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Background Epidermal growth factor receptor ( EGFR ) and v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog ( KRAS) mutations, and echinoderm microtubule-associated protein-like 4 ( EML4 ) anaplastic lymphoma kinase ( ALK ) translocation are generally considered to be mutually exclusive. However, concomitant mutations are found in a small number of patients and the effect of these on response to targeted therapy is still unknown. Patients and Methods We considered 380 non–small-cell lung cancer (NSCLC) patients who underwent nonsequential testing for EGFR and EML4-ALK translocation. KRAS mutation analysis was also performed on 282 patients. Results We found 1.6%, 1.1%, and 2.5% of patients who showed a double mutation comprising EGFR and EML4-ALK , EGFR and KRAS , and EML4-ALK and KRAS , respectively. Twenty-eight patients with EGFR mutation underwent first-line therapy with a tyrosine kinase receptor; a clinical benefit was observed in 81.8% of patients with EGFR mutations only and in 67% of those who also showed an EML4-ALK translocation. Twelve patients with an EML4-ALK translocation received crizotinib and 7 of these had disease progression within 3 months (2 had a concomitant KRAS mutation and 1 had a concomitant EGFR mutation). Two patients showed stable disease, 1 of whom also had a KRAS mutation. Two patients obtained a partial response and 1 had a complete response; all harbored an EML4-ALK translocation only. The median overall survival of patients who carried an EML4-ALK translocation alone or concomitant with a KRAS mutation was 57.1 (range, 10.7-not reached) and 10.7 (range, 4.6-not reached) months, respectively. Conclusion Concomitant EGFR, EML4-ALK , or KRAS mutations can occur in NSCLC. Concomitant KRAS mutation and EML4-ALK translocation represents the most common double alteration and confers a poor prognosis.

Background Ductal carcinoma in situ (DCIS) is a heterogeneous disease that has not been investigated as widely as invasive breast cancer. Thus, the search for biomarkers capable of identifying DCIS lesions that may recur or progress to invasive cancer is ongoing. Although conventional steroid hormone receptors, cell proliferation and other important tumor markers have been extensively studied in invasive tumors, little is known about the role played by androgen receptors (ARs), widely expressed in breast cancer, in DCIS. Methods We performed a retrospective study in a series of 43 DCIS patients treated with quadrantectomy only and followed up for a period ranging from 5 to 13 years, to evaluate the prognostic relevance of conventional biomarkers (estrogen receptor [ER], progesterone receptor [PgR], Ki67, human epidermal growth factor receptor 2 [HER2]) and AR. Results Our findings showed that AR and ER were not independent prognostic variables and that an AR/ER ratio cutoff of 1.13 showed a sensitivity of 75% and a specificity of 94% in predicting in situ relapse or progression to the invasive phenotype. Moreover, while the variables considered singly showed area under the curve (AUC) values ranging from 0.52% to 0.77%, the AR/ER ratio reached a very high AUC (0.92%). Conclusions These preliminary results highlight the potentially important role of AR and ER and, in particular, of their ratio, as prognostic indicators of DCIS evolution.

To the Editors: Mutations in the epidermal growth factor receptor (EGFR) gene in patients with nonsmall cell lung cancer (NSCLC) have been correlated with tumour response to treatment with targeted tyrosine kinase inhibitors (TKIs), especially gefitinib and erlotinib [1, 2]. A large number of studies have reported a significantly higher overall response rate, overall survival and time-to-progression in patients with EGFR-activating mutations compared with those with wild-type tumours [3–5]. It has thus become mandatory to perform EGFR mutation analysis in all adenocarcinoma patients to improve treatment opportunities. In addition to EGFR alterations, K-ras mutations are found in ∼30% of adenocarcinomas [6] and each is usually mutually exclusive. About 70% of NSCLC patients present at first diagnosis with advanced disease, and in such cases, morphologic and molecular diagnoses are necessarily based on cytologic samples obtained by different methodological procedures [7]. It has been demonstrated that EGFR mutations can be detected in cytologic specimens containing >50% of neoplastic cells, and that analysis can be performed in samples with as little as 25% tumour cellularity [8]. Moreover, although the most reliable results have been obtained in samples from which ≥100 cells were analysed, it has also been noted that mutation analysis can be performed in as few as 30 isolated cells [9]. In the present study, 66 consecutive NSCLC patients diagnosed between …

