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Targeting Kirsten Rat Sarcoma (KRAS) has been deemed impossible for long time, but new drugs have recently demonstrated promising results. Evidence on the outcome of KRAS-mutant advanced-NSCLC treated with new standard regimens are still scarce. Thus, we aimed at assessing the incidence and clinical impact of KRAS mutations in a real-life population of advanced-NSCLC, exploring the prognostic significance of distinct alterations.The present multicenter retrospective study, conducted by 5 Italian Centers from January 2018 to February 2020, involved 297 advanced KRAS mutant NSCLC. Complete clinico-pathological data were evaluated.Out of 297 patients, 130 carried KRAS_G12C mutation, while 167 presented with mutations other than G12C. Within KRAS_non-G12C group, 73%, 16.8% and 8.9% harboured G12X, codon 13 and Q61H alterations, respectively. No significant differences in survival outcome and treatment response were documented according to KRAS_G12C versus non-G12C, nor KRAS_G12C versus G12X versus other mutations. On univariate analysis ECOG PS, number and sites of metastatic lesions and PD-L1 status significantly impacted on survival. A clear trend towards worse prognosis was apparent in chemotherapy-treated patients, while immunotherapy-based regimens were associated to prolonged survival. Investigating the outcome of PD-L1 ≥ 50% population, we did not detect any significant difference between KRAS_G12C and non-G12C subsets.Here, we report on real-life data from a large retrospective cohort of advanced NSCLC harbouring KRAS alterations, with particular attention to G12C mutation. Our study offers useful clues on survival outcome, therapeutic response and clinico-pathological correlations in KRAS-mutant setting, especially in the upcoming era of KRAS G12C targeting therapy.

ROS proto-oncogene 1 (ROS1) rearrangements are reported in about 1–2% of non-squamous non-small-cell lung cancer (NSCLC). After efficacy of crizotinib was demonstrated, identification of ROS1 translocations in advanced disease became fundamental to give patients the chance of specific and effective treatment. Different methods are available for detection of rearrangements, and probably the real prevalence of ROS1 rearrangements is higher than that reported in literature, as our capacity to detect gene rearrangements is improving. In particular, with next generation sequencing (NGS) techniques, we are currently able to assess multiple genes simultaneously with increasing sensitivity. This is leading to overcome the “single oncogenic driver” paradigm, and in the very near future, the co-existence of multiple drivers will probably emerge more frequently and represent a therapeutic issue. Since recently, crizotinib has been the only available therapy, but today, many other tyrosine kinase inhibitors (TKI) are emerging and seem promising both in first and subsequent lines of treatment. Indeed, novel inhibitors are also able to overcome resistance mutations to crizotinib, hypothesizing a possible sequential strategy also in ROS1-rearranged disease. In this review, we will focus on ROS1 rearrangements, dealing with diagnostic aspects, new therapeutic options, resistance issues and the coexistence of ROS1 translocations with other molecular alterations.

AIMS Spitzoid tumours have been shown to harbour exclusive kinase fusions. Few studies have analysed substantial numbers of ROS1-rearranged lesions. The aim of the present study was to investigate also their immunohistochemical profile. METHODS AND RESULTS Among a group of 35 spitzoid tumours, of which 34 were consecutively diagnosed in a 3-year period, we found 11 ROS1 cases that were immunohistochemically positive, from 10 patients, eight of whom were female and two of whom were male, and who were aged 3-52 years (median, 29 years); most lesions (eight) were localized on the lower extremities. Four patterns of immunostaining were observed: cytoplasmic granular diffuse (six cases), sparse cytoplasmic granules (three cases), paranuclear dots (one case), and nuclear (one case). Fluorescence in-situ hybridisation (FISH) analysis showed all cases to be rearranged (cut-off of >15%). RNA next-generation sequencing (NGS) analysis showed specific fusions of ROS1 in four cases: two with PWWP2A, one with PPFIBP1, and one with ZCCHC8. DNA NGS analysis showed in five cases, specific mutations of AKT, EGFR, NRAS, MYC, ALK, and KIT. ROS1 lesions belonged predominantly to the 'atypical Spitz tumour' group, and showed mainly a nested histological pattern. Interestingly, one patient developed two ROS1-positive lesions. CONCLUSIONS Immunohistochemistry showed 100% sensitivity and specificity as compared with the FISH results, corresponding to ROS1 rearrangement in 31% of cases studied. These observations shed new light on the value of immunohistochemical evaluation of ROS1 in spitzoid tumours. ROS1 patterns of immunostaining probably reflect different subcellular localisations of ROS1 fusions, although no specific correlations were found in the cases studied. Immunohistochemistry and FISH were the most sensitive techniques for detecting ROS1 rearrangement in this subset of neoplasms.

ROS proto-oncogene 1 (ROS1) rearrangements are reported in about 1–2% of non-squamous non-small-cell lung cancer (NSCLC). After efficacy of crizotinib was demonstrated, identification of ROS1 translocations in advanced disease became fundamental to give patients the chance of specific and effective treatment. Different methods are available for detection of rearrangements, and probably the real prevalence of ROS1 rearrangements is higher than that reported in literature, as our capacity to detect gene rearrangements is improving. In particular, with next generation sequencing (NGS) techniques, we are currently able to assess multiple genes simultaneously with increasing sensitivity. This is leading to overcome the “single oncogenic driver” paradigm, and in the very near future, the co-existence of multiple drivers will probably emerge more frequently and represent a therapeutic issue. Since recently, crizotinib has been the only available therapy, but today, many other tyrosine kinase inhibitors (TKI) are emerging and seem promising both in first and subsequent lines of treatment. Indeed, novel inhibitors are also able to overcome resistance mutations to crizotinib, hypothesizing a possible sequential strategy also in ROS1-rearranged disease. In this review, we will focus on ROS1 rearrangements, dealing with diagnostic aspects, new therapeutic options, resistance issues and the coexistence of ROS1 translocations with other molecular alterations.

