Luca Mazzucchelli

Wild-Type BRAF Is Required for Response to Panitumumab or Cetuximab in Metastatic Colorectal Cancer

PURPOSE Cetuximab or panitumumab are effective in 10% to 20% unselected metastatic colorectal cancer (CRC) patients. KRAS mutations account for approximately 30% to 40% patients who are not responsive. The serine-threonine kinase BRAF is the principal effector of KRAS. We hypothesized that, in KRAS wild-type patients, BRAF mutations could have a predictive/prognostic value. PATIENTS AND METHODS We retrospectively analyzed objective tumor responses, time to progression, overall survival (OS), and the mutational status of KRAS and BRAF in 113 tumors from cetuximab- or panitumumab-treated metastatic CRC patients. The effect of the BRAF V600E mutation on cetuximab or panitumumab response was also assessed using cellular models of CRC. Results KRAS mutations were present in 30% of the patients and were associated with resistance to cetuximab or panitumumab (P = .011). The BRAF V600E mutation was detected in 11 of 79 patients who had wild-type KRAS. None of the BRAF-mutated patients responded to treatment, whereas none of the responders carried BRAF mutations (P = .029). BRAF-mutated patients had significantly shorter progression-free survival (P = .011) and OS (P < .0001) than wild-type patients. In CRC cells, the introduction of BRAF V600E allele impaired the therapeutic effect of cetuximab or panitumumab. Treatment with the BRAF inhibitor sorafenib restored sensitivity to panitumumab or cetuximab of CRC cells carrying the V600E allele. CONCLUSION BRAF wild-type is required for response to panitumumab or cetuximab and could be used to select patients who are eligible for the treatment. Double-hit therapies aimed at simultaneous inhibition of epidermal growth factor receptor and BRAF warrant exploration in CRC patients carrying the V600E oncogenic mutation.

PIK3CA Mutations in Colorectal Cancer Are Associated with Clinical Resistance to EGFR-Targeted Monoclonal Antibodies

The monoclonal antibodies (moAb) panitumumab and cetuximab target the epidermal growth factor receptor (EGFR) and have proven valuable for the treatment of metastatic colorectal cancer (mCRC). EGFR-mediated signaling involves two main intracellular cascades: on one side KRAS activates BRAF, which in turn triggers the mitogen-activated protein kinases. On the other, membrane localization of the lipid kinase PIK3CA counteracts PTEN and promotes AKT1 phosphorylation, thereby activating a parallel intracellular axis. Constitutive activation of KRAS bypasses the corresponding signaling cascade and, accordingly, patients with mCRC bearing KRAS mutations are clinically resistant to therapy with panitumumab or cetuximab. We hypothesized that mutations activating PIK3CA could also preclude responsiveness to EGFR-targeted moAbs through a similar mechanism. Here, we present the mutational analysis of PIK3CA and KRAS and evaluation of the PTEN protein status in a cohort of 110 patients with mCRC treated with anti-EGFR moAbs. We observed 15 (13.6%) PIK3CA and 32 (29.0%) KRAS mutations. PIK3CA mutations were significantly associated with clinical resistance to panitumumab or cetuximab; none of the mutated patients achieved objective response (P = 0.038). When only KRAS wild-type tumors were analyzed, the statistical correlation was stronger (P = 0.016). Patients with PIK3CA mutations displayed a worse clinical outcome also in terms of progression-free survival (P = 0.035). Our data indicate that PIK3CA mutations can independently hamper the therapeutic response to panitumumab or cetuximab in mCRC. When the molecular status of the PIK3CA/PTEN and KRAS pathways are concomitantly ascertained, up to 70% of mCRC patients unlikely to respond to EGFR moAbs can be identified.

