Full cost of diagnostic pathology for lung carcinoma in Italy: results from four Pathology Units.

Objective: To calculate the full cost of diagnostic pathology tests for Non-Small Cell Lung Cancer (NSCLC) across four Italian Pathology Units. Methods: Pathology Units were located in private (2) and public (2) hospitals distributed across the Italian territory (North: 2; Centre: 1; South: 1). Pathologists provided via questionnaire data on tests on NSCLC samples along with the identification and quantification of the necessary healthcare resources (diagnostic technologies, laboratory instruments and personnel). Resources were valued according to hospital-specific unit, yearly and hourly costs (disposables; technologies; professional clusters). Results: The full cost per NSCLC tissue sample included histopathological immunophenotypic and required molecular analysis. Overall, it reached € 659.77 and it was mainly composed of direct costs (77.69%). The processing of a NSCLC tissue sample was labour intensive, as a relevant share of the full cost (44.98%) was actually due to personnel costs, with laboratory technicians, biologists and pathologist driving this finding (17.09%,12.43% and 10.81%, respectively). Conclusions: The results of this research can facilitate the negotiation of new dedicated tariffs for NSCLC sample processing with the national or local third party-payers.

BRCA gene amplification in primary peritoneal high-grade serous carcinoma patient with intrinsic resistance to platinum treatment: a case report.

Platinum-based chemotherapy is the standard chemotherapy for high grade serous ovarian cancer and primary peritoneal high-grade serous carcinoma. PARP inhibitors have changed the paradigm of the treatment in platinum-sensitive ovarian cancers and primary peritoneal high-grade serous carcinoma with BRCA1/2 mutation or homologous recombination deficiency (HRD). Platinum-resistant ovarian and primary peritoneal high-grade serous carcinoma have a lower chance to treat and have worse outcomes. We described a case of patient with a platinum resistant primary peritoneal high-grade serous carcinoma with a rare somatic BRCA2 amplification. There are no guidelines for the treatment of ovarian cancer or primary peritoneal high-grade serous carcinoma with BRCA2 amplification. BRCA2 amplification could result in extreme homologous recombination repair (HRR) pathway efficiency and in less platinum sensitivity, which could be a molecular signature for platinum resistance. Free platinum chemotherapy regimens could be more effective in cases with BRCA2 amplification. Further studies are necessary to establish better approaches and strategies for oncological management and treatment in BRCA2 amplification high grade ovarian cancer and primary peritoneal high-grade serous carcinoma.

POLE exonuclease domain mutations in endometrial carcinoma: a case report.

Endometrial carcinoma (EC) harboring POLE exonuclease domain mutations occurs in 5-15% of ECs and frequently affects young women with low body mass index (BMI). It presents at early stage as high grade endometrioid histotype with intense tumor infiltrating lymphocytes and has good clinical outcomes and favorable prognosis. In this article we report the case of a 32-year-old woman with endometriod EC (EEC) exhibiting a "ultramutated" molecular profile and an excellent prognosis despite tumor size and grading. Herein, to highlight the importance of defining POLE status in ECs for both clinical and therapeutic implications for patients.

NTRK gene fusions in solid tumors: agnostic relevance, prevalence and diagnostic strategies.

A number of innovative drugs, developed for precision medicine, have shown impressive activity in neoplastic patients with rare molecular targets, independently from the site and type of tumor. This gave rise to the concept of agnostic treatments in oncology. The detection of such rare targets is a prerequisite for these treatments and is nowadays one of the main challenges in diagnostic molecular pathology. Various algorithms, new diagnostic strategies and pathological workflows have been suggested to help pathologists in the detection of these rare molecular alterations. An emblematic example of biological targets for agnostic treatments is represented by genetic rearrangements affecting members of the Neurotrophic Tyrosine Receptor Kinase (NTRK) gene family. These gene rearrangements have an unusual dual mode of distribution: the first, at high frequency in some very rare neoplasms, and the second with extremely lower frequencies in more common tumors. Even in the context of an agnostic approach, knowledge of site, histotype and prevalence of the tumors carrying these genetic lesions may be helpful to guide the pathologist in the daily effort in search of these molecular alterations. This review examines the prevalence of NTRK gene fusions in different forms of solid tumors, based on the largest studies to date, reports a comprehensive diagnostic algorithm and an innovative pathological workflow for rapid screening.