Background Mismatch-repair-deficiency resulting in microsatellite instability (MSI) may confer increased radiosensitivity in locally advanced/metastatic tumors and thus radiotherapy (RT) potentially might have a changing role in treating this subset of patients, alone or in combination with checkpoint inhibitors. Case presentation We report a 76 year-old Italian male patient presenting with locally advanced undifferentiated prostate cancer (LAPC), infiltrating bladder and rectum. Molecular analysis revealed high-MSI with an altered expression of MSH2 and MSH6 at immunohistochemistry. Two months after 6 chemotherapy cycles with Docetaxel associated to an LHRH analogue, a computed tomography scan showed stable disease. After palliative RT (30 Gy/10 fractions) directed to the tumor mass with a 3D-conformal setup, a follow-up computed tomography scan at 8 weeks revealed an impressive response that remained stable at computed tomography after 9 months, with sustained biochemical response. To our knowledge, this is the first case of such a sustained response to low dose RT alone in high-MSI LAPC. Conclusions Routine evaluation of MSI in patients with locally problematic advanced tumors might change treatment strategy and treatment aim in this setting, from a purely palliative approach to a quasi-curative paradigm.

The prognosis of patients with colorectal liver metastases (LMs) is mostly established on clinical variables or on the anatomic extent of colorectal cancer (CRC). Histopathological factors of LMs which may actually reflect the biological aggressiveness of the tumor are not routinely considered to define the risk of worse clinical outcome in those patients. The number of poorly differentiated clusters (PDCs) of neoplastic cells in primary CRC is associated with metastatic risk and bad prognosis, but PDC presence in LMs has been barely analyzed thus far. We assessed PDC presence in the histological slides of surgically resected and synchronous LMs in 63 patients with CRC who had been not submitted to any neoadjuvant treatments. Then, we analyzed its association with patients' cancer-specific survival (CSS) or progression-free survival. The presence of PDCs (P = .016) and PDC localization at tumor edge of LMs (P = .0004) were significantly associated with shorter CSS. PDC presence at the periphery of LMs and positive resection margin were independent prognostic variables for CSS. PDC localization at the tumor edge of LMs was a significant (P = .0079) and independent prognosticator of shorter progression-free survival. Our data suggest that PDC presence and peripheral localization in LMs may be relevant to predict outcome and useful for clinical decision making in patients with colorectal synchronous LMs.

Stage I colorectal carcinoma has excellent prognosis, with 5-year survival rate up to 95%. The occurrence of lymphovascular invasion, tumor budding, high number of PDC, or lymph node micrometastases is associated with tumor progression. The aim of this study was to evaluate the mutational status of 62 stage I colorectal carcinomas (CRC) (taken from 37 patients surviving more than five years since the initial diagnosis and from 25 patients who died of disease) and to correlate it with histopathological features and the clinical outcome. Mutations of KRAS, NRAS, BRAF, and PIK3CA genes were analyzed through Myriapod Colon Status Kit, using the high-throughput genotyping platform Sequenom MassARRAY System. Mutations in those genes were found in 31 cases (50%) and mainly in those with poor prognosis. The most frequent mutations occurred at codons 12 and 13 of the KRAS gene (40% of cases). We found concomitant PIK3CA mutations in 5 cases (8%). The presence of PIK3CA mutations was mainly observed in tumors with poor prognosis and with unfavorable histopathological prognostic features. High PDC grade (P=0.0112), the presence of tumor budding (P=0.0334), LVI (P<0.0001), KRAS mutations (P=0.0228), PIK3CA mutations (P=0.0214), multiple genetic mutations in KRAS and PIK3CA genes (P=0.039), and nodal micrometastases (P<0.0001) were significant prognostic variables for CSS. The presence of LVI was the only independent and statistically significant prognostic variable for CSS in our cohort of pTNM stage I CRCs. The analysis of KRAS/PIK3CA mutational status may be used to identify patients with stage I CRC at high risk of bad outcome and who may need additional treatments, including biological therapies.

Surrogate molecular classification identifies different subtypes of invasive breast carcinoma on the basis of their immunohistochemical markers. The purpose of the study is to verify whether the immunohistochemical markers and surrogate molecular subtypes can be correctly assessed on the core needle biopsy (CNB) when compared with the corresponding surgical excision (SE), with or without neoadjuvant treatment (NAT). Cases with invasive carcinomas identified on both CNB and SE were retrospectively selected. With immunohistochemistry for estrogen receptors (ER), progesterone receptors (PgR), Ki67, human epidermal growth factor receptor 2 (Her2), and molecular analysis for Her2, surrogate molecular classification was determined in 4 and 5 groups, according to the 2013 St Gallen consensus. A total of 1067 cases was considered and complete data for surrogate molecular classification were available for 988 cases (655 without NAT, 333 with NAT). Without NAT, concordance was strong for ER and Her2, moderate for PgR, and weak for Ki67; concordance for surrogate molecular classification was moderate. After NAT, lower concordance rates were recorded, with significant reduction of PgR (P

Background Liquid biopsy consists in the quantification and qualification of circulating cell-free DNA (cfDNA) and tumor-derived DNA (ctDNA) for cancer recognition. Recently, the characterization of seminal cfDNA (scfDNA) has been reported as a possible biomarker for prostate cancer (PCa) diagnosis. Methods Thirty patients with histologically proven PCa, 33 with benign prostate hyperplasia (BPH) and 21 healthy controls were enrolled. cfDNA was extracted from seminal fluid samples. cfDNA quantification and analysis were performed using Qubit ssDNA Kit and Agilent 2100 Bioanalyzer. Statistical analysis included: Levene's test, Shapiro-Wilk, Kolmogorov-Smirnov and Kruskal Wallis tests. Results Median cfDNA was significantly higher in PCa patients 428.45 ng/mL (173.93–1159.62) compared to BPH patients 77.4 ng/mL (18.23–501) and healthy controls 25.4 ng/mL (15.37–76.62). scfDNA fragments longer than 1000 base-pairs were more common in patients with PCa compared to those with BPH and controls. Conclusions scfDNA concentration and fragment size differed significantly in the three groups of PCa, BPH and healthy controls. Both parameters are potential clinical biomarkers for PCa and can be used in both early diagnosis and follow-up. Using automated systems for high-throughput cfDNA quantification could improve the reproducibility of the method and facilitate the implementation of liquid biopsies in the clinical setting.