The Bethesda System for Reporting Thyroid Cytopathology: A Meta-Analysis

Objective: We aimed to investigate the validity of the Bethesda System for Reporting Thyroid Cytopathology (TBSRTC) through meta-analysis. Study Design: All publications between January 1, 2008 and September 1, 2011 that studied TBSRTC and had available histological follow-up data were retrieved. To calculate the sensitivity, specificity and diagnostic accuracy, the cases diagnosed as follicular neoplasm, suspicious for malignancy and malignant which were histopathologically confirmed as malignant were defined as true-positive. True-negative included benign cases confirmed as benign on histopathology. The nondiagnostic category was excluded from the statistical calculation. The correlations between the 6 diagnostic categories were investigated. Results: The publications review resulted in a case cohort of 25,445 thyroid fine-needle aspirations, 6,362 (25%) of which underwent surgical excision; this group constituted the basis of the study. The sensitivity, specificity and diagnostic accuracy were 97, 50.7 and 68.8%, respectively. The positive predictive value and negative predictive value were 55.9 and 96.3%, respectively. The rates of false negatives and false positives were low: 3 and 0.5%, respectively. Conclusions: The results of meta-analysis showed high overall accuracy, indicating that TBSRTC represents a reliable and valid reporting system for thyroid cytology.

BCA-1 is highly expressed in Helicobacter pylori–induced mucosa-associated lymphoid tissue and gastric lymphoma

Infection with Helicobacter pylori (Hp) induces the formation of lymphoid tissue in the stomach and the occasional development of primary gastric B-cell lymphomas. We have studied the expression of 2 chemokines that attract B lymphocytes, BCA-1 and SLC, in gastric tissue samples obtained from patients with chronic gastritis induced by Hp infection or nonsteroidal anti-inflammatory drugs, as well as from patients with Hp-associated low-grade and high-grade gastric lymphomas. High-level expression of BCA-1 and its receptor, CXCR5, was observed in all mucosal lymphoid aggregates and in the mantle zone of all secondary lymphoid follicles in Hp-induced gastric mucosa-associated lymphoid tissue (MALT). Follicular dendritic cells and B lymphocytes are possible sources of BCA-1, which is not expressed by T lymphocytes, macrophages, or CD1a(+) dendritic cells. Strong expression of BCA-1 and CXCR5 was also detected in the transformed B cells of gastric MALT lymphomas. By contrast, SLC was confined almost exclusively to endothelial cells in and outside the lymphoid tissue. Only scant, occasional SLC expression was observed in the marginal zone of MALT follicles. Our findings indicate that BCA-1, which functions as a homing chemokine in normal lymphoid tissue, is induced in chronic Hp gastritis and is involved in the formation of lymphoid follicles and gastric lymphomas of the MALT type.

Definition, Diagnosis, and Management of Intravascular Large B-Cell Lymphoma: Proposals and Perspectives From an International Consensus Meeting

Intravascular large B-cell lymphoma (IVLBCL) is a rare form of diffuse LBCL characterized by preferential intravascular growth of malignant lymphocytes, aggressive behavior, and an often fatal course. IVLBCL usually affects elderly patients with poor performance status, elevated lactic dehydrogenase serum levels, anemia, and B symptoms. It displays some differences in clinical presentation among diverse geographical areas, mostly between patients diagnosed in Western countries and Japan. In addition, data from the literature suggest that pathologic diagnostic criteria as well as clinical features of this disease may be broader than described in current classification scheme(s). Under the sponsorship of the International Extranodal Lymphoma Study Group, clinicians and pathologists with interest in IVLBCL, coming from Western and Eastern countries, joined to reach a consensus on defining features as well as to focus on the most urgent unresolved issues in IVLBCL. To this end, a representative group of IVLBCL patients coming from both the aforementioned geographical areas were collectively analyzed. Additional features of IVLBCL were proposed both under clinical and pathologic stand points. At the meeting, it emerged that IVLBCL may have additional histopathologic/cytologic definition criteria with respect to those currently recommended, some clinical features are not randomly distributed worldwide, recent therapeutic approaches, such as anti-CD20-containing regimens, may improve outcome, and kidney, spleen, and liver involvement may show peculiar histopathologic features. Finally, a provisional practical diagnostic approach to hemophagocytosis-associated patients and a proposal for the most useful criteria in the settings of differential diagnosis are included.