Real-World Data on NGS Diagnostics: a survey from the Italian Society of Pathology (SIAPeC) NGS Network.

Next Generation Sequencing (NGS) is increasingly used in diagnostic centers for the assessment of genomic alterations to select patients for precision oncology. The Italian Society of Anatomic Pathology and Diagnostic Cytopathology (SIAPEC) through the Molecular Pathology and Predictive Medicine Study Group (PMMP) has been following the progressive development of centers that have adopted NGS technology in diagnostics over time. In July 2017, a study network on massive parallel sequencing was activated in Italy and recognized as the NGS SIAPeC National Network by the SIAPeC Scientific Society Board. Since then, activities have been implemented within the network that provide for alignment of laboratories through diagnostic concordance analysis and monitoring of centers adhering to the Network. Recently, considering the growing need for extended genomic analyses, the PMMP distributed a national survey to assess activities related to the use of genomic diagnostics in oncology within the NGS SIAPEC National Network.Thirty centers participated in the survey. Eighty percent of the centers are laboratories within Pathology Departments. The distribution of laboratories in the country, the diagnostic laboratory/population ratio, the staff dedicated, the type and number of sequencing and mechatronics platforms available, the genomic panels utilized, and the type and number of diagnostic tests carried out in the last year in each center, are reported.The centers were also asked whether they participated in a multidisciplinary Molecular Tumor Board (MTB) for management of patients. Thirty percent of the centers had a MTB that was ratified by regional decree. The professionals most frequently involved in the core team of the MTB are the pathologist, oncologist, molecular biologist, geneticist, pharmacologist, and bioinformatician.The data from this survey indicate that NGS diagnostics in Italy is still heterogeneous in terms of geographical distribution and the characteristics of laboratories and diagnostic test performed. The implementation of activities that favors harmonization, the logistics and the convergence of biological material in reference centers for molecular analyses is a priority for the development of a functional laboratory network.

Reduction of atherothrombotic burden before stent deployment in non-ST elevation acute coronary syndromes: Reduction of myocardial necrosis achieved with nose-dive manual thrombus aspiration (REMNANT) trial. A volumetric intravascular ultrasound study.

OBJECTIVES: To test whether thrombus aspiration (TA) reduces the atherosclerotic burden in culprit lesions and "facilitate" percutaneous coronary intervention with stent (S-PCI) among patients with non-ST elevation acute coronary syndromes (NSTE-ACS). BACKGROUND: Evidence on the effects of TA adjunctive to S-PCI in NSTE-ACS is limited and controversial. METHODS: TA was defined "aggressive" when using 7F devices or a catheter/artery ratio >0.6, "conservative" with 6F, and a catheter/artery ratio ≤0.6. Angiography and intravascular ultrasound (IVUS) were performed at baseline, after TA and after stent deployment. RESULTS: TA was accomplished in 61/76 patients (80%) with NSTE-ACS. The aspirated material was red thrombus in 23% and plaque fragments in 49% of cases. Compared with baseline, TA was associated with an 82% increase in minimal lumen diameter and a 15% reduction in diameter stenosis (P < 0.001 for both). After TA, IVUS documented a 24 and 16% increase in minimal lumen area and lumen volume, respectively (P < 0.001 for both), a 7% decrease in area stenosis through an 11% reduction of plaque + media volume (P < 0.001). When compared with "conservative", an "aggressive" TA was associated with a more pronounced reduction in percent area stenosis (P < 0.05) and an increase in percent stent expansion (P < 0.001). The plaque + media volume reduction after TA was correlated with stent expansion (r = 0.261, P = 0.046). CONCLUSIONS: Manual TA reduces atherothrombotic burden in culprit lesions of NSTE-ACS patients before S-PCI and, when deep plaque removal is obtained, TA optimizes subsequent stent expansion. © 2015 Wiley Periodicals, Inc.

Case Report: The molecular fingerprint and the clinical implication of an exceptional response to neoadjuvant therapy in a metastatic cardia adenocarcinoma.