Background: HER2+ metastatic breast cancer (MBC) is a poor prognosis disease, unusually curable. To date, no predictive factors have been clearly correlated with long-term response to anti-HER2 agents. Methods: 54 HER2+ MBC patients treated with HER2 targeted therapy as first line treatment were analysed: 40 with a time to progression longer than 3 years in Long Responders (LR) group and 14 with a progression disease within one year of anti-HER2 therapy in a control group named Early Progressors (EP). The expression of 770 genes and 13 molecular pathways were evaluated using Nanostring PanCancer pathway panel performed on FFPE BC tissues. Results: Considering baseline patients and tumor characteristics, EP women had more CNS spread and more metastatic burden of disease compared to LR (p > 0.05). Gene expression analysis identified 30 genes with significantly different expression in the two cohorts; five were driver genes (BRCA1, PDGFRA, AR, PHF6 and MSH2). The majority of these genes were over-expressed, mainly in LR patients, and encoded growth factors, pro- or anti-inflammatory interleukins and DNA repair factors. Only four genes were down regulated, all in EP group (TNFSF10, CACNG1, IL20RB and BRCA1). Most of these genes were involved in MAPK and PI3K pathways. MAPK pathway was differently expressed between LR and EP (p = 0.05). PI3K was the only pathway overexpressed in EP patients. Conclusions: Whole genome expression analysis comparing LR vs. EP identified a group of genes that may predict more favourable long-term outcomes. Up-regulation of MAPK and down-regulation of PI3K pathway could be a positive predictive factors. Further clinical implications are warranted. Abbreviations: BC: breast cancer; MBC: metastatic breast cancer; LR: long responder; EP: early progressor; FFPE: formalin-fixed paraffin-embedded; CNS: central nervous system; PFS: progression free survival; OS: overall survival.

Background.Everolimus has been shown to overcome endocrine resistance in hormone receptor positive advanced breast cancer patients. Predictive biomarkers of everolimus efficacy have been investigated in primary breast cancer tissue without finding univocal results. The goal of this study was to investigate the mutational burden in the metastatic site of endocrine-resistant tumors treated with everolimus plus exemestane.Patients and Methods.Mass Array Sequenom platform was used to analyse genetic status of 18 cancer-related genes in 25 archival tumor specimens from metastatic lesions and available primary matched breast cancer tissue of patients treated with everolimus and exemestane for advanced disease. An exploratory analysis of everolimus efficacy in terms of progression free survival benefit and single gene mutation was carried out.Results. The overall detection rate of mutation was 30% and 38% from metastatic and primary breast cancer samples, respectively.AKT1E17Kwas the most frequent mutated gene. No primary breast cancer and matched relapse maintained the same mutation profile. Considering molecular pathways, the most of the genes belong to PI3K pathway (AKT1E17K,PI3KCAE545K, andKITG565R,S709F). In patients with detected mutations in breast and/or recurrence tissue the median PFS was 5,6 months while in the subgroup of patients with no mutations the median PFS was 7,5 months.Conclusions. The mutational status of breast cancer recurrence allows the identification of some genes potentially correlating tumor response/resistance to everolimus. The most frequently mutated genes were involved in the PI3K/AKT/mTOR pathway highlighting that the deregulation of this pathway in the relapse plays a crucial role in the mechanisms of everolimus resistance/sensitivity. Owing to the small sample size and the retrospective nature of the study, these correlations need to be validated in a large prospective study.

Background Emerging literature data are showing that a change in human epidermal growth factor receptor (HER2) status adversely affects breast cancer patient's prognosis. The aim of this study was to evaluate the prognostic impact of HER2 loss in patients with HER2-positive disease treated with neoadjuvant therapy with or without anti-HER2 agents. Methods One hundred and seven consecutive HER2-positive patients were identified from a prospectively maintained database. The first cohort includes 40 patients treated with chemotherapy (CT) alone. The second cohort includes 67 patients treated with neoadjuvant CT plus anti-HER2 agents (trastuzumab and/or lapatinib). HER2 expression was evaluated by immunihistochemistry or fluorescence in situ hybridization on pretreatment core biopsy and on surgical specimen after therapy. Results The rates of pathologic complete response (pCR) and breast-conserving surgery were higher in the CT + anti-HER2 cohort. A loss of HER2 expression was observed in 40% of the patients with residual disease after CT alone versus 14.7% of the patients after CT + anti-HER2 agents (P = 0.019). Patients not achieving a pCR have a significant increase in the risk of relapse when compared with those achieving a pCR (hazard ratio [HR] 9.55, P = 0.028). Patients with HER2 loss tended to have a higher risk of relapse as comparing to patients with maintained HER2 positivity (HR 2.41, P = 0.063). Conclusion The pCR is confirmed as a powerful predictor of long-term outcome. The rate of HER2 loss is higher in patients receiving neoadjuvant CT without anti-HER2 agents. HER2 status on residual disease after preoperative therapy can be helpful in selecting patients at different risk of relapse, to be included in prospective trial exploring further adjuvant therapy.

The occurrence of secondary EGFR mutation T790M in exon 20 and histologic "transformation" are common mechanisms underlying resistance to EGFR first- or second-generation tyrosine kinase inhibitors (TKI). We describe here on a hitherto unreported mechanism of EGFR TKI resistance synchronously combining squamous-cell carcinoma change and occurrence of the EGFR exon 20 S768I secondary mutation in a 43 year-old woman with stage IV adenocarcinoma harbouring EGFR exon 21 L858R mutation. After 8 months of response to gefitinib, the patient experienced EGFR TKI resistance and died of leptomeningeal neoplastic dissemination.

