PD-L1 quantification across tumor types using the reverse phase protein microarray: implications for precision medicine.

BACKGROUND: Anti-programmed cell death protein 1 and programmed cell death ligand 1 (PD-L1) agents are broadly used in first-line and second-line treatment across different tumor types. While immunohistochemistry-based assays are routinely used to assess PD-L1 expression, their clinical utility remains controversial due to the partial predictive value and lack of standardized cut-offs across antibody clones. Using a high throughput immunoassay, the reverse phase protein microarray (RPPA), coupled with a fluorescence-based detection system, this study compared the performance of six anti-PD-L1 antibody clones on 666 tumor samples. METHODS: PD-L1 expression was measured using five antibody clones (22C3, 28-8, CAL10, E1L3N and SP142) and the therapeutic antibody atezolizumab on 222 lung, 71 ovarian, 52 prostate and 267 breast cancers, and 54 metastatic lesions. To capture clinically relevant variables, our cohort included frozen and formalin-fixed paraffin-embedded samples, surgical specimens and core needle biopsies. Pure tumor epithelia were isolated using laser capture microdissection from 602 samples. Correlation coefficients were calculated to assess concordance between antibody clones. For two independent cohorts of patients with lung cancer treated with nivolumab, RPPA-based PD-L1 measurements were examined along with response to treatment. RESULTS: Median-center PD-L1 dynamic ranged from 0.01 to 39.37 across antibody clones. Correlation coefficients between the six antibody clones were heterogeneous (range: -0.48 to 0.95) and below 0.50 in 61% of the comparisons. In nivolumab-treated patients, RPPA-based measurement identified a subgroup of tumors, where low PD-L1 expression equated to lack of response. CONCLUSIONS: Continuous RPPA-based measurements capture a broad dynamic range of PD-L1 expression in human specimens and heterogeneous concordance levels between antibody clones. This high throughput immunoassay can potentially identify subgroups of tumors in which low expression of PD-L1 equates to lack of response to treatment.

Novel Epigenetic Eight-Gene Signature Predictive of Poor Prognosis and MSI-Like Phenotype in Human Metastatic Colorectal Carcinomas.

Epigenetics is involved in tumor progression and drug resistance in human colorectal carcinoma (CRC). This study addressed the hypothesis that the DNA methylation profiling may predict the clinical behavior of metastatic CRCs (mCRCs). The global methylation profile of two human mCRC subgroups with significantly different outcome was analyzed and compared with gene expression and methylation data from The Cancer Genome Atlas COlon ADenocarcinoma (TCGA COAD) and the NCBI GENE expression Omnibus repository (GEO) GSE48684 mCRCs datasets to identify a prognostic signature of functionally methylated genes. A novel epigenetic signature of eight hypermethylated genes was characterized that was able to identify mCRCs with poor prognosis, which had a CpG-island methylator phenotype (CIMP)-high and microsatellite instability (MSI)-like phenotype. Interestingly, methylation events were enriched in genes located on the q-arm of chromosomes 13 and 20, two chromosomal regions with gain/loss alterations associated with adenoma-to-carcinoma progression. Finally, the expression of the eight-genes signature and MSI-enriching genes was confirmed in oxaliplatin- and irinotecan-resistant CRC cell lines. These data reveal that the hypermethylation of specific genes may provide prognostic information that is able to identify a subgroup of mCRCs with poor prognosis.

Uncommon frame-shift exon 19 EGFR mutations are sensitive to EGFR tyrosine kinase inhibitors in non-small cell lung carcinoma.

Exons 19-21 EGFR activating mutations are predictive biomarkers of response to EGFR tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC). However, uncommon exon 19 EGFR mutations, due to their low frequency, have an uncertain biological and clinical significance and very little is known about their TKI sensitivity. This study was designed to describe the TKI sensitivity of a small cohort of lung adenocarcinomas bearing uncommon exon 19 mutations and to evaluate in silico the correlation between frame-shift exon 19 mutations and EGFR sequence/structure modification. Among 1168 NSCLCs screened for EGFR mutational status in our Institutions between 2011 and 2016, seven uncommon exon 19 EGFR mutations were further evaluated: five complex mutations, characterized by a deletion followed by a single-nucleotide insertion, a macrodeletion of 25 bp, and a 19 bp duplication. Interestingly, three patients harboring frame-shift mutations (i.e., one complex mutation, the macrodeletion, and the duplication) showed disease stability and considerably long PFS and OS upon TKI therapy. By contrast, three patients with in-frame complex deletions, independently of the mutation starting point, showed poor/lack of response to TKI therapy. In silico structural analysis showed that sensitivity to TKIs correlates with structural changes in the length and conformation of EGFR C-helix in frame-shift mutations. These data suggest that not all uncommon exon 19 EGFR mutations have the same TKI sensitivity and that frame-shift mutations are responsive to TKIs therapy.