Background: Globally, gastric cancer holds the fifth position in terms of prevalence among malignant tumors and is the fourth leading cause of cancer-related mortality. Particular attention should be paid to cardia adenocarcinoma (CA) due to its increasing incidence and poor prognosis. Diagnosis of CA frequently occurs in advanced stages because of its late symptoms. In such cases, neoadjuvant chemotherapy is the primary treatment option. The response to chemotherapy depends on multiple variables including the tumor's molecular profile, the patient's performance status, and the feasibility of using targeted therapy. Patients exhibiting an exceptional response, defined as a complete response to medical therapy lasting more than 1 year, or a partial response or stable disease lasting more than 2 years, are rarely described. This case report presents one of the longest-lasting exceptional responses to chemotherapy in metastatic cardia adenocarcinoma and discusses its clinical implications. Case presentation: A 49-year-old male patient presented with cardia adenocarcinoma (human epidermal growth factor receptor 2 negative, mismatch repair proficient) and liver metastases. Molecular profiling identified a pathogenic mutation in the TP53 gene (R123W; Arg123Trp) as the sole alteration found. Five months after initiating the neoadjuvant chemotherapy with fluorouracil-leucovorin-oxaliplatin-docetaxel, the patient achieved a complete clinical response. The molecular profile was compared with others previously documented in an international data portal, revealing a similar pattern. At 4 years and 3 months from diagnosis, the exceptional response was still confirmed. The patient underwent a cumulative number of 33 cycles of chemotherapy, leading to chemotherapy-induced liver damage. Conclusions: Exceptional responses to neoadjuvant chemotherapy in cardia adenocarcinomas are rarely reported. The documentation of exceptional responses to cancer therapies should be included in large data repositories to explore the molecular fingerprint of these tumors. In such cases, the clinical implications of long-term chemotherapy should always be taken into account.

MDM4 (MDMX) localizes at the mitochondria and facilitates the p53-mediated intrinsic-apoptotic pathway.

MDM4 is a key regulator of p53, whose biological activities depend on both transcriptional activity and transcription-independent mitochondrial functions. MDM4 binds to p53 and blocks its transcriptional activity; however, the main cytoplasmic localization of MDM4 might also imply a regulation of p53-mitochondrial function. Here, we show that MDM4 stably localizes at the mitochondria, in which it (i) binds BCL2, (ii) facilitates mitochondrial localization of p53 phosphorylated at Ser46 (p53Ser46(P)) and (iii) promotes binding between p53Ser46(P) and BCL2, release of cytochrome C and apoptosis. In agreement with these observations, MDM4 reduction by RNA interference increases resistance to DNA-damage-induced apoptosis in a p53-dependent manner and independently of transcription. Consistent with these findings, a significant downregulation of MDM4 expression associates with cisplatin resistance in human ovarian cancers, and MDM4 modulation affects cisplatin sensitivity of ovarian cancer cells. These data define a new localization and function of MDM4 that, by acting as a docking site for p53Ser46(P) to BCL2, facilitates the p53-mediated intrinsic-apoptotic pathway. Overall, our results point to MDM4 as a double-faced regulator of p53.

Ovarian high-grade serous carcinoma with transitional-like (SET) morphology: a homologous recombination-deficient tumor.