Recently, a grading system based on the counting of poorly differentiated clusters (PDCs) of neoplastic cells was shown to be a strong predictor of nodal metastases and negative prognosis in colon cancer (CC). In this study, we assessed and compared the mutational status of KRAS, NRAS, and PIK3CA in PDCs and corresponding main tumor tissue of 25 CCs with KRAS mutations. For each tumor, PDC and main tumor tissue were distinctly analyzed by using laser microdissection and mass spectrometry. In 3 CCs, the main tumor tissue had also PIK3CA mutations (C420R: 1; E545K: 1; H1047R: 1), and in 1, it showed NRAS mutation (codon 12). In 20 cases, PDCs had the same biomolecular profile as the main tumor, but in 5, they had different biomolecular profiles. In detail, PDCs had KRAS wild type in 2 cases and additional PIK3CA mutations (E542K: 1; H1047Y: 1; E545Q: 1) in 3. All 3 cases with additional PIK3CA mutations in PDCs had nodal metastases, high pathological TNM stage, and lymphatic invasion. In 1 of 3 cases, additional PIK3CA mutation detected in PDC, but not in the main tumor, was also found in the corresponding nodal metastases. Our findings show for the first time that heterogeneous biomolecular profile previously observed in CC may depend on different histologic aspects of the lesion. Because PDCs may represent the tumor cells with the highest potential to metastatize, their molecular status may be relevant for the prediction of response to targeted therapies.

Background The counting of poorly differentiated clusters of 5 or more cancer cells lacking a gland-like structure in a tumor mass has recently been identified among the histological features predictive of poor prognosis in colorectal cancer. Main body Poorly differentiated clusters can easily be recognized in the histological sections of colorectal cancer routinely stained with haematoxylin and eosin. Despite some limitations related to specimen fragmentation, counting can also be assessed in endoscopic biopsies. Based on the number of poorly differentiated clusters that appear under a microscopic field of a ×20 objective lens (i.e., a microscopic field with a major axis of 1 mm), colorectal cancer can be graded into malignancies as follows: tumors with

Objectives Optimal procedures for adjuvant treatment and post-surgical surveillance of resected non-small-cell lung cancer remain under discussion. Pathological features are the main determinant of follow-up therapy but have limited ability to identify patients at risk of recurrence. Increasingly, molecular markers are incorporated into clinical decision-making, including measures of tumor growth. The CCP score is a quantitative, molecular measure of proliferation derived from the RNA expression of 31 cell cycle genes and a component of the molecular prognostic score (mPS). The mPS score is a linear combination of CCP score and pathological stage. CCP score and mPS are independent predictors of survival in resected lung adenocarcinoma. Materials and methods CCP scores were determined by RT-qPCR for 318 patients diagnosed with stage I–II lung adenocarcinoma. Association of mPS and CCP score with distant recurrence and lung-cancer specific survival was assessed in Cox proportional hazards regression models adjusted for age, gender, tumor size, pathological stage and pleural invasion. Distant recurrence-free survival and lung-cancer specific survival by mPS risk group were calculated by Kaplan-Meier survival analysis. Results CCP scores were obtained for 205 stage I and 84 stage II patients. CCP score and mPS were independent markers of distant recurrence (CCP: HR 1.62, 95%CI 1.15-2.29, pÂ=Â0.0055; mPS: HR 2.22, 95%CI 1.11-4.44, pÂ=Â0.023). Patients with low mPS tumors were at significantly reduced risk of distant recurrence (log-rank pÂ=Â4.2Â×Â10−5). Among stage I patients, stratification by mPS identified a patient group with increased risk of distant recurrence (36%, 95%CI 28–46%, log-rank pÂ=Â0.0011) Conclusions The molecular prognostic score stratifies early-stage, resected lung cancer patients for risk of distant recurrence and could be useful to inform treatment and surveillance decisions.

Mantle cell lymphoma rarely affects the skin and is usually a secondary involvement. The present case illustrates a primary cutaneous mantle cell lymphoma of the leg, with blastoid morphology and aberrant expression of CD10 and bcl-6, which was misinterpreted at the beginning as diffuse large B-cell lymphoma. A larger panel of immunohistochemical markers, including cyclin-D1, and molecular investigation showing the typical translocation (t11;14), pointed toward the correct diagnosis. Cutaneous diffuse B-cell lymphomas with unusual morphology should be interpreted cautiously, and the diagnosis made on the basis of an appropriate panel of antibodies and molecular studies.

Sclerosing hemangioma (SH) is an uncommon pulmonary tumor thought to derive from primitive respiratory epithelium consisting of 2 cell populations (cuboidal surface and polygonal stromal cells) and sharing some clinical characteristics (frequent occurrence in nonsmoking women of Asian ethnicity) with bronchioloalveolar carcinoma with which it has been suggested a possible common origin. We investigated 11 cases of SH by immunohistochemistry, fluorescence in situ hybridization, and polymerase chain reaction-based microsatellite and mutational analyses with particular emphasis on possible alterations of microsatellite loci located at tumor suppressor genes (FHIT, p16, Rb, and p53) involved in lung adenocarcinoma genesis and EGFR, HER2, and K-RAS genes. Although EGFR expression was observed in all tested cases, none showed HER2 immunostaining. Fluorescence in situ hybridization and mutational analysis of EGFR and HER2 and also K-RAS sequencing did not reveal molecular alterations, whereas allelic losses at p16 and Rb loci (4 and 2 out of 9 tested cases, respectively) with an identical microsatellite allelic loss pattern in both cuboidal and polygonal cells were observed. The finding of microsatellite alterations in chromosomal regions related to genes deeply involved in early stage lung adenocarcinoma could suggest a possible link between SH and bronchioloalveolar carcinoma, but tumor pathway promoted by EGFR, HER2, and K-RAS does not represent a common molecular mechanism of tumorigenesis. Microsatellite alterations identified in cuboidal and polygonal cells further confirm the clonal and neoplastic nature of both components of SH.