[F-18] FDG-PET/CT parameters as predictors of outcome in inoperable NSCLC patients.

BACKGROUND: We evaluated the prognostic significance of standardized uptake value (SUVmax), metabolic tumour volume (MTV), and total lesion glycolysis (TLG) in [F-18] FDG PET/CT findings in patients with inoperable non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS: One hundred and three patients (mean age, 65.6 ± 16 years) underwent [F-18] FDG PET/CT before the chemotherapy. The SUVmax value, the MTV (cm(3); 42% threshold) and the TLG (g) were registered. The patients were followed up to 18 months thereafter (range 12-55 months). Failure to respond without progression, progression and/or disease-related death constituted surrogate end-points. The optimal SUVmax, MTV and TLG cut-off to predict the patients' outcome were estimated. PET/CT results were then related to disease outcome (progression free survival; PFS). RESULTS: The Kaplan-Meier survival analysis for SUVmax showed a significant shorter PFS in patients presenting with lower values as compared to those with higher (p < 0.05, log-rank test). MTV and TLG were not suitable for predicting PFS apart from the subset of patients with mediastinal nodal involvement. CONCLUSIONS: Despite the availability of new tools for the quantitative assessment of disease activity on PET/CT, the SUVmax rather than MTV and TLG remains the only predictor for PFS in NSCLC patients. MTV holds a value only when concomitant nodal involvement occurs.

Pyrosequencing evaluation of low-frequency KRAS mutant alleles for EGF receptor therapy selection in metastatic colorectal carcinoma.

AIM: To evaluate whether pyrosequencing (PS) improves the KRAS mutational status predictive value. PATIENTS & METHODS: A retrospective analysis of KRAS mutations by PS and direct sequencing (DS) in 192 metastatic colorectal carcinomas (mCRCs), subgrouped in 51 KRAS mutated at PS and 141 KRAS wild-type at DS. RESULTS: DS failed to detect low-frequency KRAS mutations in four out of 51 mCRCs, whereas PS detected 12 additional low-frequency KRAS mutations in 141 mCRCs KRAS wild-type at DS. After reanalyzing by PS 97 KRAS wild-type tumors treated with anti-EGF receptor (EGFR) antibodies, nine additional mutations were revealed in nonresponders, whereas none of responders exhibited a KRAS-mutated genotype. Of note, KRAS-mutated tumors upon PS showed a worst progression-free survival after EGFR therapy. Finally, PS allowed the detection of additional NRAS, BRAF and exon 20 PIK3CA mutations mostly in KRAS wild-type mCRCs resistant to EGFR therapy. CONCLUSION: PS detection of low-frequency mutations may improve the KRAS predictive value for EGFR therapy selection.

Mechanisms of resistance to EGFR tyrosine kinase inhibitors gefitinib/erlotinib and to ALK inhibitor crizotinib.

The discovery of several molecular alterations that underlie non-small cell lung cancer (NSCLC) pathogenesis has led to the development of targeted therapies. In particular, gefitinib and erlotinib have become the standard of care in patients harboring epidermal growth factor receptor mutations, while crizotinib showed an impressive efficacy in patients with ALK-positive NSCLC. Nevertheless, the occurrence of clinical resistance limits the long term results of these novel agents. The identification of the molecular mechanisms responsible for acquired resistance to targeted therapy is crucial in order to pursue the creation of rational strategies to overcome resistance. In the current review, we will focus on the acquired resistance mechanisms to EGFR-TKIs and crizotinib and the therapeutic strategies currently under study to overcome resistance.

Coexistence of two different mutations in codon 12 of the Kras gene in colorectal cancer: Report of a case supporting the concept of tumoral heterogeneity.

