Liquid biopsy in non-small cell lung cancer: a meta-analysis of state-of-the-art and future perspectives.

Introduction: To date, tissue biopsy represents the gold standard for characterizing non-small-cell lung cancer (NSCLC), however, the complex architecture of the disease has introduced the need for new investigative approaches, such as liquid biopsy. Indeed, DNA analyzed in liquid biopsy is much more representative of tumour heterogeneity. Materials and methods: We performed a meta-analysis of 17 selected papers, to attest to the diagnostic performance of liquid biopsy in identifying EGFR mutations in NSCLC. Results: In the overall studies, we found a sensitivity of 0.59, specificity of 0.96 and diagnostic odds ratio of 24,69. Since we noticed a high heterogeneity among different papers, we also performed the meta-analysis in separate subsets of papers, divided by 1) stage of disease, 2) experimental design and 3) method of mutation detection. Liquid biopsy has the highest sensitivity/specificity in high-stage tumours, and prospective studies are more reliable than retrospective ones in terms of sensitivity and specificity, both NGS and PCR-based techniques can be used to detect tumour DNA in liquid biopsy. Discussion: Overall, liquid biopsy has the potential to help the management of NSCLC, but at present the non-homogeneous literature data, lack of optimal detection methods, together with relatively high costs make its applicability in routine diagnostics still challenging.

Circulating and tissue biomarkers in early-stage non-small cell lung cancer.

OBJECTIVE: We sought to characterise circulating and tissue tumour biomarkers of patients who developed early-stage non-small cell lung cancer (NSCLC) during long-term follow-up of a chemoprevention trial (NCT00321893). MATERIALS AND METHODS: Blood and sputum samples were collected from 202 high-risk asymptomatic individuals with CT-detected stable lung nodules. Real-time PCR was performed on plasma to quantify free circulating DNA. Baseline serum was investigated with a previously validated test based on 13 circulating miRNAs (miR-Test). Promoter methylation status of p16, RASSF1a and RARß2 and telomerase activity were assessed in sputum samples. DNA was extracted from each tumour developed during follow-up and subjected to a mutation survey using the LungCarta panel on the Sequenom MassARRAY platform. RESULTS: During follow-up (9 years) six individuals underwent surgery for stage I NSCLC with a median time of disease onset of 20.5 months. MiR-Test scores were positive (range: 0.14-7.24) in four out of six baseline pre-disease onset sera. No association was identified between free circulating DNA or sputum biomarkers and disease onset. All tumours harboured at least one somatic mutation in well-known cancer genes, including KRAS (n = 4), BRAF (n = 1), and TP53 (n = 3). CONCLUSION: Circulating miRNA tests may represent valuable tools to detect clinically-silent tumours. Early-stage lung adenocarcinomas harbour recurrent genetic events similar to those described in advanced-stage NSCLCs.

Functional characterization of a novel FGFR1OP-RET rearrangement in hematopoietic malignancies.

The RET (REarranged during Transfection) receptor tyrosine kinase is targeted by oncogenic rearrangements in thyroid and lung adenocarcinoma. Recently, a RET (exon 12) rearrangement with FGFR1OP [fibroblast growth factor receptor 1 (FGFR1) oncogene partner] (exon 12) was identified in one chronic myelomonocytic leukemia (CMML) patient. We report the molecular cloning and functional characterization of a novel FGFR1OP (exon 11)-RET (exon 11) gene fusion event (named FGFR1OP-RET), mediated by a reciprocal translocation t(6; 10)(q27; q11), in a patient affected by primary myelofibrosis (PMF) with secondary acute myeloid leukemia (AML). The FGFR1OP-RET fusion protein displayed constitutive tyrosine kinase and transforming activity in NIH3T3 fibroblasts, and induced IL3-independent growth and activation of PI3K/STAT signaling in hematopoietic Ba/F3 cells. FGFR1OP-RET supported cytokine-independent growth, protection from stress and enhanced self-renewal of primary murine hematopoietic progenitor and stem cells in vitro. In vivo, FGFR1OP-RET caused a spectrum of disease phenotypes, with >50% of mice showing a fatal myeloproliferative disorder (MPD). Other phenotypes were leukemia transplantable in secondary recipients, dramatic expansion of the mast cell lineage, and reduction of repopulating activity upon lethal irradiation. In conclusion, FGFR1OP-RET chimeric oncogenes are endowed with leukemogenic potential and associated to myeloid neoplasms (CMML and PMF/AML).