A novel grading system based on the counting of poorly differentiated clusters (PDC) of neoplastic cells at the invasive margin and in the tumour stroma was recently introduced among the histological parameters predictive of adverse clinical outcome in colorectal cancer (CRC). The aim of this study was to correlate the histological grade based on PDC and the mutational status of KRAS, NRAS and BRAF genes in 175 consecutive CRCs. The highest PDC count under the objective lens of a ×20 microscopic field in each tumour was considered for grading assessment, so that PDC counts

Background. The CHER-LOB randomized phase II study showed that the combination of lapatinib and trastuzumab plus chemotherapy increases the pathologic complete remission (pCR) rate compared with chemotherapy plus either trastuzumab or lapatinib. A biomarker program was prospectively planned to identify potential predictors of sensitivity to different treatments and to evaluate treatment effect on tumor biomarkers. Materials and Methods. Overall, 121 breast cancer patients positive for human epidermal growth factor 2 (HER2) were randomly assigned to neoadjuvant chemotherapy plus trastuzumab, lapatinib, or both trastuzumab and lapatinib. Pre- and post-treatment samples were centrally evaluated for HER2, p95-HER2, phosphorylated AKT (pAKT), phosphatase and tensin homolog, Ki67, apoptosis, and PIK3CA mutations. Fresh-frozen tissue samples were collected for genomic analyses. Results. A mutation in PIK3CA exon 20 or 9 was documented in 20% of cases. Overall, the pCR rates were similar in PIK3CA wild-type and PIK3CA-mutated patients (33.3% vs. 22.7%; p = .323). For patients receiving trastuzumab plus lapatinib, the probability of pCR was higher in PIK3CA wild-type tumors (48.4% vs. 12.5%; p = .06). Ki67, pAKT, and apoptosis measured on the residual disease were significantly reduced from baseline. The degree of Ki67 inhibition was significantly higher in patients receiving the dual anti-HER2 blockade. The integrated analysis of gene expression and copy number data demonstrated that a 50-gene signature specifically predicted the lapatinib-induced pCR. Conclusion. PIK3CA mutations seem to identify patients who are less likely to benefit from dual anti-HER2 inhibition. p95-HER2 and markers of phosphoinositide 3-kinase pathway deregulation are not confirmed as markers of different sensitivity to trastuzumab or lapatinib. Implications for Practice: HER2 is currently the only validated marker to select breast cancer patients for anti-HER2 treatment; however, it is becoming evident that HER2-positive breast cancer is a heterogeneous disease. In addition, more and more new anti-HER2 treatments are becoming available. There is a need to identify markers of sensitivity to different treatments to move in the direction of treatment personalization. This study identified PIK3CA mutations as a potential predictive marker of resistance to dual anti-HER2 treatment that should be further studied in breast cancer.

Ameloblastomas are epithelial tumors of odontogenic origin, biologically characterized by local recurrence. Among different etiologic factors, HPV infection has been recently postulated to be somehow involved in ameloblastoma etiopathogenesis. To address this issue, we studied 18 ameloblastomas by means of immunohistochemistry, in situ hybridization (conventional and amplified), polymerase chain reaction and nested-polymerase chain reaction analyses using laser capture microdissection in order to detect the occurrence of HPV in this setting. No evidence of HPV infection was detected by morphological examination, immunohistochemistry, in situ hybridization and conventional polymerase chain reaction, while nested-polymerase chain reaction showed a weak positive band in two cases. However, the subsequent restriction enzyme analysis carried out from the nested-polymerase chain reaction amplification products of these two samples excluded the presence of HPV subtypes 16, 18, 31, 33, 35, 52, and 58. The search for HPV 6 and 11 in the same specimens was also negative. In conclusion, our data do not support an etiopathogenetic evidence for HPV in ameloblastoma.

Purpose This is a randomized, double-blind, placebo-controlled study aimed to evaluate the clinical and biologic effects of letrozole plus lapatinib or placebo as neoadjuvant therapy in hormone receptor (HR) –positive/human epidermal growth factor receptor 2 (HER2) –negative operable breast cancer. Methods Ninety-two postmenopausal women with stage II to IIIA primary breast cancer were randomly assigned to preoperative therapy consisting of 6 months of letrozole 2.5 mg orally daily plus lapatinib 1,500 mg orally daily or placebo. Surgery was performed within 2 weeks from the last study medication. Clinical response was assessed by ultrasonography. Pre- and post-treatment samples were evaluated for selected biomarkers. Fresh-frozen tissue samples were collected for genomic analyses. Results Numerically similar clinical response rates (partial + complete response) were observed (70% for letrozole-lapatinib and 63% for letrozole-placebo). Toxicities were generally mild and manageable. A significant decrease in Ki-67 and pAKT expression from baseline to surgery was observed in both arms. Overall, 34 patients (37%) had a mutation in PIK3CA exon 9 or 20. In the letrozole-lapatinib arm, the probability of achieving a clinical response was significantly higher in the presence of PIK3CA mutation (objective response rate, 93% v 63% in PIK3CA wild type; P = .040). Conclusion The combination of letrozole-lapatinib in early breast cancer was feasible, with expected and manageable toxicities. In unselected estrogen receptor–positive/HER2-negative patients, letrozole-lapatinib and letrozole-placebo resulted in a similar overall clinical response rate and similar effect on Ki-67 and pAKT. Our secondary end point findings of a significant correlation between PIK3CA mutation and response to letrozole-lapatinib in HR-positive/HER2-negative early breast cancer must now be independently confirmed.