1042 Background: The androgen receptor (AR) is widely expressed in breast cancers but its role in estrogen receptor (ER)-positive tumors is still controversial. However, the AR/ER ratio may impact prognosis and the response to antiestrogen endocrine therapy (ET). Methods: We assessed whether AR in primary tumors and/or matched metastases is a predictor of efficacy of first-line ET in advanced breast cancer (ABC). We evaluated patients treated with first-line ET (2002–2011), excluding those receiving concomitant chemotherapy or trastuzumab or pretreated with > 2 lines of chemotherapy. ER, progesterone receptor (PgR), Her2, Ki67 and AR expression was determined by immunohistochemistry. A cut-off of < 1% immunostained cells was used to categorize AR expression. AR expression was analyzed in relation to the other conventional biomarkers (ER, PgR, Her2 and Ki67), best response (CR, PR, SD, PD), and time to progression (TTP) (months). TTP was estimated using the Kaplan-Meier method and compared with the log-rank test. Hazard ratios and their 95% confidence intervals (95% CI) were estimated using the Cox regression model. The Chi-square test was used to evaluate correlations between categorical variables and best response. p values < 0.05 were considered statistically significant. Results: Of the 102 evaluable patients (93% were treated with an aromatase inhibitor), biomarkers were assessed in primary tumors in 70 cases, in metastases in 49 and in 17 in both). Median TTP was 17 months (95% CI 14-21.5, median follow-up 75 months). The overall concordance rate between primary tumors and metastases was 64.7% (95% CI 42%-87.4%) for AR expression. Differences in TTP according to AR status were not statistically significant. AR/PgR ≥ 0.96 was associated with a significantly shorter TTP (HR = 1.65, 95% CI 1.05-2.61, p = 0.030). AR status in primary tumors or metastases was not associated with PD as best response. In contrast, Ki67 > 20% and PgR < 10% showed a significant association with PD as best response. Using a cut off of ≤10% for AR expression, results did not change. Conclusions: AR expression does not appear to be useful to predict the efficacy of ET in ABC. Ki67 and PgR exert a greater impact on the efficacy of hormone therapy than AR.

Tyrosine kinase inhibitors (TKIs) are very efficacious in non-small-cell lung cancer (NSCLC) patients harboring activating Epidermal Growth Factor Receptor (EGFR) mutations. However, about 10% of EGFR wild type (wt) patients respond to TKI, with unknown molecular mechanisms of sensitivity. We considered a case series of 34 EGFR wt NSCLC patients responsive to erlotinib after at least one line of therapy. Responsive patients were matched with an equal number of non-responsive EGFR wt patients. A panel of 26 genes, for a total of 214 somatic mutations, was analyzed by MassARRAY® System (Sequenom, San Diego, CA, USA). A 15% KRAS mutation was observed in both groups, with a prevalence of G12C in non-responders (80% vs. 40% in responders). NOTCH1, p53 and EGFR-resistance-related mutations were found more frequently in non-responders, whereas EGFR-sensitizing mutations and alterations in genes involved in proliferation pathways were more frequent in responders. In conclusion, our findings indicate that p53, NOTCH1 and exon 20 EGFR mutations seem to be related to TKI resistance. KRAS mutations do not appear to influence the TKI response, although G12C mutation is more frequent in non-responders. Finally, the use of highly sensitive methodologies could lead to the identification of under-represented EGFR mutations potentially associated with TKI sensitivity.

