High frequency of BCL2 gene rearrangement-negative follicular lymphoma in northwestern Italy.

BCL2 rearrangement is reported to be an early pathogenetic event in follicular lymphoma (FL) and it is considered as a reliable marker in the follow up of the disease. We aimed to investigate the frequency of BCL2 rearrangement in FLs from northwestern Italy, to evaluate their clinicopathological features, and to investigate alternative genetic aberrations in BCL2-negative FLs. We collected a series of 76 consecutive FLs diagnosed between 2013 and 2016. All lymphomas underwent histopathological review. Interphasic fluorescent in situ hybridization (FISH) was performed with break apart probes targeting BCL2, IGH, BCL6 and MYC on paraffin embedded (PE) and fresh frozen (FF) specimens. 1p36 region and p53 locus in BLC2-negative cases were investigated using dual color probes. Karyotype analysis was available in a subset of cases. BCL2 rearrangements were detected in 39 cases (51,3%). Of the remaining 37, 6 showed IGH rearrangement, and were further tested: 1 showed variant BCL2 translocation, 1 had BCL6 rearrangement, and the other 4 were negative for further gene rearrangements. FISH on FF specimens detected small BCL2+ clones in cases otherwise categorized as BCL2-. 1p36 and p53 deletion were observed in 1 and 8 BCL2- FLs, respectively. Karyotype analysis documented 3q, 1p and BCL6 alternative abnormalities in 3 cases. In conclusion, BCL2 rearrangement is not a constant finding in FL, its frequency being probably affected by geographical factors. Thus, it should not be considered as a reliable molecular marker in the follow up of the disease, unless it is found to be present at the initial diagnosis of FL. Alternative genetic aberrations exist in BCL2-negative cases.

Correlation of KRAS G12C Mutation and High PD-L1 Expression with Clinical Outcome in NSCLC Patients Treated with Anti-PD1 Immunotherapy.

BACKGROUND: Immune checkpoint inhibitors (ICIs) targeting PD-1 or PD-L1 improved the survival of non-small cell lung cancer (NSCLC) patients with PD-L1 expression ≥50% and without alterations in EGFR, ALK, ROS1, RET. However, markers able to predict the efficacy of ICIs, in combination with PD-L1 expression are still lacking. Our aim in this hypothesis-generating pilot study was to evaluate whether the KRAS G12C variant may predict the efficacy of ICIs in advanced NSCLC patients with PD-L1 ≥ 50%. METHODS: Genomic DNA or tissue sections of 44 advanced ICI-treated NSCLC cases with PD-L1 ≥ 50% without EGFR, ALK, ROS1, RET alterations were tested using Next Generation Sequencing, Fluorescence in Situ Hybridization and immunohistochemistry. Statistical analyses were carried out fitting univariate and multivariate time to event models. RESULTS: KRAS G12C mutant patients (N = 11/44) showed a significantly longer progression-free survival (PFS) at univariate and multivariate analyses (p = 0.03). The Kaplan-Meier plot of the PFS time-to-event supports that G12C positive patients have a longer time to progress. PFS improvement was not observed when any KRAS mutations were compared to wild-type cases. CONCLUSIONS: Given the limitations due to the small sample size and exploratory nature of this study, we tentatively conclude the KRAS G12C mutation should be considered in future trials as a predictive marker of prolonged response to first-line ICIs in NSCLC patients overexpressing PD-L1. This finding could be relevant as anti-KRAS G12C therapies enter the therapeutic landscape of NSCLC.

Correction to: Functional and clinical significance of ROR1 in lung adenocarcinoma.

An amendment to this paper has been published and can be accessed via the original article.

Functional and clinical significance of ROR1 in lung adenocarcinoma.