Evaluation of the mutational status of KRAS is a crucial step for the correct therapeutic approach in treating advanced colorectal cancer as the identification of wild-type KRAS tumors leads to more specific and less toxic treatments for patients. Although several studies have highlighted the differences between primary and metastatic tumors, the possibility of two or more mutations in the same codon has seldom been reported. The present study reports an additional case of an advanced adenocarcinoma of the colon showing two somatic mutations (p.G12D and p.G12V) in the same codon (codon 12) of exon 2 of the KRAS gene, thus supporting the possibility of two differing clonal origins of the tumor. Although the clinical significance of multiple mutations remains unknown at present, based on the limited data available in the literature, this rare event appears to be associated with a more aggressive disease, as in the present case. This case report demonstrates the existence of intratumoral heterogeneity and the coexistence of distinct clones within a tumor that may have profound clinical implications for disease progression and therapeutic responses.

Role of preeclampsia-related angiogenic factors in sunitinib cardiotoxicity: two cases and review of the literature.

Sunitinib is a multi-targeted tyrosine kinase inhibitor widely used in clear cell renal carcinoma and in imatinib-resistant gastrointestinal stromal tumors. Sunitinib-associated cardiotoxicity has been recognized and includes hypertension, left ventricular dysfunction and congestive heart failure; nevertheless, few data exist in the literature regarding the role of preeclampsia-related angiogenic factors in sunitinib cardiotoxicity. We report a case of sunitinib-induced severe left ventricular dysfunction that occurred in a hypertensive woman with metastatic renal carcinoma and a history of preeclampsia, and a case of sunitinib-induced preeclampsia-like syndrome in a normotensive patient with an imatinib-resistant gastrointestinal stromal tumor. Our experience confirms that inhibition of angiogenic factors to treat cancer is a novel challenge for the oncologist and requires the cardiologist's support.

A pilot characterization of human lung NSCLC by protein pathway activation mapping.

BACKGROUND: An understanding of the activated protein signaling architecture in non-small-cell lung cancer (NSCLC) is of critical importance to the development of new therapeutic approaches and identification of predictive and prognostic biomarkers for patient stratification. METHODS: We used reverse-phase protein microarrays to map the activated protein signaling networks of 47 NSCLC tumors, 28 of which were node negative, which were subjected to tumor cellular enrichment using laser capture microdissection. The phosphorylation/cleavage levels of 111 key signaling proteins and total levels of 17 proteins were measured for broadscale signaling analysis. RESULTS: Pathway activation mapping of NSCLC revealed distinct subgroups composed of epidermal growth factor receptor (ERBB1), v-erb-b2 erythroblastic leukemia viral oncogene homolog 2 (ERBB2), v-erb-b2 erythroblastic leukemia viral oncogene homolog 3 (ERBB3), v-erb-a erythroblastic leukemia viral oncogene homolog 4 (ERBB4), v-akt murine thymoma viral oncogene homolog 1- mammalian target of rapamycin (AKT-mTOR), protein kinase, AMP-activated, alpha 2 catalytic subunit (AMPK), and autophagy-related signaling, along with transforming growth factor-beta-signaling protein 1 (SMAD), insulin-line growth factor receptor (IGFR), rearranged during transfection proto-oncogene (RET), and activated CDC42-associated kinase (ACK) activation. Investigation of epidermal growth factor receptor (EGFR)-driven signaling identified a unique cohort of tumors with low EGFR protein expression yet high relative levels of phosphorylated EGFR and high EGFR total protein with low relative levels of phosphorylation. Last, mapping analysis of patients with NSCLC with N0 disease revealed a pilot pathway activation signature composed of linked epidermal growth factor receptor family (HER)-AMPK-AKT-mTOR signaling network along with focal adhesion kinase- LIM domain kinase-1 (FAK-LIMK) and janus kinase (JAK)-signal transducers and activators of transcription (STAT) pathways that correlated with short-term survival and aggressive disease. CONCLUSIONS: Functional protein pathway activation mapping of NSCLC reveals distinct activation subgroups that are underpinned by important therapeutic targets and that patients with early-stage node negative disease and poor prognosis may be identified by activation of defined, biochemically linked protein signaling events. Such findings, if confirmed in larger study sets, could help select and stratify patients for personalized targeted therapies.

