Cristian Lolli

A molecular portrait of gastrointestinal stromal tumors: an integrative analysis of gene expression profiling and high-resolution genomic copy number

In addition to KIT and PDGFRA mutations, sequential accumulation of other genetic events is involved in the development and progression of gastrointestinal stromal tumors (GISTs). Until recently, the significance of these other alterations has not been thoroughly investigated. We report the first study that integrates gene expression profiling and high-resolution genomic copy number analyses in GIST. Fresh tissue specimens from 25 patients with GIST were collected, and gene expression profiling and high-resolution genomic copy number analyses were performed, using Affymetrix U133Plus and SNP array 6.0. We found that all 21 mutant GIST patients showed both macroscopic cytogenetic alterations and cryptic microdeletions or amplifications, whereas 75% (three of four) of wild-type patients with GIST did not show genomic imbalances. The most frequently observed chromosomal alterations in patients with mutant GIST included 14q complete or partial deletion (17 of 25), 1p deletion (14 of 25) and 22q deletion (10 of 25). Genetic targets of the chromosomal aberrations were selected by integrated analysis of copy number and gene expression data. We detected the involvement of known oncogenes and tumor suppressors including KRAS in chr 12p amplification and KIF1B, PPM1A, NF2 in chr 1p, 14q and 22p deletions, respectively. The genomic segment most frequently altered in mutated samples was the 14q23.1 region, which contains potentially novel tumor suppressors, including DAAM1, RTN1 and DACT1. siRNA-mediated RTN1 downregulation showed evidence for the potential role in GIST pathogenesis. The combination of gene expression profiling and high-resolution genomic copy number analysis offers a detailed molecular portrait of GISTs, providing an essential comprehensive knowledge necessary to guide the discovery of novel target genes involved in tumor development and progression.

Insulin-like growth factor 1 receptor expression in wild-type GISTs: A potential novel therapeutic target

Aberrations of the Insulin‐like Growth Factor (IGF) system have been found in association with a variety of cancer types. The potential role of IGF1R has been postulated in a small subset of GISTs, but until now the implications of its aberrations have not been defined. The aim of the study was to examine the IGF1R status in patients with gastric GIST in regard to KIT and PDGFRA genotype. Fresh resection specimens were collected from 8 primary tumours [2 wild‐type (WT) and 6 mutant cases]. IGF1R was studied as gene expression profiling with Affymetrix GeneChip HG‐U133Plus 2.0 arrays and as genomic copy number with SNP array analysis Affymetrix Genome Wide Human SNP 6.0 arrays, and at protein level with western blotting (WB) and immunohistochemistry (IHC). The unsupervised analysis of gene expression profiling of our patients merged with a data set from gastric GISTs identified 2 patients out of 8 with different expression of IGF1R. The data were confirmed by WB and IHC. In particular, IGF1R was upregulated in 2 young patients (<30‐years old), who had both WT disease and metastases at diagnosis. The SNP array analysis showed that none of the tumours had IGF1R amplification. GISTs are characterized by abnormalities of the KIT and PDGFRA receptors that affect prognosis and response to tyrosine kinase inhibitors. Both young adult with WT GIST had the over‐expression of IGF1R at mRNA and protein level. These results further confirm the hypothesis that IGF1R may be a potential therapeutic target in GISTs lacking KIT and PDGFRA mutations.

Integrated genomic study of quadruple-WT GIST ( KIT/PDGFRA/SDH/RAS pathway wild-type GIST)