BACKGROUND: Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is normally detectable in embryonic tissues and absent in adult tissues. ROR1 was shown to inhibit apoptosis, potentiate EGFR signaling and reported to be overexpressed and associated with poor prognosis in several tumor models. This study aimed to assess the expression of ROR1 in lung adenocarcinoma (AC) patients. METHODS: We analyzed ROR1 expression by quantitative real-time PCR (qRT-PCR) in 56 histologically confirmed lung AC, stage I to IV, in addition we evaluated its association with TTF-1 (thyroid transcription factor-1) expression and the main molecular alterations involved in lung cancerogenesis. RESULTS: ROR1 overexpression was observed in 28.6% of the entire cohort, using a cut-off of 1, or in 51.8% of the cases using the median value as threshold. Among patients without any genetic alteration, ROR1 overexpression was observed in 34.8% considering a cut-off of 1 and 52.2% considering the median value. The distribution of ROR1 was homogeneous among the different molecular categories: we found no association of ROR1 expression and the presence of gene mutations/rearrangements or the expression of TTF-1. CONCLUSIONS: ROR1 overexpression could constitute a potential therapeutic target because altered in a consistent number of lung AC, especially in cases without druggable genetic alterations. ROR1 expression is independent of classical lung cancer molecular alterations and not correlated, in a Caucasian cohort, to TTF-1 expression.

Prognostic and predictive role of EGFR pathway alterations in biliary cancer patients treated with chemotherapy and anti-EGFR.

The association of anti-EGFR to gemcitabine and oxaliplatin (GEMOX) chemotherapy did not improve survival in biliary tract carcinoma (BTC) patients. Multiple mechanisms might be involved in the resistance to anti-EGFR. Here, we explored the mutation profile of EGFR extracellular domain (ECD), of tyrosine kinase domain (TKD), and its amplification status. EGFR mutational status of exons 12, 18-21 was analyzed in 57 tumors by Sanger sequencing. EGFR amplification was evaluated in 37 tumors by Fluorescent In Situ Hybridization (FISH). Kaplan-Meier curves were calculated using the log-rank test. Six patients had mutations in exon 12 of EGFR ECD and 7 in EGFR TKD. Neither EGFR ECD nor TKD mutations affected progression free survival (PFS) or overall survival (OS) in the entire population. In the panitumumab plus GEMOX (P-GEMOX) arm, ECD mutated patients had a worse OS, while EGFR TKD mutated patients had a trend towards shorter PFS and OS. Overall, the presence of mutations in EGFR or in its transducers did not affect PFS or OS, while the extrahepatic cholangiocarcinoma (ECC) mutated patients had a worse prognosis compared to WT. Nineteen out of 37 tumors were EGFR amplified, but the amplification did not correlate with survival. ECC EGFR amplified patients had improved OS, whereas the amplification significantly correlated with poor PFS (p = 0.03) in gallbladder carcinoma patients. The high molecular heterogeneity is a predominant feature of BTC: the alterations found in this work seem to have a prognostic impact rather than a predictive role towards anti-EGFR therapy.

Multicenter Phase II Study of Panitumumab in Platinum Pretreated, Advanced Head and Neck Squamous Cell Cancer.