Deregulation of the PI3K and KRAS signaling pathways in human cancer cells determines their response to everolimus

Personalized cancer medicine is based on the concept that targeted therapies are effective on subsets of patients whose tumors carry specific molecular alterations. Several mammalian target of rapamycin (mTOR) inhibitors are in preclinical or clinical trials for cancers, but the molecular basis of sensitivity or resistance to these inhibitors among patients is largely unknown. Here we have identified oncogenic variants of phosphoinositide-3-kinase, catalytic, alpha polypeptide (PIK3CA) and KRAS as determinants of response to the mTOR inhibitor everolimus. Human cancer cells carrying alterations in the PI3K pathway were responsive to everolimus, both in vitro and in vivo, except when KRAS mutations occurred concomitantly or were exogenously introduced. In human cancer cells with mutations in both PIK3CA and KRAS, genetic ablation of mutant KRAS reinstated response to the drug. Consistent with these data, PIK3CA mutant cells, but not KRAS mutant cells, displayed everolimus-sensitive translation. Importantly, in a cohort of metastatic cancer patients, the presence of oncogenic KRAS mutations was associated with lack of benefit after everolimus therapy. Thus, our results demonstrate that alterations in the KRAS and PIK3CA genes may represent biomarkers to optimize treatment of patients with mTOR inhibitors.

Disseminated and sustained HIV infection in CD34+ cord blood cell-transplanted Rag2-/-gamma c-/- mice.

Because of species selectivity, HIV research is largely restricted to in vitro or clinical studies, both limited in their ability to rapidly assess new strategies to fight the virus. To prospectively study some aspects of HIV in vivo, immunodeficient mice, transplanted with either human peripheral blood leukocytes or human fetal tissues, have been developed. Although these are susceptible to HIV infection, xenoreactivity, and short infection spans, resource and ethical constraints, as well as biased HIV coreceptor tropic strain infection, pose substantial problems in their use. Rag2−/−γc−/− mice, transplanted as newborns with human CD34+ cells, were recently shown to develop human B, T, and dendritic cells, constituting lymphoid organs in situ. Here we tested these mice as a model system for HIV-1 infection. HIV RNA levels peaked to up to 2 × 106 copies per milliliter of plasma early after infection, and viremia was observed for up to 190 days, the longest time followed. A marked relative CD4+ T cell depletion in peripheral blood occurred in CXCR4-tropic strain-infected mice, whereas this was less pronounced in CCR5-tropic strain-infected animals. Thymus infection was almost exclusively observed in CXCR4-tropic strain-infected mice, whereas spleen and lymph node HIV infection occurred irrespective of coreceptor selectivity, consistent with respective coreceptor expression on human CD4+ T cells. Thus, this straightforward to generate and cost-effective in vivo model closely resembles HIV infection in man and therefore should be valuable to study virus-induced pathology and to rapidly evaluate new approaches aiming to prevent or treat HIV infection.

Multi-Determinants Analysis of Molecular Alterations for Predicting Clinical Benefit to EGFR-Targeted Monoclonal Antibodies in Colorectal Cancer

Background KRAS mutations occur in 35–45% of metastatic colorectal cancers (mCRC) and preclude responsiveness to EGFR-targeted therapy with cetuximab or panitumumab. However, less than 20% patients displaying wild-type KRAS tumors achieve objective response. Alterations in other effectors downstream of the EGFR, such as BRAF, and deregulation of the PIK3CA/PTEN pathway have independently been found to give rise to resistance. We present a comprehensive analysis of KRAS, BRAF, PIK3CA mutations, and PTEN expression in mCRC patients treated with cetuximab or panitumumab, with the aim of clarifying the relative contribution of these molecular alterations to resistance. Methodology/Principal Findings We retrospectively analyzed objective tumor response, progression-free (PFS) and overall survival (OS) together with the mutational status of KRAS, BRAF, PIK3CA and expression of PTEN in 132 tumors from cetuximab or panitumumab treated mCRC patients. Among the 106 non-responsive patients, 74 (70%) had tumors with at least one molecular alteration in the four markers. The probability of response was 51% (22/43) among patients with no alterations, 4% (2/47) among patients with 1 alteration, and 0% (0/24) for patients with ≥2 alterations (p<0.0001). Accordingly, PFS and OS were increasingly worse for patients with tumors harboring none, 1, or ≥2 molecular alteration(s) (p<0.001). Conclusions/Significance When expression of PTEN and mutations of KRAS, BRAF and PIK3CA are concomitantly ascertained, up to 70% of mCRC patients unlikely to respond to anti-EGFR therapies can be identified. We propose to define as ‘quadruple negative’, the CRCs lacking alterations in KRAS, BRAF, PTEN and PIK3CA. Comprehensive molecular dissection of the EGFR signaling pathways should be considered to select mCRC patients for cetuximab- or panitumumab-based therapies.