Immunohistochemically defined subtypes and outcome in occult breast carcinoma with axillary presentation.

The aim of this study is to evaluate the outcome of occult breast cancer (OBC) in patients with axillary presentation overall and according to the immunohistochemically defined tumour subtypes. We reviewed information on 15,490 consecutive primary breast cancer patients, who underwent surgery at the European institute of oncology between September 1997 and December 2008. Patients with OBC were compared with an equal number of patients with small invasive breast carcinomas (pT1) observed at the same institution during the same period, matched for year of surgery, age, nodal status and biological features. Eighty patients with OBC (study group) and 80 patients with early breast cancer (control group) were identified. There was no significant difference in the disease-free survival (5 years DFS 66 vs. 68% P = 0.91) and the overall survival (5 years OS 80 and 86% P = 0.99) between the OBC and control groups. A statistically significant worse outcome was observed within the group of OBC for patients with more than four involved lymph nodes and with triple negative tumours. The outcome of OBC patients is comparable with that of matched patients with small sized breast cancer. High risk of relapse and death was observed in OBC patients with triple negative tumours and extensive nodal involvement.

Sentinel lymph node biopsy in multicentric breast cancer: five-year results in a large series from a single institution.

PURPOSE: This study was designed to present the 5-year results of patients with multicentric breast cancer who underwent sentinel lymph node biopsy (SLNB) in a single institution. METHODS: Between June 1999 and December 2007, 337 patients with multicentric breast cancer and a clinically negative axilla underwent lymphatic mapping by a single periareolar/peritumoral (n = 306) or a double peritumoral or subdermal injection (n = 31) of (99m)Tc-HSA nanocolloids. The sentinel lymph node (SLN) was evaluated by intraoperative frozen section and axillary dissection was performed only in cases of positive SLNB. RESULTS: The median age of the patients was 48 (range, 22-81) years. The mean number of hot spots identified was 1.4 in the whole series, 1.3 in patients who received a single injection, and 1.7 in those who received a double injection (P < 0.001). The mean number of removed SLNs was 1.7 (median, 1; range, 1-7) with an identification rate of 100%. A total of 138 patients with negative SLNB (n = 134) or isolated tumor cells in the SLN (n = 4) did not receive completion axillary lymph node dissection (CALND). In these latter patients, a total of 27 events (19.5%) occurred with 3 patients (2.2%) developing axillary recurrences after a median follow-up of 5 years (range, 17-134 months). CONCLUSIONS: Axillary lymph node reappearance was infrequent among patients with multicentric breast cancer, having negative SLNB and no CALND. We recommend SLNB as the standard procedure for nodal staging in patients with multicentric breast cancer and a clinically negative axilla.

The lymphoma-associated NPM-ALK oncogene elicits a p16INK4a/pRb-dependent tumor-suppressive pathway.

Oncogene-induced senescence (OIS) is a barrier for tumor development. Oncogene-dependent DNA damage and activation of the ARF/p53 pathway play a central role in OIS and, accordingly, ARF and p53 are frequently mutated in human cancer. A number of leukemia/lymphoma-initiating oncogenes, however, inhibit ARF/p53 and only infrequently select for ARF or p53 mutations, suggesting the involvement of other tumor-suppressive pathways. We report that NPM-ALK, the initiating oncogene of anaplastic large cell lymphomas (ALCLs), induces DNA damage and irreversibly arrests the cell cycle of primary fibroblasts and hematopoietic progenitors. This effect is associated with inhibition of p53 and is caused by activation of the p16INK4a/pRb tumor-suppressive pathway. Analysis of NPM-ALK lymphomagenesis in transgenic mice showed p16INK4a-dependent accumulation of senescent cells in premalignant lesions and decreased tumor latency in the absence of p16INK4a. Accordingly, human ALCLs showed no expression of either p16INK4a or pRb. Up-regulation of the histone-demethylase Jmjd3 and de-methylation at the p16INK4a promoter contributed to the effect of NPM-ALK on p16INK4a, which was transcriptionally regulated. These data demonstrate that p16INK4a/pRb may function as an alternative pathway of oncogene-induced senescence, and suggest that the reactivation of p16INK4a expression might be a novel strategy to restore the senescence program in some tumors.

Rituximab in Hodgkin lymphoma: is the target always a hit?