S. Bravaccini, M. M. Tumedei, P. Ulivi, W. Zoli, D. Calistri, P. Candoli, D. Amadori and M. PuccettiALK translocation detection in non-small cell lung cancer cytological samples obtained by EBUS-TBNAObjective: As the diagnosis of non-small cell lung cancer (NSCLC) is based on cytology in around 70% ofcases, it is important to use the same material for molecular analyses. Fluorescence in situ hybridization (FISH)is the only approved test for the detection of the translocation and inversion of anaplastic lymphoma kinase(ALK), but the optimal procedures for the fixation or staining of the sample before FISH evaluation have notbeen established. We investigated whether ALK gene status determined by FISH in a prospectively enrolledcase series of patients was affected by fixation and staining.Methods: One hundred and fifteen cytological samples were obtained by transbronchial needle aspiration(TBNA) or endobronchial ultrasound (EBUS)-TBNA from 109 patients with NSCLC. All samples were evalu-ated for epidermal growth factor receptor (EGFR) mutation by pyrosequencing and for ALK rearrangement byFISH. Specimens for ALK determination had been fixed with Cytofix and/or Carnoy’s solution or 10% for-malin (cell blocks) and variously stained.Results: Sixteen (14%) of the 115 samples were mutated for EGFR and 99 (86%) showed wild-type EGFRstatus. Of these 115 samples, 79 (69%) were negative for echinoderm microtubule-associated protein like 4(EML4)-ALK translocation, nine (8%) were positive and 27 (23%) were unevaluable. In particular, 19 (26%)of the 72 Papanicolaou-stained smears fixed with Cytofix were unevaluable because of inadequate samples orcell overlapping; neither of the two May–Gr€unwald –Giemsa-stained samples were evaluable. Ten of 17 smearsused for rapid on-site evaluation (ROSE) and immediately post-fixed in Carnoy’s solution or 80% alcoholwere evaluable.Conclusions: In this series, smears were unevaluable as a result of inadequate samples, cell overlapping orlack of fixation performed immediately after FNA.Keywords: cytological samples, ALK translocation, FISH, EBUS-TBNA, cell fixation, non-small cell lungcancerIntroductionSeveral new targeted drugs have shown promisingresults for the treatment of non-small cell lung can-cer (NSCLC), and some have already been intro-duced into clinical practice. Crizotinib, an orallyactive, small-molecule inhibitor of anaplastic lym-phoma kinase (ALK) and MNNG HOS transforminggene (c-MET), has produced high overall survivalrates in patients with NSCLC whose tumours har-bour the echinoderm microtubule-associated proteinlike 4 (EML4)-ALK rearrangement, indicating theimportance of checking for the presence of thesealterations before making treatment decisions.

Background Although fine needle aspiration (FNA) is the standard diagnostic test for the characterization of a suspicious thyroid nodule, in some cases cytological evaluation is inconclusive. The aim of this study was to determine the role of BRAF mutation in aiding diagnosis and to verify whether archival cytological samples could be suitable for molecular analysis. Methods Eighty-five patients with suspicious (Thy4) or follicular (Thy3) lesions on cytology were resubmitted to a second FNA for BRAF mutation analysis. Of these, 56 subsequently underwent surgery. The usefulness of archival samples for molecular analysis was also studied in a second cohort of 42 patients with a confirmed diagnosis of papillary thyroid carcinoma for whom both archived paraffin-embedded histological samples and cytological smears were available. A further 15 patients with paired fresh FNA and archived cytological and histological samples were recruited. Results BRAF mutation was found in the fresh FNA samples from 10 of 56 patients who had surgery with previous inconclusive cytology (4/45, 9%, Thy3 and 6/11, 55%, Thy4). The BRAF test showed a specificity and positive predictive value of 100% (26/26 and 10/10, respectively), sensitivity of 33% (10/30) and negative predictive value of 57% (26/46). There was absolute concordance between the BRAF results obtained with 42 histological and cytological archived samples. BRAF analysis on 15 archived cytological samples showed absolute concordance with histology, whereas there was one false negative on the matched fresh FNA. Conclusion BRAF analysis is a highly specific test that can facilitate cytological diagnosis in some cases and can also be performed on archived cytological samples.