LESSONS LEARNED: Panitumumab shows activity in terms of disease control rate and preventing disease progression but not for tumor shrinkage in head and neck squamous cell cancer for second-line treatment. Epidermal growth factor receptor (EGFR) copy number gain, a property of tumor cells that theoretically could identify patients more likely to experience disease response, was common among patients having disease control.Our trial, given the lower toxicity with an every-2-week schedule, provides guidance for future trials, for example, in combinations of immune therapies and anti-EGFR-antibodies. BACKGROUND: The objective of this study was to investigate the efficacy and safety of panitumumab (anti-epidermal growth factor receptor [EGFR] antibody) given as a single agent in platinum-pretreated head and neck squamous cell cancer (HNSCC). METHODS: Patients with advanced HNSCC previously treated with platinum-containing therapy were included. Panitumumab was administered intravenously every 2 weeks at a dose of 6 mg/kg. Primary endpoint was overall response rate (ORR) according to Response Evaluation Criteria In Solid Tumors (RECIST) version 1.1; secondary endpoints were progression-free survival (PFS) and safety. A Simon's two-step design was chosen; 4 partial remissions (PR) in the first 32 patients were required for continuing to step two. An exploratory biomarker analysis was performed. RESULTS: Thirty-three patients were enrolled. Two patients obtained a PR for an ORR of 6%, and 15 (45%) showed stable disease (SD) for at least 2 months, resulting in a 51% disease control rate. Median PFS was 2.6 months (95% confidence interval [CI]: 1.7-3.7), while median OS was 9.7 months (95% CI: 6.3-17.2). The most frequent adverse drug reactions were cutaneous rash (64%) and hypomagnesemia (55%). Overall, 30% of patients experienced grade 3/4 adverse events. No infusion-related reactions occurred. EGFR copy number gain (CNG) was more frequent in patients who benefitted from panitumumab. Two uncommon KRAS mutations (G48E, T50I) and 3 canonical PIK3CA mutations (all E545K) were detected. High-risk HPV16 was found in 10 patients and EGFR CNG in 13 treated patients. EGFR CNG seems to be more frequent in individuals with at least SD compared with patients with progressive disease (59% vs. 30%). PFS for patients with EGFR CNG was 4.6 months (95% CI: 1.0-9.2 months) and 1.9 months (95% CI: 1.0-3.2 months) for patients without CNG (p = .02). CONCLUSION: Panitumumab monotherapy in pretreated HNSCC patients was well tolerated but moderately active. We observed a considerable disease control rate. Future strategies with this agent comprise right patient selection through the identification of reliable biomarkers and gene signatures predicting response and, considering good tolerability and convenience, combination strategies with novel agents and immune therapeutic agents.

Synovial sarcoma metastatic to the parotid gland: A possible pitfall in salivary gland fine needle aspiration cytology. Report of a case with immunocytochemical and fluorescence in situ hybridization findings.

We report one case of metastatic synovial sarcoma (SS) to the parotid gland in a 93-year-old male. The patient had undergone upper left pulmonary lobectomy with mediastinal lymphadenectomy for SS of the lung 5 years before. The cytopathologic presentation and the immunocytochemical findings on the FNA sample were suggestive of a spindle cell myoepithelioma, while a SYT rearrangement was identified by a FISH performed on a cytological smear of the lesion. The diagnosis was further confirmed also by positive immunocytochemical expression of TLE1 on a section from the obtained cell block. The cytologic and immunophenotypic findings are shortly discussed in view of the reported immunophenotypic inconsistency of SS and of its differential diagnosis with spindle cell myoepithelioma of the salivary glands.The importance of the recently described TLE1 staining and its close correlation to SYT rearrangement is briefly discussed.

Definition of MYC genetic heteroclonality in diffuse large B-cell lymphoma with 8q24 rearrangement and its impact on protein expression.

MYC rearrangement can be detected in a subgroup of diffuse large B-cell lymphoma characterized by unfavorable prognosis. In contrast to Burkitt lymphoma, the correlation between MYC rearrangement and MYC protein expression in diffuse large B-cell lymphoma is less clear, as approximately one-third of rearranged cases show negative or low expression by immunohistochemistry. To better understand whether specific characteristics of the MYC rearrangement may influence its protein expression, we investigated 43 de novo diffuse large B-cell lymphoma positive for 8q24 rearrangement by FISH, using 14 Burkitt lymphoma for comparison. Different cell populations (clones), breakpoints (classical vs non-classical FISH patterns), partner genes (IGH vs non-IGH) and immunostaining were detected and analyzed using computerized image systems. In a subgroup of diffuse large B-cell lymphoma, we observed different clones within the same tumor distinguishing the founder clone with MYC rearrangement alone from other subclones, carrying MYC rearrangement coupled with loss/extra copies of derivatives/normal alleles. This picture, which we defined MYC genetic heteroclonality, was found in 42% of cases and correlated to negative MYC expression (P=0.026). Non-classical FISH breakpoints were detected in 16% of diffuse large B-cell lymphoma without affecting expression (P=0.040). Non-IGH gene was the preferential partner of rearrangement in those diffuse large B-cell lymphoma showing MYC heteroclonality (P=0.016) and/or non-classical FISH breakpoints (P=0.058). MYC heteroclonality was not observed in Burkitt lymphoma and all cases had positive MYC expression. Non-classical FISH MYC breakpoint and non-IGH partner were found in 29 and 20% of Burkitt lymphoma, respectively. In conclusion, MYC genetic heteroclonality is a frequent event in diffuse large B-cell lymphoma and may have a relevant role in modulating MYC expression.

