Immune Phenotype and Response to Neoadjuvant Therapy in Triple-Negative Breast Cancer.

PURPOSE: Increasing tumor-infiltrating lymphocytes (TIL) is associated with higher rates of pathologic complete response (pCR) to neoadjuvant therapy (NAT) in patients with triple-negative breast cancer (TNBC). However, the presence of TILs does not consistently predict pCR, therefore, the current study was undertaken to more fully characterize the immune cell response and its association with pCR. EXPERIMENTAL DESIGN: We obtained pretreatment core-needle biopsies from 105 patients with stage I-III TNBC enrolled in ARTEMIS (NCT02276443) who received NAT from Oct 22, 2015 through July 24, 2018. The tumor-immune microenvironment was comprehensively profiled by performing T-cell receptor (TCR) sequencing, programmed death-ligand 1 (PD-L1) IHC, multiplex immunofluorescence, and RNA sequencing on pretreatment tumor samples. The primary endpoint was pathologic response to NAT. RESULTS: The pCR rate was 40% (42/105). Higher TCR clonality (median = 0.2 vs. 0.1, P = 0.03), PD-L1 positivity (OR: 2.91, P = 0.020), higher CD3+:CD68+ ratio (median = 14.70 vs. 8.20, P = 0.0128), and closer spatial proximity of T cells to tumor cells (median = 19.26 vs. 21.94 µm, P = 0.0169) were associated with pCR. In a multivariable model, closer spatial proximity of T cells to tumor cells and PD-L1 expression enhanced prediction of pCR when considered in conjunction with clinical stage. CONCLUSIONS: In patients receiving NAT for TNBC, deep immune profiling through detailed phenotypic characterization and spatial analysis can improve prediction of pCR in patients receiving NAT for TNBC when considered with traditional clinical parameters.

Relevance of PD-L1 Non-Coding Polymorphisms on the Prognosis of a Genetically Admixed NSCLC Cohort.

PURPOSE: Although non-small cell lung cancer (NSCLC) remains a deadly disease, new predictive biomarkers have emerged to assist in managing the disease, of which one of the most promising is the programmed death-ligand 1 (PD-L1). Each, PD-L1 variant seem to modulate the function of immune checkpoints differently and affect response to adjuvant treatment and outcome in NSCLC patients. We thus investigated the influence of these PD-L1 genetic variations in genetically admixed NSCLC tissue samples, and correlated these values with clinicopathological characteristics, including prognosis. MATERIALS AND METHODS: We evaluated PD-L1 non-coding genetic variants and protein expression in lung adenocarcinomas (ADC), squamous cell carcinomas (SqCC), and large cell carcinomas (LCC) in silico. Microarray paraffin blocks from 70 samples of ADC (N=33), SqCC (N=24), and LCC (N=13) were used to create PD-L1 multiplex immunofluorescence assays with a Cell Signaling E1L3N clone. Fifteen polymorphisms of the PD-L1 gene were investigated by targeted sequencing and evaluated in silico using dedicated tools. RESULTS: Although PD-L1 polymorphisms seemed not to interfere with protein expression, PD-L1 expression varied among different histological subtypes, as did clinical outcomes, with the rs4742098A>G, rs4143815G>C, and rs7041009G>A variants being associated with relapse (P=0.01; P=0.05; P=0.02, respectively). The rs7041009 GG genotype showed a significant correlation with younger and alive patients compared to carriers of the A allele (P=0.02 and P<0.01, respectively). The Cox regression model showed that the rs7041009 GG genotype may influence OS (P<0.01) as a co-dependent factor associated with radiotherapy and recurrence in NSCLC patients. Furthermore, the Kaplan-Meier survival curves showed that rs7041009 and rs4742098 might impact PPS in relapsed patients. In silico approaches identified the variants as benign. CONCLUSION: PD-L1 non-coding variants play an important role in modulating immune checkpoint function and may be explored as immunotherapy biomarkers. We highlight the rs7041009 variant, which impacts OS and PPS in NSCLC patients.

Development and Validation of a Predictive Radiomics Model for Clinical Outcomes in Stage I Non-small Cell Lung Cancer.