Background: Epigenetic alterations of specific genes have been reported to be related to colorectal cancer (CRC) transformation and would also appear to be involved in the early stages of colorectal carcinogenesis. Little data are available on the role of these alterations in determining a different risk of colorectal lesion recurrence. The aim of the present study was to verify whether epigenetic alterations present in pre-neoplastic colorectal lesions detected by colonoscopy can predict disease recurrence. Methods. A retrospective series of 78 adenomas were collected and classified as low (35) or high-risk (43) for recurrence according to National Comprehensive Cancer Network guidelines. Methylation alterations were analyzed by the methylation-specific multiplex ligation probe assay (MS-MLPA) which is capable of quantifying methylation levels simultaneously in 24 different gene promoters. MS-MLPA results were confirmed by pyrosequencing and immunohistochemistry. Results: Higher levels of methylation were associated with disease recurrence. In particular, MLH1, ATM and FHIT gene promoters were found to be significantly hypermethylated in recurring adenomas. Unconditional logistic regression analysis used to evaluate the relative risk (RR) of recurrence showed that FHIT and MLH1 were independent variables with an RR of 35.30 (95% CI 4.15-300.06, P = 0.001) and 17.68 (95% CI 1.91-163.54, P = 0.011), respectively. Conclusions: Histopathological classification does not permit an accurate evaluation of the risk of recurrence of colorectal lesions. Conversely, results from our methylation analysis suggest that a classification based on molecular parameters could help to define the mechanisms involved in carcinogenesis and prove an effective method for identifying patients at high risk of recurrence.

Background.Genomic studies have shown that gene expression profiles are similar inin situ(CIS) and invasive breast cancers, suggesting that several biofunctional modifications of the transformation process occur before or during the development of CIS lesion.Methods.We investigated 3 biomarkers in 44 patients with CIS: TG2 (transglutaminase 2), HJURP (Holliday junction recognition protein), and HIF-1α (hypoxia inducible factor-1 alpha).Results.TG2 was more highly expressed than the other two markers and significantly more so in stromal than in tumor cells. HIF-1α evaluation showed a higher expression in both tumor and stromal cells in patients with relapsed G3 tumors, indicating a potential role of this marker in CIS evolution. A greater than sevenfold higher risk of relapse(P=0.050)was observed in patients highly expressing HJURP in stroma and a tenfold higher recurrence risk(P=0.026)was seen in those with a higher stromal HIF-1α expression. An important increase in risk accuracy (AUC 0.80) was obtained when HIF-1α and HJURP were evaluated together.Conclusions.Despite the limited number of relapsed patients, we formulated some hypotheses on the factors responsible for malignant evolution and recurrence which are now being tested in a large case series with a longer follow-up.

Information on hormone receptor and human epidermal growth factor receptor-2 (HER2) expression in breast cancer is acknowledged as mandatory for prognostic stratification and treatment planning. Data on the biological features of African breast cancers are poor. We decided to compare histopathological and biomolecular characteristics (estrogen and progesterone receptor—ER, PgR, and HER2) of Tanzanian and Italian breast cancers. Differences in proliferating index and androgen receptor (AR) expression in triple-negative patients from the two case series were also assessed. Of the 103 consecutive patients seen at the Bugando Medical Center (Mwanza, Tanzania) from 2003 to 2010, who underwent biopsy or surgical resection of primary breast cancer, 69 patients had tissue samples that were evaluable for estrogen receptor (ER), progesterone receptor (PgR), and HER2. Histopathological assessment and biomolecular determinations were performed at the Cancer Institute of Romagna (IRST IRCCS, Meldola, Italy). Caucasian breast cancers were randomly extracted from an electronic database and matched (1:2 ratio) for year of diagnosis and age at diagnosis. Median age of both populations was 51 years (range 27–84). With respect to Caucasian tumors, Tanzanian breast cancers at diagnosis more frequently showed high histological grade (mainly grade 3) (P = 0.03), advanced clinical stage (III or IV) (P\0.001), ER negativity (52.2 %, P\0.001) and high proliferation (P = 0.0002). Triple-negative tumors were over-represented in Tanzanian women. AR was positive in 38.5 and 38 %of triple-negative Tanzanian and Italian breast cancers, respectively. Our results show that histopathological and biomolecular characteristics in Tanzanian and Italian breast cancers differ substantially. The high frequency of poorly differentiated, ER-negative, highly proliferating tumors, together with advanced stage at presentation, could be considered as the main prognostic factors linked to the high mortality rates for breast cancer in the African population.