BackgroundAbout 10-15% of adult gastrointestinal stromal tumors (GIST) and the vast majority of pediatric GIST do not harbour KIT or platelet-derived growth factor receptor alpha (PDGFRA) mutations (J Clin Oncol 22:3813–3825, 2004; Hematol Oncol Clin North Am 23:15–34, 2009). The molecular biology of these GIST, originally defined as KIT/PDGFRA wild-type (WT), is complex due to the existence of different subgroups with distinct molecular hallmarks, including defects in the succinate dehydrogenase (SDH) complex and mutations of neurofibromatosis type 1 (NF1), BRAF, or KRAS genes (RAS-pathway or RAS-P).In this extremely heterogeneous landscape, the clinical profile and molecular abnormalities of the small subgroup of WT GIST suitably referred to as quadruple wild-type GIST (quadrupleWT or KITWT/PDGFRAWT/SDHWT/RAS-PWT) remains undefined. The aim of this study is to investigate the genomic profile of KITWT/PDGFRAWT/SDHWT/RAS-PWT GIST, by using a massively parallel sequencing and microarray approach, and compare it with the genomic profile of other GIST subtypes.MethodsWe performed a whole genome analysis using a massively parallel sequencing approach on a total of 16 GIST cases (2 KITWT/PDGFRAWT/SDHWT and SDHBIHC+/SDHAIHC+, 2 KITWT/PDGFRAWT/SDHAmut and SDHBIHC-/SDHAIHC- and 12 cases of KITmut or PDGFRAmut GIST). To confirm and extend the results, whole-genome gene expression analysis by microarray was performed on 9 out 16 patients analyzed by RNAseq and an additional 20 GIST patients (1 KITWT/PDGFRAWTSDHAmut GIST and 19 KITmut or PDGFRAmut GIST). The most impressive data were validated by quantitave PCR and Western Blot analysis.ResultsWe found that both cases of quadrupleWT GIST had a genomic profile profoundly different from both either KIT/PDGFRA mutated or SDHA-mutated GIST. In particular, the quadrupleWT GIST tumors are characterized by the overexpression of molecular markers (CALCRL and COL22A1) and of specific oncogenes including tyrosine and cyclin- dependent kinases (NTRK2 and CDK6) and one member of the ETS-transcription factor family (ERG).ConclusionWe report for the first time an integrated genomic picture of KITWT/PDGFRAWT/SDHWT/RAS-PWT GIST, using massively parallel sequencing and gene expression analyses, and found that quadrupleWT GIST have an expression signature that is distinct from SDH-mutant GIST as well as GIST harbouring mutations in KIT or PDGFRA. Our findings suggest that quadrupleWT GIST represent another unique group within the family of gastrointestintal stromal tumors.

Androgen receptor gene status in plasma DNA associates with worse outcome on enzalutamide or abiraterone for castration-resistant prostate cancer: a multi-institution correlative biomarker study.

Abstract Background There is an urgent need to identify biomarkers to guide personalized therapy in castration-resistant prostate cancer (CRPC). We aimed to clinically qualify androgen receptor (AR) gene status measurement in plasma DNA using multiplex droplet digital PCR (ddPCR) in pre- and post-chemotherapy CRPC. Methods We optimized ddPCR assays for AR copy number and mutations and retrospectively analyzed plasma DNA from patients recruited to one of the three biomarker protocols with prospectively collected clinical data. We evaluated associations between plasma AR and overall survival (OS) and progression-free survival (PFS) in 73 chemotherapy-naïve and 98 post-docetaxel CRPC patients treated with enzalutamide or abiraterone (Primary cohort) and 94 chemotherapy-naïve patients treated with enzalutamide (Secondary cohort; PREMIERE trial). Results In the primary cohort, AR gain was observed in 10 (14%) chemotherapy-naïve and 33 (34%) post-docetaxel patients and associated with worse OS [hazard ratio (HR), 3.98; 95% CI 1.74–9.10; P < 0.001 and HR 3.81; 95% CI 2.28–6.37; P < 0.001, respectively], PFS (HR 2.18; 95% CI 1.08–4.39; P = 0.03, and HR 1.95; 95% CI 1.23–3.11; P = 0.01, respectively) and rate of PSA decline ≥50% [odds ratio (OR), 4.7; 95% CI 1.17–19.17; P = 0.035 and OR, 5.0; 95% CI 1.70–14.91; P = 0.003, respectively]. AR mutations [2105T>A (p.L702H) and 2632A>G (p.T878A)] were observed in eight (11%) post-docetaxel but no chemotherapy-naïve abiraterone-treated patients and were also associated with worse OS (HR 3.26; 95% CI 1.47–not reached; P = 0.004). There was no interaction between AR and docetaxel status (P = 0.83 for OS, P = 0.99 for PFS). In the PREMIERE trial, 11 patients (12%) with AR gain had worse PSA-PFS (sPFS) (HR 4.33; 95% CI 1.94–9.68; P < 0.001), radiographic-PFS (rPFS) (HR 8.06; 95% CI 3.26–19.93; P < 0.001) and OS (HR 11.08; 95% CI 2.16–56.95; P = 0.004). Plasma AR was an independent predictor of outcome on multivariable analyses in both cohorts. Conclusion Plasma AR status assessment using ddPCR identifies CRPC with worse outcome to enzalutamide or abiraterone. Prospective evaluation of treatment decisions based on plasma AR is now required. Clinical Trial number NCT02288936 (PREMIERE trial).