PURPOSE: To develop and validate a radiomics signature that can predict the clinical outcomes for patients with stage I non-small cell lung cancer (NSCLC). METHODS AND MATERIALS: We retrospectively analyzed contrast-enhanced computed tomography images of patients from a training cohort (n = 147) treated with surgery and an independent validation cohort (n = 295) treated with stereotactic ablative radiation therapy. Twelve radiomics features with established strategies for filtering and preprocessing were extracted. The random survival forests (RSF) method was used to build models from subsets of the 12 candidate features based on their survival relevance and generate a mortality risk index for each observation in the training set. An optimal model was selected, and its ability to predict clinical outcomes was evaluated in the validation set using predicted mortality risk indexes. RESULTS: The optimal RSF model, consisting of 2 predictive features, kurtosis and the gray level co-occurrence matrix feature homogeneity2, allowed for significant risk stratification (log-rank P < .0001) and remained an independent predictor of overall survival after adjusting for age, tumor volume and histologic type, and Karnofsky performance status (hazard ratio [HR] 1.27; P < 2e-16) in the training set. The resultant mortality risk indexes were significantly associated with overall survival in the validation set (log-rank P = .0173; HR 1.02, P = .0438). They were also significant for distant metastasis (log-rank P < .05; HR 1.04, P = .0407) and were borderline significant for regional recurrence on univariate analysis (log-rank P < .05; HR 1.04, P = .0617). CONCLUSIONS: Our radiomics model accurately predicted several clinical outcomes and allowed pretreatment risk stratification in stage I NSCLC, allowing the choice of treatment to be tailored to each patient's individual risk profile.

Cancer-Associated Fibroblasts Induce a Collagen Cross-link Switch in Tumor Stroma.

UNLABELLED: Intratumoral collagen cross-links heighten stromal stiffness and stimulate tumor cell invasion, but it is unclear how collagen cross-linking is regulated in epithelial tumors. To address this question, we used Kras(LA1) mice, which develop lung adenocarcinomas from somatic activation of a Kras(G12D) allele. The lung tumors in Kras(LA1) mice were highly fibrotic and contained cancer-associated fibroblasts (CAF) that produced collagen and generated stiffness in collagen gels. In xenograft tumors generated by injection of wild-type mice with lung adenocarcinoma cells alone or in combination with CAFs, the total concentration of collagen cross-links was the same in tumors generated with or without CAFs, but coinjected tumors had higher hydroxylysine aldehyde-derived collagen cross-links (HLCC) and lower lysine-aldehyde-derived collagen cross-links (LCCs). Therefore, we postulated that an LCC-to-HLCC switch induced by CAFs promotes the migratory and invasive properties of lung adenocarcinoma cells. To test this hypothesis, we created coculture models in which CAFs are positioned interstitially or peripherally in tumor cell aggregates, mimicking distinct spatial orientations of CAFs in human lung cancer. In both contexts, CAFs enhanced the invasive properties of tumor cells in three-dimensional (3D) collagen gels. Tumor cell aggregates that attached to CAF networks on a Matrigel surface dissociated and migrated on the networks. Lysyl hydroxylase 2 (PLOD2/LH2), which drives HLCC formation, was expressed in CAFs, and LH2 depletion abrogated the ability of CAFs to promote tumor cell invasion and migration. IMPLICATIONS: CAFs induce a collagen cross-link switch in tumor stroma to influence the invasive properties of tumor cells.

Effects of cilostazol in kidney and skeletal striated muscle of Wistar rats submitted to acute ischemia and reperfusion of hind limbs.

PURPOSE: To investigate the effect of cilostazol, in kidney and skeletal muscle of rats submitted to acute ischemia and reperfusion. METHODS: Fourty three animals were randomized and divided into two groups. Group I received a solution of cilostazol (10 mg/Kg) and group II received saline solution 0.9% (SS) by orogastric tube after ligature of the abdominal aorta. After four hours of ischemia the animals were divided into four subgroups: group IA (Cilostazol): two hours of reperfusion. Group IIA (SS): two hours of reperfusion. Group IB (Cilostazol): six hours of reperfusion. Group IIB (SS) six hours of reperfusion. After reperfusion, a left nephrectomy was performed and removal of the muscles of the hind limb. The histological parameters were studied. In kidney cylinders of myoglobin, vacuolar degeneration and acute tubular necrosis. In muscle interstitial edema, inflammatory infiltrate, hypereosinophilia fiber, cariopicnose and necrosis. Apoptosis was assessed by immunohistochemistry for cleaved caspase-3 and TUNEL. RESULTS: There was no statistically significant difference between groups. CONCLUSION: Cilostazol had no protective effect on the kidney and the skeletal striated muscle in rats submitted to acute ischemia and reperfusion in this model.