Thirteen years ago, we pointed out that ovarian transitional cell carcinomas (TCCs) and conventional high-grade serous carcinomas (HGSCs) had similar genetic alterations and clinical behavior. Consequently, ovarian TCC is now classified as a morphologic variant of HGSC. Defective homologous recombination, resulting from genetic or epigenetic inactivation of DNA damage repair genes, such as BRCA1/2, occurs in approximately 50% of the HGSCs. Although BRCA mutations have been associated with HGSCs with solid, pseudoendometrioid or transitional (SET) features, little is known about the role of non-BRCA homologous recombinationrepair (HRR) genes and the HRR status in these tumors. Using two commercially available assays (Myriad Genetics MyChoice CDx Plus test and SOPHiA Dx Homologous Recombination Deficiency Solution), we study mutations of BRCA1/2 and non-BRCA HRR genes (ATM, BARD1, BRIP1, CDK12, CHEK1/2, FANCL, PALB2, PPP2R2A, RAD51B, RAD51C, RAD51D, and RAD54L), and the HRR status in 19 HGSCs with SET features and 20 HGSCs with classic morphology. We also studied, as control cases, 5 endometrioid carcinomas, 1 clear cell carcinoma, 2 low-grade serous carcinomas, and 1 malignant Brenner tumor. Seven HGSCs with SET features (7/19; 37%) showed BRCA mutations (4 BRCA1, 2 BRCA2, and 1 BRCA1/2). Mutations in non-BRCA HRR genes were found in ATM (1/15; 7%), BARD1 (1/15; 7%), and BRIP1 (1/19; 5%). Most HGSCs with SET features (17/19; 90%) were considered to be homologous recombination-deficient tumors. Three HGSCs with classic morphology (3/20; 15%) showed BRCA2 mutations. Mutations in non-BRCA HRR genes were found in CDK12 (2/14; 14%), FANCL (1/14; 7%), RAD51B (1/14; 7%), and RAD54L (1/14; 7%). Eleven HGSCs with classical morphology (11/20; 55%) were considered to be homologous recombination deficient. In contrast, all ovarian carcinoma control cases (5 endometrioid carcinomas, 1 clear cell carcinoma, 2 low-grade serous carcinomas, and 1 malignant Brenner tumor) were homologous recombination proficient and did not have BRCA mutations. Our results show that the majority of HGSCs with SET features are homologous recombination-deficient tumors independently of the BRCA status and highlight the importance of the HRR tumor testing, especially in BRCA wild-type tumors. Recognition of transitional cell variant of HGSCs may help to identify patients most likely to benefit from PARP inhibitors.

Adjuvant osimertinib treatment in patients with early stage NSCLC (IB-IIIA): pathological pathway adaptations.

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer. Around 30% of patients are diagnosed with early disease and 60% after the tumour has spread to a different part of the body. The earlier NSCLC is diagnosed, the better the chances of prolonging survival. Recent years have seen striking improvements in cancer treatment outcomes through increased use of molecular diagnostics. Therapy decisions are now based on a combination of genetic testing and genetically matched targeted therapies. The positive results obtained with the use of tyrosine kinase inhibitors (TKIs), including osimertinib, in the metastatic disease, coupled with recent data in early stage disease support the importance of molecular testing in this setting. In this overview we discuss factors paramount in pathological pathways to ensure optimal management of early stage NSCLC and also provide an overview of requirements/recommendations. Critical issues in the pre-analytical phases regarding both cytology/biopsy samples and surgically resected tissues are highlighted and solutions are proposed to guarantee accuracy, adequacy and sustainability in the innovative approach to be introduced in clinical practice for NSCLC patients.

A unique microRNA signature associated with plaque instability in humans.

BACKGROUND AND PURPOSE: Atherosclerotic plaque rupture is considered the most important mechanism that underlies the onset of stroke, myocardial infarction, and sudden death. Several evidences demonstrated the pivotal role of inflammatory processes in plaque destabilization. MicroRNAs (miRNAs) are small endogenous RNAs and represent a new important class of gene regulators. Nevertheless, no data exist about the expression profile of miRNAs in atherosclerotic plaques. Thus, the aim of this study was to investigate the expression level of miRNAs in human plaques and to correlate it with clinical features of plaque destabilization. METHODS: Two separate groups of plaques were collected from patients who underwent carotid endarterectomy in Chieti (n=15) and Ancona (n=38) Hospitals. All the plaques were subdivided in symptomatic (n=22) and asymptomatic (n=31) according to the presence/absence of stroke. RESULTS: First, on the plaques collected at Chieti Hospital, we performed large-scale analysis of miRNA expression. Between the 41 miRNAs examined, we discovered profound differences in the expression of 5 miRNAs (miRNA-100, miRNA-127, miRNA-145, miRNA-133a, and miRNA-133b) in symptomatic versus asymptomatic plaques. Remarkably, when we repeated the analysis on the Ancona plaque subset, all these 5 miRNAs confirmed to be significantly more expressed in the symptomatic plaques. Finally, in vitro experiments on endothelial cells transfected with miRNA-145 and miRNA-133a confirmed the importance of these miRNAs in the modulation of stroke-related proteins. CONCLUSIONS: These results are the first to report alterations in the expression of specific miRNAs in human atherosclerotic plaques and suggest that miRNAs may have an important role in regulating the evolution of atherosclerotic plaque toward instability and rupture. Furthermore, by identifying the specific miRNA signature for stroke now, we are able to use computer algorithms to identify previously unrecognized molecular targets.