Circulating AR copy number and outcome to enzalutamide in docetaxel-treated metastatic castration-resistant prostate cancer

In the present study, we aimed to evaluate the association of circulating AR copy number (CN) and outcome in a cohort of patients with advanced castration-resistant prostate cancer (CRPC) treated with enzalutamide after docetaxel. Fifty-nine CRPC patients were evaluated. AR CN was analyzed with real-time and digital PCR in the serum collected at starting of treatment. Progressive disease was defined on the basis of Prostate Cancer Working Group 2 criteria. AR CN gain was found in 21 of 59 (36%) patients. Median baseline PSA, alkaline phosphatase and lactate dehydrogenase levels were higher in the AR CN gained group (p = 0.007, p = 0.003, p = 0.0009, respectively). Median PFS of patients with AR CN gain was 2.4 (95%CI: 1.9−3.2) vs. 4.0 months (95%CI: 3.0−6.5) of those with no gain (p = 0.0004). Median OS of patients with AR CN gain was 6.1 (95%CI: 3.4−8.6) vs. 14.1 months (95%CI: 8.2−20.5) of those with no gain (p = 0.0003). At multivariate analysis, PSA decline ≥ 50% and AR CN showed a significant association with PFS (p = 0.008 and p = 0.002, respectively) and OS (p = 0.009 and p = 0.001, respectively). These findings indicate that the detection of circulating AR CN gain is a promising non-invasive biomarker for outcome prediction to enzalutamide treatment in CRPC patients.

Development of coronary artery stenosis in a patient with metastatic renal cell carcinoma treated with sorafenib

BackgroundTyrosine kinase inhibitors (TKIs) are currently approved for the treatment of metastatic renal cell carcinoma (mRCC). The cardiotoxic effects of sorafenib and sunitinib may cause hypertension, left ventricular ejection fraction (LVEF) dysfunction and/or congestive heart failure (CHF), and arterial thrombo-embolic events (ATE). Only three cases of coronary artery disease related to sorafenib therapy have been described in the literature, and all were due to arterial vasospasm without evidence of coronary artery stenosis on angiography. Cardiotoxicity is commonly associated with the presence of cardiovascular risk factors, such as a history of hypertension or coronary artery disease.Case presentationWe describe a patient who experienced an unusual cardiac event after 2 years of sorafenib treatment. A 58-year-old man with mRCC developed acute coronary syndrome (ischemia/infarction) associated with critical sub-occlusion of the common trunk of the left coronary artery and some of its branches, which was documented on coronary angiography. The patient underwent triple coronary artery bypass surgery, and sorafenib treatment was discontinued. He did not have any cardiovascular risk factors, and his cardiac function and morphology were normal prior to sorafenib treatment.ConclusionsFurther investigation of a larger patient population is needed to better understand cardiac damage due to TKI treatment. Understanding the usefulness of careful cardiovascular monitoring might be important for the prevention of fatal cardiovascular events, and to avoid discontinuation of therapy for the underlying cancer.

Systemic immune-inflammation index predicts the clinical outcome in patients with mCRPC treated with abiraterone

Background: A systemic immune-inflammation index (SII) based on neutrophil (N), lymphocyte (L), and platelet (P) counts has shown a prognostic impact in several solid tumors. The aim of this study is to evaluate the prognostic role of SII in metastatic castration-resistant prostate cancer (mCRPC) patients treated with abiraterone post docetaxel. Patients and Methods: We retrospectively reviewed consecutive mCRPC patients treated with abiraterone after docetaxel in our Institutions. X-tile 3.6.1 software, cut-off values of SII, neutrophil-to-lymphocyte ratio (NLR) defined as N/L and platelets-to-lymphocyte ratio (PLR) as P/L. Overall survival (OS) and their 95% Confidence Intervals (95% CI) was estimated by the Kaplan-Meier method and compared with the log-rank test. The impact of SII, PLR, and NLR on overall survival (OS) was evaluated by Cox regression analyses and on prostate-specific antigen (PSA) response rates were evaluated by binary logistic regression. Results: A total of 230 mCRPC patients treated abiraterone were included. SII ≥ 535, NLR ≥ 3 and PLR ≥ 210 were considered as elevated levels (high risk groups. The median OS was 17.3 months, 21.8 months in SII < 535 group and 14.7 months in SII ≥ 535 (p < 0.0001). At univariate analysis Eastern Cooperative Oncology Group (ECOG) performance status, previous enzalutamide, visceral metastases, SII, NLR, and PLR predicted OS. In multivariate analysis, ECOG performance status, previous enzalutamide, visceral metastases, SII, and NLR remained significant predictors of OS [hazard ratio (HR) = 5.08, p < 0.0001; HR = 2.12, p = 0.009, HR = 1.77, 95% p = 0.012; HR = 1.80, p = 0.002; and HR = 1.90, p = 0.001, respectively], whereas, PLR showed a borderline ability only (HR = 1.41, p = 0.068). Conclusion: SII and NLR might represent an early and easy prognostic marker in mCRPC patients treated with abiraterone. Further studies are needed to better define their impact and role in these patients.