Effects of cilostazol in kidney and skeletal striated muscle of Wistar rats submitted to acute ischemia and reperfusion of hind limbs / Efeitos do cilostazol em rim e musculatura estriada esquelética de ratos Wistar submetidos à isquemia aguda e reperfusão de membros posteriores

PURPOSE: To investigate the effect of cilostazol, in kidney and skeletal muscle of rats submitted to acute ischemia and reperfusion. METHODS: Fourty three animals were randomized and divided into two groups. Group I received a solution of cilostazol (10 mg/Kg) and group II received saline solution 0.9% (SS) by orogastric tube after ligature of the abdominal aorta. After four hours of ischemia the animals were divided into four subgroups: group IA (Cilostazol): two hours of reperfusion. Group IIA (SS): two hours of reperfusion. Group IB (Cilostazol): six hours of reperfusion. Group IIB (SS) six hours of reperfusion. After reperfusion, a left nephrectomy was performed and removal of the muscles of the hind limb. The histological parameters were studied. In kidney cylinders of myoglobin, vacuolar degeneration and acute tubular necrosis. In muscle interstitial edema, inflammatory infiltrate, hypereosinophilia fiber, cariopicnose and necrosis. Apoptosis was assessed by immunohistochemistry for cleaved caspase-3 and TUNEL. RESULTS: There was no statistically significant difference between groups. CONCLUSION: Cilostazol had no protective effect on the kidney and the skeletal striated muscle in rats submitted to acute ischemia and reperfusion in this model.

OBJETIVO: Investigar o efeito do cilostazol no rim e na musculatura esquelética de ratos submetidos à isquemia aguda e reperfusão. MÉTODOS: Quarenta e três animais foram aleatoriamente distribuídos em dois grupos. Grupo I recebeu solução de cilostazol (10 mg/Kg) e Grupo II recebeu solução fisiológica a 0,9% (SF), após ligadura da aorta abdominal. Decorridas quatro horas de isquemia os animais foram distribuídos em quatro subgrupos: Grupo IA (Cilostazol): duas horas de reperfusão. Grupo IIA (SF): duas horas de reperfusão. Grupo IB (Cilostazol): seis horas de reperfusão. Grupo IIB (SF): seis horas de reperfusão. Após a reperfusão, realizou-se nefrectomia esquerda e a retirada da musculatura de membro posterior. Os parâmetros histológicos estudados em rim foram cilindros de mioglobina, degeneração vacuolar e necrose tubular. Em músculo foram edema, infiltrado inflamatório, hipereosinofilia de fibras, cariopicnose e necrose. A apoptose foi avaliada por imunohistoquímica, através da caspase-3 clivada e TUNEL. RESULTADOS: Não houve diferença estatisticamente significante entre os grupos estudados. CONCLUSÃO: O cilostazol não teve efeito protetor sobre o rim e sobre a musculatura estriada esquelética em ratos Wistar submetidos à isquemia aguda e reperfusão no modelo estudado.

Pneumonias intersticiais idiopáticas: da patogênese e do remodelamento aos determinantes anátomo-clínico-radiológicos de prognóstico e sobrevida com ênfase ao componente vascular / Idiopathic interstitial pneumonias: pathogenesis and remodeling for determinants anathomic-clinical-radiologic of prognosthic and survival enfaticed of vascular component

Estudou-se por morfologia, morfometria e imuno-histoquímica o remodelamento vascular (moléculas de adesão), epitelial (moléculas de adesão) e intersticial (colágeno V e células imunes) nos três tipos maiores de pneumonias intersticiais idiopáticas: em 62 casos de IPF, 22 casos de NSIP e 25 casos de AIP. O impacto dessas alterações foi avaliado nas provas de função, sobrevida e prognóstico. Demonstrou-se que o remodelamento vascular ativo e fibroelastótico é diretamente proporcional ao grau de atividade parenquimatosa principalmente na UIP. O colágeno V, o mapeamento das células imunes, o aumento da atividade endotelial e epitelial tiveram impacto no espectro diferencial e possivelmente na patogênese das três pneumonias intersticiais estudadas. A resposta imune celular na UIP teve impacto na sobrevida dos pacientes / Studied for morphology, morphometry and immunohischemistry the vascular (adhesion molecules), epithelial (adhesion molecules) and interstitial (collagen V and immune cells) remodeling in the three major types of idiopathic interstitial pneumonias: in 62 cases of IPF, 22 cases of NSIP, and 25 cases of AIP. The impact of these alterations was evaluated in the function tests, survival and prognostic. We demonstrated that the active and fibroelastotic vascular remodeling is directly proportional to the degree of parenchymal activity, mainly in the UIP. Collagen V, mapping of the immune cells, increase of the endothelial and epithelial activity had possibly impact in the distinguishing specter and in pathogenesis of the three interstitial pneumonias studied. The cellular immune reply in the UIP it had impact in survival of the patients...