There are no validated predictors of benefit from anthracyclines. We compared cyclophosphamide, methotrexate, 5-fluorouracil (CMF), and epirubicin in different sequences with CMF alone in a phase III trial on operable breast cancers. Outcomes were analyzed in relation to tumor biological profiles to identify potential predictors of the efficacy of different treatments/drug combinations. Patients with N− or 1–3N+ tumors, were randomized to receive (a) epirubicin (4 cycles) followed by CMF (4 cycles); (b) CMF (4 cycles) followed by epirubicin (4 cycles), or (c) CMF (6 cycles) alone. Immunohistochemical assessments of estrogen (ER) and progesterone (PgR) receptors, HER2 and Ki67 were available for 705 patients (arm A/B/C: 276/269/160). Prognostic and predictive relevance was analyzed by log-rank tests and Cox models. Ki67 > 20 % and absent/low expression of ER and PgR were associated with worsen disease-free (DFS) and overall survival (OS). In patients with triple negative tumors (ER−, PgR−, HER2−), epirubicin-containing regimens yielded better DFS (HR 0.33, 95 % CI 0.17–0.62, P = 0.0007) and OS (HR 0.24, 95 % CI 0.10–0.57, P = 0.001) compared with CMF alone, whereas no differences were found in patients with HER2-positive (HER2+, ER−, PgR−) subtype. Treatment by subtype interaction (HER2-positive vs. others) was significant for DFS (χ2 = 6.72, P = 0.009). In triple unfavorable (ER−, PgR−, Ki67 > 20 %) tumors, the use of epirubicin yielded better DFS (HR 0.45,95 % CI 0.26–0.78, P = 0.005) and OS (HR 0.30, 95 % CI 0.15–0.63, P = 0.001). Epirubicin-containing regimens seem to be superior to CMF alone in patients with highly proliferating, triple negative or triple unfavorable tumors .

Introduction Tyrosine kinase inhibitors (TKIs) and anti-anaplastic lymphoma kinase (ALK) agents are highly effective for the treatment of non-small cell lung cancer (NSCLC) patients harbouring specific alterations, and molecular characterisation of the tumour is needed even when limited tumour material is available. Methods 20 patients with a known epidermal growth factor receptor ( EGFR ) gene status were enrolled: 10 had mutated and 10 had wild type tumours. FISH analysis was performed on one cytological or histological sample to determine EML4-ALK status, after which the same cells scraped off each slide were used to evaluate the EGFR status. Results In the 10 EGFR mutated patients, molecular analysis showed the same results as those obtained before the FISH test. One patient with an EGFR mutation also showed an EML4-ALK translocation, and both FISH-positive and FISH-negative cells maintained the EGFR mutation. Conclusions EGFR mutation analysis can be performed on the same sample previously submitted to the EML4-ALK FISH procedure.