A Retrospective Analysis of 153 Patients Treated With or Without Intravesical Bacillus Calmette-Guerin for Primary Stage T1 Grade 3 Bladder Cancer: Recurrence, Progression and Survival

PURPOSE We retrospectively evaluated the long-term outcome in patients with newly diagnosed stage T1 grade 3 bladder cancer treated with transurethral resection with or without intravesical bacillus Calmette-Guerin (BCG). MATERIALS AND METHODS Of 153 patients with a median age of 67 years (range 36 to 88) and a male-to-female ratio of 4:1 we treated 92 with transurethral bladder resection and additional BCG, and 61 with transurethral bladder resection alone. BCG was administered intravesically as 120 mg. BCG Pasteur F dissolved in 50 ml. saline, retained for up to 2 hours weekly for 6 weeks and repeated as necessary. RESULTS Median followup was 5.3 years (range 0.4 to 18.2). Disease recurred in 70% of the patients treated with BCG and in 75% treated with transurethral resection alone. Median time to recurrence was 38 and 22 months for BCG and resection alone (p = 0.19). Tumor progressed in 33% of patients with BCG and in 36% with resection alone. Deferred cystectomy was performed in 29% of the patients with BCG and in 31% with resection alone. Overall and disease specific survival did not differ significantly. CONCLUSIONS Our results suggest that intravesical BCG therapy after transurethral bladder resection for stage T1 grade 3 bladder cancer may delay the time to recurrence and cystectomy but it does not substantially alter the final outcome. Our findings reflect the rule of 30% for stage T1 grade 3 cancer, namely approximately 30% of patients never have recurrence, 30% ultimately die of metastatic disease and 30% require deferred cystectomy.

DIFFERENTIAL IN SITU EXPRESSION OF THE GENES ENCODING THE CHEMOKINES MCP-1 AND RANTES IN HUMAN INFLAMMATORY BOWEL DISEASE

Two chemotactic cytokines, monocyte chemoattractant protein‐1 (MCP‐1) and RANTES, possibly contribute to the recruitment and activation of leukocytes in inflamed tissues. The expression of these cytokine genes was evaluated in tissue sections from resected bowel segments of 14 patients with inflammatory bowel disease (IBD) and seven control patients by use of 35S‐labelled antisense RNA probes. MCP‐1 and RANTES transcripts were generally increased in the intestinal mucosa of patients with IBD, compared with controls. Whereas MCP‐1 gene expression in the mucosa was restricted to the lamina propria, the gene coding for RANTES was expressed in intraepithelial lymphocytes and in the subepithelial lamina propria. Furthermore, MCP‐1 mRNA, but not RANTES mRNA, was abundant in vessel‐associated cells, such as endothelial cells, medial smooth muscle cells, and intraluminal cells; in smooth muscle cells of the intestinal tunica muscularis; and in cells of the myenteric plexus. Compared with controls, a significant increase of MCP‐1‐expressing cells was observed in tissue specimens from patients with IBD, in endothelial cells of venules, and in cells present in the lumen of intestinal vessels. Conversely, the expression of MCP‐1 mRNA in smooth muscle cells and myenteric plexus cells appeared to be comparable in control and diseased intestines. The increased number of MCP‐1 and RANTES mRNA‐expressing cells in mucosa from patients with IBD suggests that these cytokines play a role in the pathogenesis of mucosal inflammation. Furthermore, the expression of the MCP‐1 gene in vessel‐associated cells may indicate its involvement in mechanisms regulating the adhesion of blood monocytes to endothelial cells.