Extragenital Bowen's disease (EBD) has rarely been studied for the presence of human papillomaviruses (HPVs). Twenty consecutive patients with EBD were investigated for the presence of HPVs using in situ hybridization with a generic probe that can detect HPV DNA types 6, 11, 16, 18, 30, 31, 33, 35, 45, 51, and 52 and specific probes for HPV DNA types 6/11, 16/18, and 31/33/35. All cases were tested with the polymerase chain reaction (PCR) technique employing the L1 consensus primer pair, MY11 (primer for the positive strand) and MY9 (primer for the negative strand) complementary to genital and dermal HPV types. Seven caucasian patients, five males and two females, with an average age of 70.4 years, showed positive in situ hybridization (ISH) for HPV DNA. The positivity varied from 5 to 40% of neoplastic cells. Three of seven of the ISH DNA-positive cases showed a positive PCR for DNA HPVs. The role of HPVs in human tumors is not fully understood since oncogenic types of HPVs have been found in normal tissue and the actions of cofactors have been postulated. Bowen's disease usually occurs in elderly people in whom the efficiency of the immune systems may be compromised. The association between HPV infection and low efficiency of the immune response may be responsible for HPV-related Bowen's disease in elderly people.

Background Tumor phenotype may change during breast cancer progression. This study evaluates the prognostic impact of receptor discordance between paired primaries and recurrences. Patients and methods One hundred and thirty-nine patients underwent histological sampling of suspected breast cancer recurrence. All the pathology assessments [ER, PgR and human epidermal growth factor receptor 2 (HER2)] on both primaries and confirmed recurrences were performed at the same laboratory. Results A breast cancer recurrence was confirmed in 119 cases. Rates of discordance were 13.4%, 39% and 11.8% for ER, PgR and HER2, respectively. Ninety-two patients maintained the same tumor phenotype [i.e. the same hormone receptors (HR) and HER2 status], whereas 27 (22.7%) changed during progression. The loss of HR positivity and the loss of HER2 positivity resulted in a worse post-recurrence survival (P = 0.01 and P = 0.008, respectively) and overall survival (OS; P = 0.06 and P = 0.0002, respectively), compared with the corresponding concordant-positive cases. Tumor phenotype discordance was associated with worse post-recurrence and OS (P = 0.006 and P = 0.002, respectively); those cases who turned into triple-negative experienced the poorest outcome, respect to the concordant group (P = 0.001, OS). Conclusions We demonstrated for the first time an impact on OS of phenotype discordance between primary breast cancer and relapse. Among discordant cases, receptor loss resulted in the main determinant of poorer outcome.

The study reviews the endoscopic and histological features of human cytomegalovirus (HCMV) infections of the upper gastrointestinal (UGI) tract.Clinical histories, endoscopic findings and bioptic specimens of 30 cases of HCMV infection of the UGI tract, diagnosed in a University Hospital in a 10-year period, were reviewed. In all cases, viral inclusion bodies were detected in routine histopathological sections and the diagnosis was confirmed with immunohistochemistry.Six patients were HIV+, whereas four had received organ transplantations, one was affected by common variable immunodeficiency and four had a recent history of malignancy. No other pathologic condition was evidenced in the remaining 15 cases. Mucosal alterations were endoscopically observed in the stomach (19 cases), esophagus (9), cardias (6) and duodenum (1), and multiple organs being synchronously affected in five patients (3 HIV+, 2 with history of malignancy). The antropyloric area was the most frequently affected site. Single ulcers were detected in 11 cases and multiple ulcers in 8, whereas mucosal thickenings (in the form of localized thickenings, polyps or rugal hypertrophy) were present in 13 patients. Thickenings of the mucosa were detected only in the stomach. At histology, necrotic material and granulation tissue were associated with moderate or marked lympho-plasmacytic infiltrate and foveolar hyperplasia in ulcerative lesions, whereas lesions labeled as mucosal thickenings showed mild or moderate chronic inflammatory infiltrate and foveolar hyperplasia.Endoscopic manifestations of UGI tract involvement in HCMV infection are not specific, varying from erythematous mucosa to ulcers to mucosal thickenings.

Background: Identification of the clinical behavior of atypical Spitzoid tumors with conflicting histopathologic features remains controversial. Objective: We sought to assess whether molecular findings may be helpful in the diagnostic and prognostic assessment of atypical Spitzoid tumors. Methods: A total of 38 controversial, atypical Spitzoid lesions (1 mm in thickness) were analyzed for clinicopathological features, chromosomal alterations by fluorescence in situ hybridization (FISH) analysis (RREB1/MYB/CCND1/CEP6), BRAF(V600E) mutation by allele-specific real-time polymerase chain reaction confirmed by sequencing, and H-RAS gene mutation by direct sequencing. Results: Atypical Spitzoid lesions developed in 21 female and 17 male patients (mean age 22 years). Nine patients underwent sentinel lymph node biopsy and a sentinel lymph node micrometastasis was detected in 4 of these 9 cases. Four additional patients, who did not receive a sentinel lymph node biopsy, experienced bulky lymph node metastases and one experienced visceral metastases and death. Lesions from patients with lymph node involvement showed more deep mitoses (P < .01), less inflammation = .05), and more plasma cells (P = .04). FISH analysis demonstrated the presence of chromosomal alterations in 6 of 25 cases. Correlation with follow-up data showed that the only case with fatal outcome showed multiple chromosomal alterations by FISH analysis. BRAF(V600E) mutation was detected in 12 of 16 cases (75%) and H-RAS mutation on exon 3 was found in 3 of 11 cases (27%). Limitations: Our results require validation in a larger series with longer follow-up information. Conclusions: FISH assay may be of help in the prognostic evaluation of atypical Spitzoid tumors. Diagnostic significance of BRAF(V600E) and H-RAS mutations in this setting remains unclear. (J Am Acad Dermatol 2011;64:919-35)

Background: Epidermal growth factor receptor (EGFR), evaluated by immunohistochemistry, has been shown to have prognostic significance in patients with colorectal cancer. Gene copy number (GCN) of EGFR and KRAS status predict response and outcome in patients treated with anti-EGFR therapy, but their prognostic significance in colorectal cancer patients is still unclear. Methods: We have retrospectively reviewed the baseline EGFR GCN, KRAS status and clinical outcome of 146 locally advanced rectal cancer (LARC) patients treated with preoperative chemoradiotherapy. Pathological response evaluated by Dworak's tumour regression grade (TRG), disease-free survival (DFS) and overall survival (OS) were analysed. Results: Tumour regression grade 4 and TRG3–4 were achieved in 14.4 and 30.8% of the patients respectively. Twenty-nine (19.9%) and 33 patients (19.2%) had an EGFR/nuclei ratio >2.9 and CEP7 polisomy >50% respectively; 28 patients (19.2%) had a KRAS mutation. Neither EGFR GCN nor KRAS status was statistically correlated to TRG. 5-year DFS and OS were 63.3 and 71.5%, respectively, and no significant relation with EGFR GCN or KRAS status was found. Conclusion: Our data show that EGFR GCN and KRAS status are not prognostic factors in LARC treated with preoperative chemoradiation.