Systemic immune-inflammation index predicts the clinical outcome in patients with metastatic renal cell cancer treated with sunitinib

Background In this retrospective analysis, we explored the prognostic and predictive value of the systemic immune-inflammation index (SII), based on lymphocyte, neutrophil, and platelet counts, at baseline and changes at week 6 during first-line sunitinib in patients with metastatic renal cell cancer (RCC). Results Patients were stratified into high SII (≥ 730) and low SII (< 730) groups. SII was associated with objective response, p < 0.0001. The median PFS was 6.3 months (95% CI 5.5–8.9) in patients with SII ≥ 730 and 18.7 months (95% CI 14.7–22.8) in those with SII < 730, p < 0.0001. The median OS was 43.6 months (95% CI 35.3–52.1) in patients with SII < 730, and 13.5 months (95% CI 9.8–18.5) in those with SII ≥ 730, p < 0.0001. In multivariate analysis, performance status, IMDC score and SII were able to predict OS (HR = 3.29, HR = 1.71 and HR = 1.79, respectively). Materials and Methods We included 335 consecutive RCC patients treated with first-line sunitinib. The X-tile 3.6.1 software (Yale University, New Haven, CT) was used for bioinformatic analysis of the data to determine the cutoff value of SII. Progression-free survival (PFS), overall survival (OS) and their 95% confidence interval (95% CI) were estimated by Kaplan-Meier method and compared with logrank test. The impact of SII conversion at week 6 of treatment on PFS and OS was evaluated by Cox regression analyses. Conclusions The SII and its changes during treatment represent a powerful prognostic indicator of clinical outcome in patients with metastatic RCC.

Three cases of bone metastases in patients with gastrointestinal stromal tumors.

Gastrointestinal stromal tumors (GISTs) are rare, but represent the most common mesenchymal neoplasms of the gastrointestinal tract. Tumor resection is the treatment of choice for localized disease. Tyrosine kinase inhibitors (imatinib, sunitinib) are the standard therapy for metastatic or unresectable GISTs. GISTs usually metastasize to the liver and peritoneum. Bone metastases are uncommon. We describe three cases of bone metastases in patients with advanced GISTs: two women (82 and 54 years of age), and one man (62 years of age). Bones metastases involved the spine, pelvis and ribs in one patient, multiple vertebral bodies and pelvis in one, and the spine and iliac wings in the third case. The lesions presented a lytic pattern in all cases. Two patients presented with multiple bone metastases at the time of initial diagnosis and one patient after seven years during the follow-up period. This report describes the diagnosis and treatment of the lesions and may help clinicians to manage bones metastases in GIST patients.

Good survival outcome of metastatic SDH-deficient gastrointestinal stromal tumors harboring SDHA mutations

Purpose:A subset of patients with KIT/PDGFRA wild-type gastrointestinal stromal tumors show loss of function of succinate dehydrogenase, mostly due to germ-line mutations of succinate dehydrogenase subunits, with a predominance of succinate dehydrogenase subunit A. The clinical outcome of these patients seems favorable, as reported in small series in which patients were individually described. This work evaluates a retrospective survival analysis of a series of patients with metastatic KIT/PDGFRA wild-type succinate dehydrogenase–deficient gastrointestinal stromal tumors.Methods:Sixty-nine patients with metastatic gastrointestinal stromal tumors were included in the study (11 KIT/PDGFRA wild-type, of whom 6 were succinate dehydrogenase deficient, 5 were non–succinate dehydrogenase deficient, and 58 were KIT/PDGFRA mutant). All six succinate dehydrogenase–deficient patients harbored SDHA mutations. Kaplan–Meier curves and log-rank tests were used to compare the survival of patients with succinate dehydrogenase subunit A–mutant gastrointestinal stromal tumors with that of KIT/PDGFRA wild-type patients without succinate dehydrogenase deficiency and patients with KIT/PDGFRA-mutant gastrointestinal stromal tumors.Results:Follow-up ranged from 8.5 to 200.7 months. The difference between succinate dehydrogenase subunit A–mutant gastrointestinal stromal tumors and KIT/PDGFRA-mutant or KIT/PDGFRA wild-type non–succinate dehydrogenase deficient gastrointestinal stromal tumors was significant considering different analyses (P = 0.007 and P = 0.033, respectively, from diagnosis of gastrointestinal stromal tumor for the whole study population; P = 0.005 and P = 0.018, respectively, from diagnosis of metastatic disease for the whole study population; P = 0.007 for only patients who were metastatic at diagnosis).Conclusion:Patients with metastatic KIT/PDGFRA wild-type succinate dehydrogenase–deficient gastrointestinal stromal tumors harboring succinate dehydrogenase subunit A mutations present an impressively long survival. These patients should be identified in clinical practice to better tailor treatments and follow-up over time.Genet Med 17 5, 391–395.