EGFR and KRAS Mutations in ALK-Positive Lung Adenocarcinomas: Biological and Clinical Effect.

INTRODUCTION: In lung adenocarcinoma (ADC), anaplastic lymphoma receptor tyrosine kinase (ALK) rearrangements are mutually exclusive with epidermal growth factor receptor (EGFR) and Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations. However, the existence of double-positive (DP) patients have been sporadically described. We identified DP cases in therapy-naive ALK-rearranged ADC and characterized the biology of these tumors to better understand the clinical response to tyrosine kinase inhibitors (TKIs). MATERIALS AND METHODS: We selected 42 ALK-positive ADCs from a multicentric series of 301 cases of ADCs. A mutational analysis was performed using Sanger and/or pyrosequencing to address exons 18-21 of EGFR and codons 12-13 of the KRAS gene. In addition, the KRAS and EGFR copy number was investigated using fluorescent in situ hybridization. DP patients were treated with TKIs, and their response was evaluated according to the Response Evaluation Criteria in Solid Tumors criteria. RESULTS: Eight of 42 ALK-positive ADCs (19%) demonstrated a concomitant mutation in the EGFR (3 cases) or KRAS (5 cases) genes and were classified as DP. All DP cases displayed copy number gains in the EGFR or KRAS gene because of polysomy or gene amplification. In the latter cases, a mutant allele-specific imbalance was observed. Four patients were treated with TKIs. The 2 EGFR-mutant DP patients demonstrated a better response to crizotinib compared with erlotinib. The 2 KRAS-mutant DP patients experienced opposite responses to crizotinib. CONCLUSION: The incidence of DP ADC is not negligible. Patients with ALK/EGFR might benefit more from crizotinib compared with erlotinib administration, although the efficacy of TKIs in patients with ALK/KRAS remains unclear. An integrated targeted therapy should be considered for patients with DP ADC.

NEU3 activity enhances EGFR activation without affecting EGFR expression and acts on its sialylation levels.

Several studies performed over the last decade have focused on the role of sialylation in the progression of cancer and, in particular, on the association between deregulation of sialidases and tumorigenic transformation. The plasma membrane-associated sialidase NEU3 is often deregulated in colorectal cancer (CRC), and it was shown that this enzyme co-immunoprecipitates in HeLa cells with epidermal growth factor receptor (EGFR), the molecular target of most recent monoclonal antibody-based therapies against CRC. To investigate the role of NEU3 sialidase on EGFR deregulation in CRC, we first collected data on NEU3 gene expression levels from a library of commercial colon cell lines, demonstrating that NEU3 transcription is upregulated in these cell lines. We also found EGFR to be hyperphosphorylated in all cell lines, with the exception of SW620 cells and the CCD841 normal intestinal cell line. By comparing the effects induced by overexpression of either the wild-type or the inactive mutant form of NEU3 on EGFR, we demonstrated that the active form of NEU3 enhanced receptor activation without affecting EGFR mRNA or protein expression. Moreover, through western blots and mass spectrometry analysis, we found that EGFR immunoprecipitated from cells overexpressing active NEU3, unlike the receptor from mock cells and cells overexpressing inactive NEU3, is desialylated. On the whole, our data demonstrate that, besides the already reported indirect EGFR activation through GM3, sialidase NEU3 could also play a role on EGFR activation through its desialylation.

Chromosomal aberrations of cancer-testis antigens in myeloma patients.