Identification of a new insertion in exon 20 of EGFR in a woman with NSCLC.

Mutations of epidermal growth factor receptor 1 (EGFR) gene occur in about 15 % of all NSCLCs in Western Europe and are frequently located in exons 19 and 21, being associated with high sensitivity to EGFR tyrosine kinase inhibitors (TKIs). By contrast, exon 20 insertions account for up to 10 % of all EGFR mutations and are correlated to EGFR TKI resistance. Herein, we describe a novel mutation in EGFR exon 20 in a female non-smoker bearing a lung adenocarcinoma, characterized by the insertion of a nucleotide triplet GTT, which translates into a protein with an additional Valine between Proline 772 and Histidine 773 (p.P772_H773insV-c.2316_2317insGTT). The patient was treated with cisplatin/pemetrexed 1st-line and docetaxel 2nd-line chemotherapies, reporting a prolonged disease stabilization of 25 months. The identification and the biological and clinical characterization of novel EGFR mutations represent a prerequisite for their wide use as predictive biomarkers for personalized therapy in NSCLC.

Protein pathway activation mapping of brain metastasis from lung and breast cancers reveals organ type specific drug target activation.

Brain metastases are the most common fatal complication of systemic cancer, especially of lung (40-50%) and breast (20-30%) cancers. In this era of personalized therapy, there is a critical need to uncover the signaling architecture of brain metastases; however, little is known about what signaling pathways are activated in the context of the brain microenvironment. Using a unique study set of 42 brain metastases from patients with breast or nonsmall cell lung cancer (NSCLC), the phosphorylation/activation states of 128 key signaling proteins involved in cancer signaling were measured in laser capture microdissected tumor epithelium using reverse phase protein microarray (RPMA) technology. Distinct pathway activation subgroups from both breast and lung metastases were underpinned by, among others, ERBB2, AKT, mTOR, EGFR, SMAD, and ERK-p38 signaling. Breast cancer metastases showed significantly (p < 0.05) higher activation of the c-ERBB2/IGFR-AKT pathway network compared to NSCLC metastases, whereas NSCLC metastases to the brain exhibited higher relative levels of many members of the EGFR-ERK signaling network. Protein pathway activation mapping using RPMA revealed both the heterogeneity of signaling networks in brain metastases that would require a prior stratification to targeted therapies as well as the requirement of direct analysis of the metastatic lesion.

Polymorphisms of the CYP1A1, CYP2E1 and XRCC1 genes and cancer risk in a Southern Italian population: a case-control study.

BACKGROUND: Polymorphisms in genes encoding enzymes involved in xenobiotic metabolism and/or in cellular defenses against carcinogen-induced DNA damage play an important role in determining individual cancer susceptibility. However, their distribution and association with cancer susceptibility can vary in different populations. MATERIALS AND METHODS: A case-control study including 290 cancer patients (cases) and 242 controls was performed to evaluate the relationship between polymorphisms of cytochrome P450 (CYP)1A1 and CYP2E1 and X-ray repair complementing defective repair in Chinese hamster cells (XRCC)1 genes and the risk of developing cancer in a Southern Italian (Basilicata) population. Genomic DNA was isolated from 5 ml whole blood and genotyping was performed using a PCR-RFLP technique. RESULTS: No significant differences were observed in the distribution of the CYP1A1, CYP2E1 and XRCC1 gene polymorphisms between the cases and controls in the population under study. CONCLUSION: The distribution of CYP1A1, CYP2E1 and XRCC1 gene polymorphisms in the Basilicata population is not different from that of other Italian regions or from that reported in the literature for Caucasian populations, and polymorphisms in these genes do not play an important role in determining cancer risk in the population under study.

GSTM1 and NAT2 polymorphisms and colon, lung and bladder cancer risk: a case-control study.