Reliability and reproducibility among different platforms for tumour BRCA testing in ovarian cancer: a study of the Italian NGS Network.

INTRODUCTION: BRCA tumour testing is a crucial tool for personalised therapy of patients with ovarian cancer. Since different next-generation sequencing (NGS) platforms and BRCA panels are available, the NGS Italian Network proposed to assess the robustness of different technologies. METHODS: Six centres, using four different technologies, provided raw data of 284 cases, including 75 cases with pathogenic/likely pathogenic variants, for a revision blindly performed by an external bioinformatic platform. RESULTS: The third-party revision assessed that all the 284 raw data reached good quality parameters. The variant calling analysis confirmed all the 75 pathogenic/likely pathogenic variants, including challenging variants, achieving a concordance rate of 100% regardless of the panel, instrument and bioinformatic pipeline adopted. No additional variants were identified in the reanalysis of a subset of 41 cases. CONCLUSIONS: BRCA tumour testing performed with different technologies in different centres, may achieve the realibility and reproducibility required for clinical diagnostic procedures.

Survival prediction of stage I lung adenocarcinomas by expression of 10 genes.

Adenocarcinoma is the predominant histological subtype of lung cancer, the leading cause of cancer deaths in the world. At stage I, the tumor is cured by surgery alone in about 60% of cases. Markers are needed to stratify patients by prognostic outcomes and may help in devising more effective therapies for poor prognosis patients. To achieve this goal, we used an integrated strategy combining meta-analysis of published lung cancer microarray data with expression profiling from an experimental model. The resulting 80-gene model was tested on an independent cohort of patients using RT-PCR, resulting in a 10-gene predictive model that exhibited a prognostic accuracy of approximately 75% in stage I lung adenocarcinoma when tested on 2 additional independent cohorts. Thus, we have identified a predictive signature of limited size that can be analyzed by RT-PCR, a technology that is easy to implement in clinical laboratories.

Digital Pathology and PD-L1 Testing in Non Small Cell Lung Cancer: A Workshop Record.

A meeting among expert pathologists was held in 2019 in Rome to verify the results of the previous harmonization efforts on the PD-L1 immunohistochemical testing by scoring a representative series of non-small cell lung cancer (NSCLC) digital slides. The current paper shows the results of this digital experimental meeting and the expertise achieved by the community of Italian pathologists. PD-L1 protein expression was determined using tumor proportion score (TPS), i.e., the percentage of viable tumor cells showing partial or complete membrane staining at any intensity. The gold standard was defined as the final PD-L1 score formulated by a panel of seven lung committed pathologists. PD-L1 status was clustered in three categories, namely negative (TPS < 1), low (TPS 1-49%), and high (TPS ≥ 50%). In 23 cases (71.9%) PD-L1 staining was performed using the companion diagnostic 22C3 pharmDx kit on Dako Autostainer, while in nine (28.1%) cases it was performed using the SP263 Ventana kit on BenchMark platform. A complete PD-L1 scoring agreement between the panel of experts and the participants was reached in 57.1% of cases, whereas a minor disagreement in 16.1% of cases was recorded. Italian pathologists performed best in strong positive cases (i.e., tumor proportion score TPS > 50%), whereas only 10.8% of disagreement with the gold standard was observed, and 55.6% regarded a single challenging case. The worst performance was achieved in the negative cases, with 32.0% disagreement. A significant difference resulted from the analysis of the data separated by the different clones used: 22.3% and 38.1% disagreement (p = 0.01) was found in the group of cases analyzed by 22C3 and SP263 antibody clones, respectively. In conclusion, this workshop record proposed the application of a digital pathology platform to share controversial cases in educational meetings as an alternative possibility for improving the interpretation and reporting of specific histological tools. Due to the crucial role of PD-L1 TPS for the selection of patients for immunotherapy, the identification of unconventional approaches as virtual slides to focus experiences and give more detailed practical verifications of the standard quality reached may be a considerable option.

Early prediction of resistance to tyrosine kinase inhibitors by plasma monitoring of EGFR mutations in NSCLC: a new algorithm for patient selection and personalized treatment.