In 1997, the anti-CD20 monoclonal antibody (MAb) rituximab became the first MAb approved for clinical use in oncology, and ushered in a new era of rationally designed targeted agents in cancer therapeutics. It is currently approved for use in non-Hodgkin lymphoma (NHL), chronic lymphoid leukemia (CLL), and rheumatoid arthritis (RA). Rituximab is non-mutagenic, associated with low treatment-related toxicity, and few, if any, long term adverse events, making it an attractive agent to be tried in off-label settings like Hodgkin lymphoma (HL). HL consists of two distinct subtypes - classic HL (cHL) and lymphocyte predominant HL (LPHL). CD20 is present in virtually all patients with LPHL, and in a significant minority of patients with cHL. In this CD20 positive sub-population, the use of rituximab is a rational intervention strategy. Rituximab has been used in patients with cHL as well as LPHL with good efficacy. In this article, we provide a clinically-oriented overview of the use of rituximab in the different sub-types of HL, and report updated results of our series of 8 LPHL patients treated with rituximab. A systematic review of the literature is also presented.

VEGF expression correlates with microvessel density in Philadelphia chromosome-negative chronic myeloproliferative disorders.

We examined microvessel density (MVD) and immunohistochemical expression of vascular endothelial growth factor (VEGF) in the bone marrow biopsy specimens of 98 patients with Philadelphia chromosome-negative (Ph-) chronic myeloproliferative disorders (CMPDs). There were significantly more MVD "hot spots" in chronic idiopathic myelofibrosis (CIMF; mean +/- SD, 25.6 +/- 6.3) and polycythemia vera (PV; 20.7 +/- 10.2) cases than in essential thrombocythemia (ET) cases (10.1 +/- 4.5) and normal control (NC) samples (7.5 +/- 3.6) (P < .05). Similar results were found using a semiquantitative method (P < .0001). A calculated VEGF index (VEGF(i)) was higher in CIMF (0.29 +/- 0.15) and PV (0.28 +/- 0.20) cases than in ET (0.12 +/- 0.05) and NC (0.08 +/- 0.04) cases (P < .0001). MVD and VEGF(i) were higher in the myelofibrotic phases of CIMF and PV. There was a direct correlation between VEGF(i) and MVD when considering the Ph- CMPDs together (r = 0.67; P < .001) and when considering PV (r = 0.79; P < .001) and CIMF (r = 0.40; P = .013) as individual entities. Our data could provide a rationale for directly targeting VEGF or endothelial cells in CIMF and PV.

Essential thrombocythemia or chronic idiopathic myelofibrosis? A single-center study based on hematopoietic bone marrow histology.

We reviewed a large series of patients with essential thrombocythemia diagnosed on the basis of the Polycythemia Vera Study Group criteria, and reclassified them by evaluating their major morphologic features and clinical course using the World Health Organization classification. The morphologic review of the bone marrow biopsies of 116 patients (44 males and 72 females; aged 19 - 83 years, median 55 years; median follow-up 121 months) led to 22 cases (19%) being classified as essential thrombocythemia (ET), 24 (21%) as chronic idiopathic myelofibrosis (CIMF)-0, 44 (37%) as CIMF-1, 13 (12%) as CIMF-2, 9 (8%) as latent phase polycythemia vera, and four (3%) as chronic myeloproliferative disorder, unclassifiable. There was a significant difference in the median age of the ET and fibrotic CIMF patients (54.7 +/- 13.55 vs. 59.13 +/- 15.05 years; P = 0.03). Histologic analysis showed that the simultaneous presence of loose clusters of large/giant megakaryocytes and nuclear hyperlobulation was significantly different between the ET and the prefibrotic CIMF (P<0.01) and fibrotic CIMF patients (P<0.01), and that the association of dense clusters of megakaryocytes with maturation defects and bulbous nuclei also distinguished the prefibrotic CIMF (P<0.05) and fibrotic CIMF patients (P<0.001) from those with ET. The association of cellularity, granulocytic proliferation and reticulin fibers was helpful in distinguishing prefibrotic from fibrotic CIMF (P<0.001).

Bcl-6 gene mutations in primary cutaneous B-cell lymphomas.

We analyzed mutations in the 5' non-coding region of the BCL-6 gene in 46 cases of primary cutaneous B-cell lymphomas (PCBCL), using a polymerase chain reaction single strand conformation polymorphism (PCR-SSCP) method. The results indicate that PCBCL display a low frequency of mutations and support a marginal zone B-cell origin for most of these neoplasms.