Cancer-testis antigens (CTAgs) play a major role in the immune response against cancer, but their biological functions in germ and cancer cells is still unclear. MAGE-C1 and MAGE-C2 are two CTAgs located at the Xq27 region of chromosome X and frequently expressed in multiple myeloma. Chromosomal rearrangements often occur in myeloma. We therefore investigated whether numerical and structural chromosomal aberrations correlate with their protein expression in primary multiple myelomas. To this aim, we designed new fluorescence in situ hybridization probes specific for the MAGE region in the Xq27 region and evaluated simultaneously aberrations of the X chromosome centromere. The comparison of MAGE copy number and chromosome X status revealed that MAGE copy number changes occurred in 6/43 (14%) cases, independent of concomitant X chromosome alterations. These numerical aberrations are less frequent than the expression of MAGE-C1 and MAGE-C2 (63% and 27% of patients, respectively) and do not always correlate with MAGE-C1 and MAGE-C2 expressions, suggesting alternative regulatory mechanisms in the expression of these genes.

ALK testing in lung adenocarcinoma: technical aspects to improve FISH evaluation in daily practice.

INTRODUCTION: Anaplastic lymphoma kinase (ALK) gene rearrangement characterizes a subgroup of patients with lung adenocarcinoma who may benefit from ALK inhibitors. Fluorescence in situ hybridization (FISH) with a break-apart/split-signal strategy is the gold standard to investigate ALK. The cutoff to define ALK positivity has been settled at 15% or greater. A subset of patients has ALK borderline status, showing 15% ± 5% positive cells. Several aspects, both biological and technical, might influence signals evaluation, making FISH interpretation a challenging task. To improve ALK evaluation, we classified the different FISH patterns on the basis of the type of the split signals, namely short, long, far away, and deleted. METHODS: We investigated ALK gene status by FISH in 244 lung adenocarcinomas and in a series of ALK negative cell lines samples, collected in three Institutions. RESULTS: ALK positive profile was found in 12% of patients; long, deleted, and far-away splits were the primary patterns observed. ALK borderline profile characterized 10% of samples; long and deleted splits were significantly more frequent in those borderline finally classified as ALK positive, whereas short split were mostly detected in those borderline patients finally classified as ALK negative (p = 3.4 × 10). In the ALK negative control series, short split was the predominant pattern. Concordance was observed among different operators and probes for both samples and controls. CONCLUSIONS: Difficulties in ALK FISH signal interpretation might be bypassed using this detailed scoring system, which is highly reproducible, helps clarify borderline samples (according to split type), and provides experimental evidence that 15% is a reasonable cutoff to overcome the assay-dependent background noise.

Screening for the FIG-ROS1 fusion in biliary tract carcinomas by nested PCR.

ROS1 rearrangements have been detected in a variety of tumors and are considered as suitable targets of anticancer therapies. We developed a new, quick, specific, and sensitive PCR test to screen for the FIG-ROS1 fusion and applied it to a series of Italian patients with bile duct carcinoma (BTC). Formalin-fixed, paraffin-embedded tissues, derived from 65 Italian BTC patients, and six cell lines were analyzed by nested PCR to investigate the prevalence of a previously reported FIG-ROS1 fusion. The specificity and sensitivity of nested PCR were investigated in FIG-ROS1 positive U118MG cells in reconstitution experiments with peripheral blood mononuclear cells. We found that six out of 65 (9%) BTC patients were positive for the FIG-ROS1 fusion, comprising two out of 14 (14%) gallbladder carcinoma (GBC) patients and four out of 25 (16%) extrahepatic cholangiocarcinoma (ECC) patients. None of the 26 intrahepatic cholangiocarcinoma cases harbored the FIG-ROS1 fusion. All the cell lines were negative for this variant. In conclusion, 14-16% of GBC and ECC were positive for FIG-ROS1. This may have clinical implications, since these patients will potentially benefit from the treatment with specific ROS1 inhibitors.

HER2 in solid tumors: more than 10 years under the microscope; where are we now?