Angiogenesis as an indicator of metastatic potential in papillary thyroid carcinoma.

UNLABELLED: Angiogenesis is new blood vessel formation, a process that can lead to tumor development. Microvessel count has been correlated to metastasis in some neoplasias. PURPOSE: To determine if measurement of microvessel density is useful in predicting metastasis to the cervical lymph node and prognosis in patients with papillary thyroid carcinoma. METHODS: A retrospective analysis was performed in 30 patients that had undergone total thyroidectomy. They were divided in 2 groups of 15 patients--with and without metastatic disease. Immunohistochemistry was used to detect expression of CD34 in archival paraffin-embedded papillary thyroid tumors, and microvessel density was calculated based on it. Association between microvessel density and the presence of metastasis, according to histological subtype, disease recurrence, and AMES prognostic index groups was determined through statistical analysis. RESULTS: The median microvessel density for the patient group without metastasis (200.0 microvessels/mm2) was apparently, but not significantly, less than that observed among metastatic disease patients (254.4 microvessels/mm2) (P=.20). When papillary carcinoma subtypes were analyzed, this difference became significant (P=02). The follicular variant exhibited a greater microvessel density than the other subtypes, independent of metastasis presence. There was an apparent, but not significant, tendency for a larger median microvessel density in the group of patients that presented recurrence (294.4 microvessels/mm2 vs 249.6 microvessels/mm2, P=.11). There was no relationship between risk level and microvessel density: in the low- and high-risk groups, the median MVD was 304.0 microvessels/mm2 and 229.6 microvessels/mm2, respectively (P=.27). CONCLUSIONS: The results suggest that angiogenesis is more intense among metastatic tumors in the classic and the tall cell variants, indicating that microvessel count can be an indicator of the potential for metastasis in these subtypes of papillary thyroid carcinoma. Patients that developed recurrent disease had a tendency to exhibit higher angiogenesis; however, there was no association between microvessel density and the AMES prognostic index.

Angiogenesis as an indicator of metastatic potential in papillary thyroid carcinoma

O desenvolvimento dos tumores depende da formação de neovasos, a angiogênese. Em algumas neoplasias, a alta densidade de microvasos tumorais correlaciona-se com a presença de metástase. OBJETIVO: Determinar se a medida da angiogênese pode indicar o potencial de metástase e o prognóstico do carcinoma papilífero tireóideo. MÉTODO: Foi feita análise retrospectiva de 30 tireoidectomizados, divididos em dois grupos de 15 indivíduos cada, respectivamente com e sem metástase. A partir dos blocos de parafina, foi calculada a densidade de microvasos no tecido tumoral por meio da quantificação da expressão do anticorpo CD34 pela imunohistoquímica. A associação da densidade de microvasos com a presença de metástase, ocorrência de recidiva e os grupos de risco do índice prognóstico AMES foi determinada por análise estatística. RESULTADOS: A mediana da densidade de microvasos no grupo de doentes sem metástase (200,0 microvasos/mm2) foi inferior àquela dos portadores de metástase (254,4 microvasos/mm2) (p = .2), sem atingir significância estatística. Ao considerar apenas os subtipos histológicos clássico e de células altas, essa diferença tornou-se significante (p = .02), uma vez que a variante folicular exibiu maior DMV que os demais subtipos, independente da presença de metástase. Houve tendência não significativa à maior densidade de microvasos entre aqueles que apresentaram recidiva (294,4 microvasos/mm2 contra 249,6 microvasos/mm2, p = .11). Nos grupos de baixo e alto risco, a mediana da densidade de microvasos foi de 304,0 microvasos/mm2 e 229,6 microvasos/mm2 respectivamente (p = .27). CONCLUSAO: A angiogênese foi mais intensa nos tumores com metástase nos subtipos clássico e de células altas, sugerindo que a contagem de microvasos pode ser um indicador do potencial de metástase nestes subtipos histológicos do carcinoma papilífero tireóideo. Doentes que evoluíram com recidiva tenderam a exibir maior angiogênese, porém não houve associação da densidade de microvasos e o índice prognóstico.