Introduction: After an enthusiastic and promising start to the large scale use of screening programs for the detection of small in situ breast cancers, an analysis of results from different screening studies has led to disappointing and somewhat alarming conclusions. A problem of over diagnosis has emerged, involving, in many cases, unnecessary treatments, and causing a negative psychological and social impact on patients and a considerable economic burden on Health Services. Ductal carcinoma in situ (DCIS) is a heterogeneous disease that has not been investigated as widely as invasive breast cancer. Today women with in situ breast cancer are mainly treated with conservative surgery and often with radiotherapy, even though the real impact of this treatment is still not known. New diagnostic and therapeutic information is thus needed. Although conventional steroid hormone receptors, cell proliferation and other important tumor markers have been extensively studied in invasive tumors, little is known about the role played by androgen receptors (ARs), widely expressed in DCIS. We previously demonstrated in a series of DCIS patients treated with surgery alone that the AR/estrogen receptor (ER) ratio is an important prognostic marker. In the present study, we analyzed these markers in a series of DCIS patients treated with surgery and radiotherapy. Methods: We performed a retrospective study on a series of 42 DCIS patients treated with quadrantectomy and radiotherapy and followed up for a period ranging from 5 to 13 years to evaluate the prognostic relevance of ER and ARs determined by immunohistochemistry. Results: Our findings showed that AR expression was significantly higher in relapsed patients than in non relapsed patients, and that ER levels were higher in patients who did not relapse with respect to those who did. The AR/ER ratio was significantly different in the two subgroups. Moreover, when the variables were considered singly, area under the curve (AUC) values were 0.85 for ARs, 0.62 for ER and 0.79 for the AR/ER ratio. Conclusions: In agreement with our previous work on a series of DCIS patients treated with surgery alone, the preliminary results from the present study highlight the important role of AR and of the AR/ER ratio as prognostic indicators of relapse in patients treated with surgery and radiotherapy. Our findings also suggest that the treatment used was not capable of controlling disease or of eliminating the risk of recurrence. New avenues for tailored therapy must be sought. Citation Format: Sara Bravaccini, Sara Ravaioli, Maria Maddalena Tumedei, Rosella Silvestrini, Flavia Foca, Ilaria Massa, Roberta Maltoni, Andrea Rocca, Secondo Folli, Federico Buggi, Annalisa Curcio, Maurizio Puccetti, Luigi Serra, Elisabetta Pietri, Daniele Andreis, Alberto Farolfi, Dino Amadori. Clinical relevance of androgen and estrogen receptors in patients with ductal carcinoma in situ of the breast treated with surgery and radiotherapy. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B44.

This is a report of an angiolipoma of the thyroid gland, an extremely rare entity. A thorough search of the literature revealed only one previously reported example. The patient was a 77-year-old woman with a history of a nodular lesion of the thyroid in the context of a multinodular goiter. A fine needle aspiration highlighted the presence of abundant adipocytes associated with numerous dilated vessels. Histopathologic examination of the lobectomy specimen documented the presence of a tumor composed of two main elements, namely, mature adipocytes and proliferating vessels, some of the latter containing fibrin thrombi.

e22227 Background: Ductal carcinoma in situ (DCIS) is a heterogeneous disease that has not been investigated as widely as invasive breast cancer. Thus, the search for biomarkers capable of identify...

e19086 Background: Tyrosine kinase inhibitors (TKIs) are the first choice of treatment in a subset of patients with non- small cell lung cancer (NSCLC) harboring specific epidermal growth factor re...

5594 Background: In Sub-Saharan Africa cervical cancer represents 24% of all cancers and accounts for 23% of all cancer deaths in women. An early diagnosis program for breast and cervical cancer (Vanda Project) is ongoing in Mwanza and the surrounding lake area (12 districts with a population of 14,000,000). The aim of this project was to screen women aged 15-64 years living in the 12 districts. Methods: Women were invited to participate through local media and a mobile unit operating within the districts. A multidisciplinary team including medical oncologists was involved. Interventions consisted in Pap smear, clinical breast examination, breast self-examination training and training of district physicians to perform Pap smear and breast examination. Results: From May to December 2012, 2155 women from the districts of Shinyanga, Bukumbi, Kibara, Serengema and Musoma took part in the program: of these 91 (4%) had clinically evident cervical cancer. Age distribution classes were: < 18 years, 12% ; 18-35, 38%; 36-50, 41%; > 50, 9%. As expected a high stage distribution at diagnosis was observed: 30% stage III and 20% stage IV. Among the women with no clinical evidence of cancer, 408 samples were analyzed by cytology and 4% consisted of inadequate material. Of the remaining 392 samples, 85 (22%) were normal, 216 (55%) were infections (chiefly mycotic), 72 (18%) were precancerous lesions (50% H-SIL according to Bethesda classification) and 19 (5%) were positive for cancer (mainly stages III-IV). Precancerous lesions turned out to be cancer at histology in 44% of cases. 22% of precancerous lesions and 8% of clinically evident cancer were HIV-positive. Conclusions: This experience shows the high feasibility, good compliance and usefulness of a screening program for the early detection of cervical cancer in this high-risk population. [Table: see text]