In Non-Small-Cell Lung Cancer (NSCLC) patients treated with Tyrosine Kinase-Inhibitors (TKIs) therapy, the emergence of acquired resistance can be investigated by plasma monitoring of circulating tumor DNA (ctDNA). A series of 116 patients with EGFR-positive lung adenocarcinomas were treated with first/second generation EGFR TKIs. At clinical progression, 64 (55%) EGFR T790M plasma positive patients were subjected to second line-treatment with osimertinib and strictly monitored during the first month of therapy. Plasma analysis by the EGFR Cobas test showed in 57 (89%) cases a substantial decrease in the levels of the sensitizing EGFR mutant allele (sEGFRma), down to a not detectable value. These patients were defined as plasmatic good responders (PGR). In 7 (11%) patients, the sEGFRma did not drop to zero (plasmatic poor responders, PPR). In these latter cases, Massive Parallel Sequencing (MPS) analysis at the end of the first month and at clinical progression showed the presence of resistant-inducing mutations, including MET and HER2 gene amplification, KRAS and PIK3CA gene mutations. PPR showed disease progression in 5 (71%) cases, stable disease in 2 (29%) cases, and a shorter median Progression-free survival (PFS) (4.3 ± 1.1 months) than that observed in PGR (13.3 ± 1.2 months) (P < 0.0001). Our data indicate that plasma monitoring by a simple RT-PCR-based EGFR mutation test in the first month of treatment may be useful for a rapid identification of patients to be subjected to further characterization by MPS. A diagnostic algorithm for an early detection of resistance-inducing mutations and patient management is reported.

BRAF as a positive predictive biomarker: Focus on lung cancer and melanoma patients.

In the era of personalized medicine, BRAF mutational assessment is mandatory in advanced-stage melanoma and non-small cell lung cancer (NSCLC) patients. The identification of actionable mutations is crucial for the adequate management of these patients. To date various drugs have been implemented in clinical practice. Similarly, various methods may be adopted for the identification of BRAF mutations. Here, we briefly review the current literature on BRAF in melanoma and NSCLC, focusing attention in particular on the different methods and drugs adopted in these patients. In addition, an overview of the real-world practice in different Italian laboratories with high expertise in molecular predictive pathology testing is provided.

IDH1 mutations at residue p.R132 (IDH1(R132)) occur frequently in high-grade gliomas but not in other solid tumors.

Systematic sequence profiling of the Glioblastoma Multiforme (GBM) genome has recently led to the identification of somatic mutations in the isocitrate dehydrogenase 1 (IDH1) gene. Interestingly, only the evolutionarily conserved residue R132 located in the substrate binding site of IDH1 was found mutated in GBM. At present, the occurrence and the relevance of p.R132 (IDH1(R132)) variants in tumors other than GBMs is largely unknown. We searched for mutations at position R132 of the IDH1 gene in a panel of 672 tumor samples. These included high-grade glioma, gastrointestinal stromal tumors (GIST), melanoma, bladder, breast, colorectal, lung, ovarian, pancreas, prostate, and thyroid carcinoma specimens. In addition, we assessed a panel of 84 cell lines from different tumor lineages. Somatic mutations affecting the IDH1(R132) residue were detected in 20% (23 of 113) high-grade glioma samples. In addition to the previously reported p.R132H and p.R132S alleles, we identified three novel somatic mutations (p.R132C, p.R132G, and p.R132L) affecting residue IDH1(R132) in GBM. Strikingly, no IDH1 mutations were detected in the other tumor types. These data indicate that cancer mutations affecting IDH1(R132) are tissue-specific, and suggest that it plays a unique role in the development of high-grade gliomas.

The Integrated Oncology Program of the Italian Ministry of Health. Analytical and clinical validation of new biomarkers for early diagnosis: network, resources, methodology, quality control, and data analysis.