BACKGROUND: Glutathione S-transferase M1 (GSTM1) and N-acetyltransferase-2 (NAT2) are phase II enzymes involved in the metabolism of xenobiotics and whose polymorphisms have been related to individual cancer risks. PATIENTS AND METHODS: A case-control study was performed including 92 colon, 75 lung and 23 bladder cancer patients and 121 corresponding controls to verify the existence of an association between the main genetic polymorphisms of GSTM1 and NAT2 and the risk to develop cancer. Genomic DNA, isolated from 5 mL whole blood, was used to study GSTM1 and NAT2 polymorphisms using multiplex PCR and a PCR-RFLP technique, respectively. RESULTS: GSTM1 homozygous null genotype was associated with an increased risk of colon cancer, especially in females and in younger patients. For NAT2 gene, the results suggest a role for the low acetylator phenotype in the development of colon and lung cancer, especially in females. In bladder cancer patients two rare NAT2 genotypes were found at a higher frequency compared with all the other groups. CONCLUSION: The results do not suggest a different distribution of GSTM1 and NAT2 polymorphisms in the studied population compared to those reported for other Caucasian populations and warrant further studies in order to evaluate their potential relationship with individual cancer risks.

Polymorphisms of the DNA repair genes XRCC1 and XRCC3 and risk of lung and colorectal cancer: a case-control study in a Southern Italian population.

BACKGROUND: Genetic polymorphisms in DNA repair genes may influence individual variation in DNA repair capacity, which may be associated with a higher risk of developing cancer. Studies on the association between DNA repair gene polymorphisms and lung and colorectal cancer risk appear to be very limited. This study was designed to examine the polymorphisms associated with two DNA repair genes, namely XRCC1 Arg194Trp, XRCC1 Arg399Gln and XRCC3 Thr241Met, and to investigate their role as susceptibility markers for lung and colorectal cancer. MATERIALS AND METHODS: A case-control study was conducted including 94 and 109 cases of lung and colorectal cancer, respectively, and 121 hospital-based age- and sex-matched healthy controls to examine the role of XRCC1 and XRCC3 genetic polymorphisms in the context of lung and colorectal cancer risk for a Southern Italian population. Genomic DNA isolated from 5 ml whole blood was used to genotype XRCC1 Arg194Trp, XRCC1 Arg399Gln and XRCC3 Thr241Met by means of polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis. RESULTS: No differences were observed among the studied groups with regard to the genotype distribution of XRCC1 codon 194 and 399, thus the risk for lung and colorectal cancer did not appear to be significantly influenced by polymorphisms of this gene. Significant differences were observed among the studied groups with regard to the genotype distribution of XRCC3 codon 241. In particular, the XRCC3 241Met allele was associated with an increased risk of lung and colorectal cancer. CONCLUSION: Our results showed no evidence of a relationship between the XRCC1 Arg194Trp and Arg399Gln polymorphisms and the risk of lung and colorectal cancer. On the other hand, they suggested an increased risk in individuals with the XRCC3 Thr241Met polymorphism thus warranting further study to definitively evaluate the role of DNA repair mechanisms in colorectal and lung cancer susceptibility.

Glutathione S-transferase (GST) polymorphisms as risk factors for cancer in a highly homogeneous population from southern Italy.

Glutathione S-Transferase (GST) is a phase II enzyme and catalyses reactions between glutathione and a variety of electrophilic compounds, including some environmental carcinogens. In man, at least 20 isoenzymatic forms of GST have been identified and many of them show genetically-based individual variability of enzyme activity. The GSTM1 and GSTT1 isoenzymes display several polymorphisms, including a homozygotic deletion, which have been associated with an increased risk for developing neoplastic diseases. There is geographical and ethnic variation in genotype frequencies for both genes. The available data suggest that cancer incidence varies amongst Italian regions, being higher in Northern that in Southern areas, though it is unknown whether this phenomenon is to be attributed to genetic and/or environmental factors. We performed a case-control study to evaluate the GSTM1 and GSTT1 polymorphisms in a series of cancer patients in Basilicata, a Southern Italian region, and in corresponding controls. The results obtained demonstrate that the occurrence of GST polymorphisms in the Basilicata population is not different from other Italian regions and suggest that the population attributable risk associated with these genotypes may be quite high. GSTM1 homozygous null genotype was associated with an increasing risk of cancer, especially in females. The strongest association was with colon and breast cancers. For the GSTT1 gene, the results obtained were suggestive of a decreased risk of cancer associated with the null genotype. Thus, similar studies on these and other susceptibility genes are warranted since they can help to identify susceptible subgroups of people who can be targeted for cancer prevention.