The aim of this study was to investigate whether 9p21 status influence the prognosis of the spitzoid melanocytic tumours, peculiar lesions whose biological behaviour cannot be predicted by histopathological criteria alone. Twenty-eight atypical spitzoid tumours, 12 conventional Spitz's nevi and one congenital Spitz's nevus were studied by fluorescent in-situ hybridization (FISH) and multiple ligation-dependent probe amplification (MLPA) for the presence of 9p21 deletion. The 28 patients were aged 3-56years (mean 32, median 35), and follow-up ranged between 4 and 156months (mean 51, median 48). Eight patients (28.5%) experienced lymph node metastasis (three cases with macrometastasis and five with micrometastasis). Of those with macrometastasis, two are alive after 159 and 26 months, whereas a third developed widespread metastases and died after 26months. All of the other patients are alive. Statistically, the thickness (P=0.01) and the diameter (P=0.009) of the lesions significantly correlated with metastasis. Deletion of 9p21 by FISH analysis was observed in eight spitzoid tumours (28.5%), and MLPA demonstrated alterations of 9p21, particularly deletion of CDKN2A, in the same lesions, whereas all Spitz's nevi, except the congenital one, were of unaltered 9p21 status (P0.0001). Deletion of 9p21/CDKN2A did not correlate with the presence of metastasis. Alterations at 9p21 locus are significantly more frequent in spitzoid tumours than in Spitz's nevi, but do not predict their biological behaviour.

Background Cetuximab improves activity of chemotherapy in metastatic colorectal cancer (mCRC). Gene copy number (GCN) of epidermal growth factor receptor (EGFR) has been suggested to be a predictive factor of response to cetuximab in patients (pts) with mCRC; on the contrary, K-ras mutation has been associated with cetuximab resistance. Patients and methods We have conducted a phase II study with cetuximab administered weekly for 3 weeks as single agent and then with 5-fluorouracil and radiation therapy as neo-adjuvant treatment for locally advanced rectal cancer (LARC). EGFR immunohistochemistry expression, EGFR GCN and K-ras mutation were evaluated on diagnostic tumor biopsy. Dworak's tumor regression grade (TRG) was evaluated on surgical specimens. Results Forty pts have been treated; 39 pts are assessable. TRG 3 and 4 were achieved in nine (23.1%) and three pts (7.7%) respectively; TRG 3–4 rate was 55% and 5.3% in case of high and low GCN, respectively (P 0.0016). Pts with K-ras mutated tumors had lower rate of high TRG: 11% versus 36.7% (P 0.12). In pts with wild-type K-ras, TRG 3–4 rate was 58.8% versus 7.7% in case of high or low GCN, respectively (P 0.0012). Conclusions In pts with LARC, EGFR GCN is predictive of high TRG to cetuximab plus 5-FU radiotherapy. Moreover, our data suggest that a wild-type K-ras associated with a high EGFR GCN can predict sensitivity to cetuximab-based treatment.

The occurrence of hemangioma in the female genital tract, particularly in uterine cervix, is rare. The majority of them show asymptomatic behavior. Surgical excision remains curative in most of the cases. Conservative therapies such as sclerosing agents, cryotherapy, and CO(2) laser excision may be alternatively applied. We present three cases of hemangiomas of the cervix in asymptomatic women, diagnosed as cavernous hemangioma in two cases and capillary hemangioma in one. All tumors were immunoreactive for CD31, CD34, factor-VIII-related antigen. Focal expression of estrogen receptors was detected. No positivity was obtained with progesterone receptor antibodies. The presence of estrogen receptor in the endothelial cells of the hemangioma of the cervix suggests a direct role of this hormone in the hemangioma development. A possible target therapy is discussed.

Postmortem studies on still-borns and miscarriages are important to define the sex and eventually the morphologic anomalies correlated to chromosomal aberrations. When the conditions for carrying out a cytogenetic study do not exist, these chromosomal alterations can be investigated by nucleic acid fluorescent in situ hybridization (FISH), which can be performed on interphase nuclei, usually on formalin-fixed paraffin embedded tissues or on fresh cytological specimens. The objective of the present study is to prove whether this technique can be successfully applied to formalin-fixed cell touch preparations and cytological specimens obtained from foetal autopsies. The study was carried out 12 abortions some of which were spontaneous and some of which were therapeutic. The materials were formalin-fixed. Cell touch preparations and cytological specimens were obtained. The FISH was performed using X/Y probes (Vysis) and the Aneuvysion Kit (05J38-030, Vysis), the probes being for chromosomes 13/21 and X/Y/18. To verify the reliability of the technique, the same reactions were also performed on fresh analogous materials. The slides were evaluable, and the probes hybridized to interphase nuclei showed distinct signals. All the samples were adequate for FISH analysis without any notable difference in the results. Moreover, it is technically possible to perform this analysis not only on fresh but particularly on formalin-fixed cytological specimens. On the other hand, the use of this type of cytological samples, as compared to formalin-fixed and paraffin embedded tissue sections, has the advantage of presenting intact, noncut nuclei with preserved cytomorphology, avoiding the problems of overlapping nuclei and making the identification of the real chromosomal arrangement easier. Diagn. Cytopathol. 2008;36:633–636. © 2008 Wiley-Liss, Inc.