In 2007, an Italian cancer research group proposed a specific concerted action aimed at the "analytical and clinica validation of new biomarkers for early diagnosis: Network, resources, methodology, quality control, and data analysis." The proposal united 37 national operative units involved in different biomarker studies and it created a strong coordinative body with the necessary expertise in methodologies, statistical analysis, quality control, and biological resources to perform ad hoc validation studies for new biomarkers of early cancer diagnosis. The action, financed by the Italian Ministry of Health within the Integrated Oncology Program (PIO) coordinated by NCI-Istituto Tumori Bari, started in 2007 and activated 7 projects, each of which focused on disease-specific biomarker studies. Overall, the 37 participating units proposed studies on 50 biomarkers, including analytical and clinical validation procedures. Clusters of units were specifically involved in research of early-detection biomarkers for cancers of the lung, digestive tract, prostate/bladder, and nervous system, as well as female cancers. Furthermore, a cluster involved in biomarkers for bioimaging and infection-related cancers was created. The first investigators' meeting, "Analytical and clinical validation of new biomarkers for early diagnosis," was held on 9 September 2008 in Bari. During this meeting, methodological aspects, scientific programs and preliminary results were presented and discussed.

An innovative diagnostic strategy for the detection of rare molecular targets to select cancer patients for tumor-agnostic treatments.

Targeted therapies are playing an increasing role in oncology. Among them, particular attention is nowadays reserved to histology-agnostic treatments. Rare molecular alterations affecting different neoplastic forms, such as Microsatellite Instability (MSI), Neurotropic Tyrosine Receptor Kinase (NTRK) gene fusions, etc., can allow efficient treatments, irrespective of the histologic type. Developing an effective testing strategy for the detection of rare molecular alterations is challenging. We report an innovative diagnostic strategy for a rapid and economically affordable detection of this uncommon targets. Malignant tumor samples are selected at the time of histopathological diagnosis and further processed for simultaneous analysis of multiple samples on Tissue Micro Arrays (TMAs) and Tissue Slice Arrays (TSAs). The TSA approach was specifically designed for large scale screening of small biopsies. TMA sections and TSA were first screened by immunohistochemistry (IHC) for the expression of mismatch repair and TRK proteins. Positive cases were subjected to confirmation tests (fragment analysis/FISH/NGS). In a series of 1865 malignant tumors, 48 (2.6%) MSI cases and 6 (0.3%) NTRK fusion cases were detected in 9 and 4 different tumor forms, respectively. On average, the TMA/TSA screening approach enabled IHC analysis of about 20 patients simultaneously with significant saving of time and costs. In addition, we have shown that multiplex IHC can further increment the throughput. A detailed procedure for application of this diagnostic approach in clinical practice is reported. The strategy described may allow an efficient and sustainable selection of tumors carrying rare molecular targets, not to leave behind patients for effective agnostic treatments.

Weekly alternate intensive regimen FIrB/FOx in metastatic colorectal cancer patients: an update from clinical practice.

BACKGROUND: Several trials evaluated the role of intensive regimens, made of triplet chemotherapies plus bevacizumab, as first-line treatment for patients with metastatic colorectal cancer (mCRC). We previously reported, in a Phase II prospective study, the efficacy and the tolerability of FIrB/FOx regimen, reporting interesting results in terms of received dose intensities (rDIs) and safety. METHODS: We reported a retrospective update of 85 patients treated with FIrB/FOx, an intensive regimen of 5-fluorouracil, bevacizumab, and weekly alternate irinotecan and oxaliplatin, to confirm its feasibility in "real life". Subgroup analyses were performed, particularly among patients treated with standard and modified FIrB/FOx (based on age, performance status, and/or comorbidities). RESULTS: Overall, 3-month objective response rate (ORR) and 6-month ORR were 75.9% and 55.3%, respectively. Median progression-free survival (PFS) and median overall survival (OS) were 14.4 and 34.9 months, respectively. Among the patients treated with standard and modified regimens, 3-month ORR, PFS, and OS were 75.8% and 76% (P=1.0000), 14.4 and 14.4 months (P=0.8589), and 37.8 and 26.6 months (P=0.7746), respectively. Among the K/NRAS wild-type and K/NRAS mutant patients, 3-month ORR, PFS, and OS were 95.2% and 74.5% (P=0.0526), 15.3 and 14.4 months (P=0.8753), and 37.8 and 51.4 months (P=0.8527), respectively. The rDIs were ≥80% of full doses both in the standard and in the modified regimens subgroups. Cumulative G3/4 toxicities were neutropenia (14.1%), diarrhea (17.6%), asthenia (9.4%), vomiting (5.6%), and hypertension (16.5%). CONCLUSION: This update shows that intensive regimens such as FIrB/FOx are also feasible options for first-line treatment of mCRC patients in the "real-life" setting.