HER2 is a well-recognized mediator of the cancerogenic process. It is dysregulated in a wide range of solid tumors, mainly via protein overexpression and/or gene amplification, thus making HER2 an attractive target for tailored treatment. The anti-HER2 therapy trastuzumab was approved for the treatment of HER2-positive metastatic breast cancer patients more than 10 years ago. Since then, trastuzumab and other HER2-inhibitors have been entered into clinical practice for the treatment of breast cancer and, more recently, have been approved to treat HER2-positive metastatic gastric cancers. Currently, HER2-targeted therapies are under evaluation in other tumor types. Due to the relevance of proper patient selection, the accurate assessment of HER2 status is fundamental. This review will discuss the established knowledge and novel insights into the HER2 story, mainly focusing on breast, gastric and colorectal cancers, as well as providing a brief overview of salivary gland, bladder, ovarian and lung tumors.

The combined expression of VPREB3 and ID3 represents a new helpful tool for the routine diagnosis of mature aggressive B-cell lymphomas.

Genomic studies, such as gene expression profiling and next-generation sequencing studies, have provided new insights into the phenotypic characteristics and pathogenesis of mature aggressive B-cell lymphomas. In particular, mutations in the transcription factors ID3 and TCF3, leading to overexpression of B-cell receptor components such as VPREB3, have been shown to be specific for Burkitt lymphoma (BL) and play an important tumourigenic role by mediating the activation of the pro-survival phosphatidylinositol-3-OH kinase pathway. We performed immunohistochemical analysis by applying commercially available anti-VPREB3 antibody to a large cohort of 185 genetically and immunophenotypically characterized mature aggressive B-cell lymphomas and analyzed these results together with recent data on ID3 expression. The combined expression of both VPREB3 and ID3 was associated with a diagnosis of BL with high sensitivity (0.77), high specificity (0.75) and high negative predictive values (0.96), however, with lower positive predictive value (0.30). Double negative cases were absent in the group of BLs but could be found in approximately one third of the remaining cases of mature aggressive B-cell lymphomas. Further, we could not identify a correlation with MYC, BCL2 or BCL6 aberrations with neither VPREB3 nor ID3 expression in each of the diagnostic groups analyzed. Our results, which are in line with recently discovered mutations in next-generation sequencing studies, suggest that the combined immunohistochemical detection of VPREB3 and ID3 is applicable to the routine diagnostic in case of mature aggressive B-cell lymphomas. In particular, it represents a useful and routinely applicable diagnostic tool to exclude BL diagnosis in case of single positive or double negative cases.

EGFR, KRAS, BRAF, and PIK3CA characterization in squamous cell anal cancer.

BACKGROUND: Combined chemoradiation therapy is the gold standard in the treatment of squamous cell anal cancer (SCAC). However, even if the response rate is very high, many patients eventually relapse or experience a reccurrence, thus requiring an invasive surgical procedure that has severe side effects. Most SCAC tumors overexpress epidermal growth factor receptor (EGFR); therefore, it is reasonable to consider anti-EGFR drugs as a new treatment option, as demonstrated by anecdotal reports. Promising results obtained in other solid tumors, both squamous and non-squamous, have revealed that an increase in the EGFR gene copy number may predict the efficacy of anti-EGFR therapies, while the presence of mutations in downstream members of the EGFR pathway may confer resistance. These markers have been only sporadically considered in SCAC. METHODS: We investigated the status of the EGFR gene using FISH and examined KRAS, BRAF, and PIK3CA hot-spots mutations using sequencing analysis in a cohort of 84 patients affected by SCAC. RESULTS: Twenty-eight patients (34%) showed an increase in EGFR gene copy number due to amplification (4%) or to polysomy (30%). KRAS and PIK3CA gene mutations were found in 4 (5%) and 13 patients (16%), respectively. No mutations were found in the BRAF gene. CONCLUSIONS: The characterization of the EGFR pathway may help in identifying different subgroups of SCAC that have specific molecular features, which may have implications in what targeted therapies are used to treat each patient.