Introduction. The assessment of hormone receptors (HRs) and human epidermal growth factor receptor (HER)-2 is necessary to select patients who are candidates for hormonal and anti–HER-2 therapy. The evaluation of these parameters is generally carried out in primary tumors and it is not clear if reassessment in metastatic lesions might have an impact on patient management. The primary aim of this analysis was to compare HER-2 and HR status in primary tumors versus metastatic sites in breast cancer patients. Patients and Methods. Seventy-five patients with available samples from primary tumors and paired metastases were included. HER-2 status was evaluated by immunohistochemistry (IHC) and/or fluorescence in situ hybridization (FISH); HR status was assessed by IHC. Results. Nineteen percent of primary tumors were HER-2 positive; 77% were HR positive. Sites of biopsied or resected metastases were: locoregional soft tissues (n = 30), liver (n = 20), central nervous system (n = 5), bone (n = 5), pleura (n = 4), distant soft tissues (n = 3), abdomen (stomach, colon, peritoneum) (n = 3), bronchus (n = 3), and bone marrow (n = 2). For paired metastases, the HER-2 status was unchanged in 84% of cases; two patients changed from positive to negative, while 10 patients converted from negative to positive (agreement, 84%; κ = 0.5681). A change in HR status was observed in 16 cases (21%): nine cases from positive to negative and seven cases from negative to positive (agreement, 78.7%; κ = 0.4158). Conclusions. Further studies are necessary to better define the level of discordance in HER-2 or HR status between primary tumors and paired metastases. However, a biopsy of metastatic disease can be recommended, if feasible with minimal invasiveness, because treatment options might change for a significant proportion of patients.

Background Large cell neuroendocrine carcinoma (LCNEC) is a high-grade neuroendocrine tumor of the lung that shares some clinicopathologic and molecular features with small cell lung carcinoma (SCLC). Optimal treatment has not yet been standardized and significant prognostic factors are lacking. Because c-kit protein overexpression has been recently reported as a negative prognostic factor in SCLC we investigated its expression and prognostic value in a series of LCNEC. Methods Resected LCNEC fulfilling the morphologic criteria of the 1999 World Health Organization classification of lung tumors and showing neuroendocrine differentiation by appropriate immunohistochemical markers were retrospectively reviewed. Immunostaining for c-kit protein expression was performed using the polyclonal antibody CD117. Clinical and pathologic characteristic were reported and analyzed and a survival study was performed. Results Thirty-three patients underwent radical resection. Thirty-one were male (94%) and 32 were smokers (97%). Ten (30.3%), 11 (33.3%), 5 (15.2%), and 7 (21.2%) were at stage IA, IB, IIB, and IIIA respectively. Overall 1-, 3-, and 5-year survival rates were respectively 79%, 58%, and 51%. Survival analysis showed no differences for any of the clinicopathological features except for CD117 immunostaining: 1-year and 3-year survival rates were respectively 91% and 82% for CD117-negative LCNEC, and 72% and 44% for CD117-positive ones ( p = 0.046). Positivity of CD117 was significantly related to recurrence rate: 60% versus 23% for CD117 positive and negative LCNEC respectively ( p = 0.037). Conclusions Radical resection of large cell neuroendocrine carcinoma achieves poor outcomes. The c-kit protein is frequently expressed in this neoplasia and its expression represents a negative prognostic factor. This immunohistochemical marker may represent the basic rationale to select LCNEC for novel targeted therapy.

AIMS: To investigate the sensitivity of an in situ hybridisation system to detect human papillomavirus (HPV) infection in transitional cell bladder cancer and to evaluate the advantages of analysing multiple biopsies; to examine the correlation between HPV tumour infection detected by in situ hybridisation and the presence of serum anti-HPV antibodies detected by enzyme linked immunosorbent assay (ELISA); and to relate the presence of viral infection to grade, stage, and follow up in cases of bladder cancer. METHODS: The in situ hybridisation technique was used with broad spectrum and type specific (6/11, 16/18, 31/33/35) probes against HPV DNA in formalin fixed, paraffin embedded tissues from 43 cases of bladder cancer. The results were analysed for the presence and type of papillomavirus and correlated with clinicopathological variables. RESULTS: The presence of HPV DNA was identified by the in situ hybridisation technique in 17 of 43 cases of bladder cancer; 12 of these were serum antibody positive and 10 had had multiple biopsies. Fifteen of the cases that were negative for HPV DNA by in situ hybridisation had positive serum serology when tested by ELISA. In 14 cases, the HPV was either types 16/18 or types 31/33/35, both of which carry high oncogenic risk. The stage (p < 0.05) and grade (NS) of the tumour and the outcome on follow up (p < 0.05) were correlated with the presence of HPV infection. CONCLUSIONS: ELISA is not useful in identifying patients with HPV positive bladder cancer, but the use of several probes and multiple biopsies increases the detection rate of HPV in neoplastic tissues. The association between tumour virus infection and high grade/high stage tumours and worse outcome suggests that HPV infection of neoplastic tissue has a negative effect on the behaviour and evolution of transitional cell bladder carcinoma.

Aims and background Primary squamous cell carcinoma of the thyroid gland (PSCCT) is an uncommon malignancy characterized by a poor prognosis. A radical surgical approach combined with radiotherapy or chemotherapy is the generally accepted treatment for this tumor. The epidermal growth factor receptor (EGFR) is a transmembrane tyrosine kinase receptor modulating the cell proliferation and biological progression of many human epithelial tumors. The EGFR overexpression in PSCCT suggests an additional therapeutic option for the treatment of this tumor. Methods and study design The clinicopathological features and immunohistochemical profiles of two cases of primary squamous cell carcinoma of the thyroid in a 66-year-old and an 83-year-old woman are presented. EGFR status was valued in both cases. Results Overexpression of EGFR protein was detected in 50% and 75% of the tumor cell membranes. EGRF gene polysomy was detected in both tumors. Conclusions Pharmaceuticals targeting EGFR may help to provide the rationale for an additional, novel therapeutic option for this rare tumor, especially when other therapeutic options have been exhausted.