluate which factors influenced survival in CRC patients undergoing pulmonary metastasectomy by studying primary tumors and pulmonary metastases. METHODS: All patients treated for primary CRC who presented pulmonary metastases in a 10-year period were considered (group A). A control group treated for primary CRC who did not develop any pulmonary or extra-pulmonary metastases was taken for comparison (group B). Different prognostic factors including gender, age, tumor location, histological type, inflammatory infiltrate, BRAF, CDX2 and extra-pulmonary metastases were analyzed. Overall survival (OS) and patients' survival after pulmonary metastasectomy were also considered. RESULTS: Fifty-four patients were evaluated in group A and twenty-three in group B. In group A, BRAF immunohistochemistry did not significantly differ between primary tumors and pulmonary metastases; no difference of BRAF expression was found between group A and B. Even the expression of CDX2 was not significantly different in primary tumors and metastases. Similarly, in group B CDX2 did not significantly differ from primary CRC of group A. The most significant prognostic factor was the presence of extra-pulmonary metastases. Patients with extra-pulmonary metastases experienced a significant shorter survival compared to patients with pulmonary metastases alone (P=0.001 with log-rank test vs. P=0.003 with univariate Cox regression). Interestingly, patients with right pulmonary metastases presented a significant longer survival than those with left pulmonary metastases (P=0.027 with log-rank test vs. 0.04 with univariate Cox regression). CONCLUSIONS: The main prognostic factor associated with poor survival after lung resection of CRC metastases is a history of extra-pulmonary metastases. BRAF and CDX2 did not have a significant role in this small series of patients.