e16530 Background: In Tanzania, cervical cancer is the leading cause of death after breast cancer, with the majority of tumors diagnosed at a very advanced stage. Little information is available on the characteristics of cervical cancer in Sub-Saharan African women. We aimed to evaluate histopathological features of cervical cancers and relative margins in a group of African women and to characterize HPV infection. Methods: Tissue samples from32 Tanzanian women with invasive cervical cancer were evaluated in the Biosciences Laboratory of our Institute (IRCCS IRST, Meldola, Italy) as part of a global cancer control project currently ongoing in close cooperation with the Bugando Medical Center of Mwanza, Tanzania. Results: 24/32 (75%) women had squamous carcinoma and 4/32 (12.5%) had adenocarcinoma of the cervix. The HPV test was performed in the entire series but DNA amplification by pyrosequencing was only possible in 27/32 (84.4%). The presence of HPV infection was confirmed in 26/27 (96.3%) cases in both tumor and relative margins. Typization of the virus revealed that a high percentage of the women had HPV 16 virus of whom 12 (46.2 %) had African type 1 and 4 (15.4%) African type 2. Conclusions: A major problem in the cancer tissue samples from the Tanzanian patients was the poor evaluability of DNA amplification due to suboptimal fixation which compromised tissue morphology. Our preliminary study shows that cervical cancer in Tanzanian women is characterized by a very high frequency of HPV-positive tumors, with a high prevalence of HPV 16 African type 1 and 2 infections. The substantial number of HPV infections could explain the high rate of cervical cancer and subsequent mortality in this African population. [Table: see text]

e15055 Background: In literature three studies have described KRAS status in SCAC: they analyzed 139 patients, only 1, 4% were mutated. In this study we analyzed BRAF and PIK3CA status. The evaluation of these two genes is important for predicting the response to anti-EGFR drugs. There is one study that values the positivity of HPV in SCAC. This study with few patients shows that the positivity of HPV16 is predictive for improvement of progression free survival (PFS). Methods: 79 patients were treated for anal cancer at the Oncology center of Forlì, Ravenna and Faenza in the last ten years. Up to now we analyzed 29 patients (9 with relapse of disease). DNA was extracted from paraffin-embedded sections and KRAS, BRAF and PIK3CA gene status were assessed by Pyrosequencing methodology, using 3 different kits: anti-EGFR MoAb response (KRAS status), anti-EGFR MoAb response (BRAF status), and anti- EGFR MoAb response (PIK3CA status) (Diatech, Jesi, Ancona, Italy). Reactions were run on a PyroMark Q96 ID (Qiagen). The presence and genotyping of Human Papilloma Virus (HPV) were assessed using the AMPLIQUALITY HPV-HS Bio and AMPLIQUALITY HPV-Type (AB ANALITICA, Padova, Italy) kit. Results: KRAS and BRAF genes were wild-type in all cases. Conversely, PIK3CA gene resulted mutated in 9 of 31 cases (28.1%). In particular, 7 mutations occurred in exon 9 (4 E454K and 3 E542K) and 2 in exon 20 (H1047R and H1047L) of the PIK3CA gene. Patients without relapse of disease were positive in 94% of cases for HPV16 and in 40% for mutation of PIK3CA. Patients with relapse of disease were positive in 70% of cases for HPV16 and 37,5% for mutation of PIK3CA. Conclusions: KRAS and BRAF genes were wild type in 100% of cases. This study demonstrates that the SCAC could potentially respond to an anti-EGFR drug.The mutation of PIK3CA does not seem to be predictive for response to treatment. Positivity for HPV16 seems predictive for PFS and overall survival. Furthermore, the study shows that PIK3CA mutation may contribute to carcinogenesis in some cases of this tumor. In addition, patients with a mutation of PI3K could have benefit from specific drugs that inhibit this kinase.

10601 Background: There is little information available on the clinical and molecular epidemiology of breast cancer in Sub-Saharan African women. In Tanzania, breast cancer mortality is the second cause of death after cancer of the uterine cervix, with the majority of tumors diagnosed at a very advanced stage. We aimed to compare the distribution of histopathological and biomolecular characteristics (ER, PgR, Ki67, HER2, grading) of breast cancer in African (Tanzania) and Caucasian (Italy) patients. Methods: 142 women with invasive breast cancer, 71 from Tanzania and 71 from Italy, were considered. Histopathological classification and biomolecular determinations were performed at the Biosciences Laboratory of the Cancer Institute of Romagna (I.R.S.T., Meldola, Italy) as part of a joint Cancer Control Project between I.R.S.T and the Bugando Medical Center in Mwanza, Tanzania. All cases were evaluable for histopatological characterization, while biomolecular determination was possible in only 61 of the African patients. Hormone receptor status and Ki67 were determined by IHC; HER2 was evaluated by IHC for the African population and by FISH for the Italian one. Results: Whilst histopathological patterns were similar in the two populations, some biomolecular characteristics differed substantially. Of note, 64% of African breast cancer patients had ER negative tumors compared to only 17% of the Caucasian population. Important differences were observed in high Ki67 (>15%) values: 76% in Tanzanian women vs 58% in Italian breast cancer cases. HER2 positivity rates also differed in the two populations (25% in Tanzanian and 17% in Italian patients), as did the incidence of triple negative disease (13% and 7% in African and Caucasian populations, respectively). Conclusions: This preliminary study shows that breast cancer in African (Tanzanian) women is characterized by a high frequency of HER2 positive, ER negative, highly proliferating tumors. These biological patterns, together with very advanced stage at diagnosis, could be considered the main prognostic factors related to the high mortality rates for breast cancer in this African population.

e18045 Background: Guidelines do not recommend surgical resection of metastatic non-small cell lung cancer (NSCLC), with the exception of single brain or adrenal gland lesions, and further resection in the event of a new lung metastasis is generally not indicated. A different therapeutic approach is used in patients with multiple primary lesions. Can molecular alterations help to distinguish between independent primary tumors and metastatic lesions? Methods: A 74-year old woman underwent bilateral inferior lobectomy in May 2008 for two different synchronous NSCLC (adenocarcinoma and adenocarcinoma in situ, formally known as “bronchioloalveolar carcinoma” – J Thorac Oncol 2011;6:244–285). Three months after surgery the patient developed a single symptomatic brain metastasis. A CT scan also highlighted a suspect lingular lesion of the lung. Stereotaxic brain radiotherapy was carried out followed by systemic platinum-based chemotherapy. The brain metastasis showed very good radiological partial remission (PR) while the lung lesion remained stable after 4 cycles of chemotherapy. After two years’ follow-up the brain metastasis remained in PR whereas the lung lesion had progressed, and fine needle aspirate revealed adenocarcinoma. Results: Molecular analysis showed that EGFR gene status was wild type in all three tumors, whereas K-ras gene status differed. With regard to the two synchronous tumors, a barely visible G12C K-ras gene mutation was found in the bronchioloalveolar carcinoma, while a G12D K-ras mutation was detected in the adenocarcinoma. Moreover, K-ras gene status in the lingular node revealed a G12A mutation. On the basis of these results we hypothesized the presence of a third lung cancer and it was decided to proceed with surgery. The patient underwent laparoscopic lingulectomy which confirmed the diagnosis of adenocarcinoma with the same G12A K-ras mutation. Conclusions: This case highlights the importance of the molecular characterization of tumors in patients with multiple lesions. Different alterations within a single gene in different lesions are indicative of an independent clonal origin and suggestive of separate primary tumors rather than secondary lesions, thus facilitating treatment decision making.