Single Primary Tumor Research Articles

Abstract 4943: Visualization of endogenous melanoma initiation and progression using intravital microscopy

Abstract The death rates for malignant metastatic melanoma continue to rise because melanoma is largely refractory to existing therapies. Loss or inactivation of the tumor suppressor PTEN (Phosphatase and Tensin Homolog on Chromosome Ten) is observed in 40-50% of melanoma, and BRAF and PTEN mutations exist coincidentally in approximately 20% of melanoma cases. In mouse GEMM models, these mutations cooperate to promote metastatic melanomagenesis. However, existing methods to activate these lesions with Tyr-CreERT2 and topical tamoxifen application yields multifocal primary tumors whose origins cannot be accurately determined. To improve this model, we combined a tdTomatoLSL allele that serves as a visual marker of Cre recombination with Tyr-CreERT2;BrafCA;Ptenlox/lox to produce an animal where we can reproducibly initiate tumorigenesis in a spatiotemporally controllable manner by transiently applying extremely low doses (<20 nM) of tamoxifen to the dermis of the ear. Due to melanocyte-specific expression of the fluorescent protein tdTomato, we can track tumor progression from endogenous cells using two-photon intravital imaging. Starting from a single primary tumor on the ear, we can detect distant metastases in cervical lymph nodes and lungs via macroscopic fluorescent imaging, qRT-PCR and histology. We are using this model in combination with targeted therapy to investigate the effects of selective molecular inhibitors on the growth and metastasis of endogenous BRAFV600E/PTEN-/- metastatic melanoma at the cellular level. Preliminary studies using intravital imaging and a selective MEK1/2 inhibitor, GSK212 (Trametinib, FDA approved treatment for metastatic melanoma) show that targeted therapy causes heterogeneous reorganization of the tumor cells as early as 3 days post-treatment. After several weeks of continuous treatment, the remaining, resistant tumor cells are highly correlated spatially with bundled collagen structures detected by second harmonic generation signal, suggesting that cellular milieu strongly influences drug response. In addition, GSK212 treated animals still had lung macro-metastatic tumors, indicating that despite continuous MEK1/2 inhibition, metastatic tumor growth in the lung was still possible. In summary, we have developed an inducible endogenous model of melanoma metastasis that can be used to directly investigate both heterogeneous effects of targeted therapy at the cellular level and the efficacy of targeted therapy against metastasis. Citation Format: James E. Bear, Hailey E. Brighton, Norman E. Sharpless, David B. Darr, Kelly S. Clark. Visualization of endogenous melanoma initiation and progression using intravital microscopy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4943. doi:10.1158/1538-7445.AM2014-4943

Abstract NG04: Diversity of circulating tumor cells in a mouse pancreatic cancer model identified by single cell RNA sequencing

Abstract NG04: Diversity of circulating tumor cells in a mouse pancreatic cancer model identified by single cell RNA sequencing

SciClone: inferring clonal architecture and tracking the spatial and temporal patterns of tumor evolution.

The sensitivity of massively-parallel sequencing has confirmed that most cancers are oligoclonal, with subpopulations of neoplastic cells harboring distinct mutations. A fine resolution view of this clonal architecture provides insight into tumor heterogeneity, evolution, and treatment response, all of which may have clinical implications. Single tumor analysis already contributes to understanding these phenomena. However, cryptic subclones are frequently revealed by additional patient samples (e.g., collected at relapse or following treatment), indicating that accurately characterizing a tumor requires analyzing multiple samples from the same patient. To address this need, we present SciClone, a computational method that identifies the number and genetic composition of subclones by analyzing the variant allele frequencies of somatic mutations. We use it to detect subclones in acute myeloid leukemia and breast cancer samples that, though present at disease onset, are not evident from a single primary tumor sample. By doing so, we can track tumor evolution and identify the spatial origins of cells resisting therapy.

In reply to Detection of skeletal muscle metastases at the initial staging of lung cancer: a retrospective case series

To the Editor: We have carefully read the Letter by Dr. Surov [1] and wish to thank him for his interest in our paper. The letter raises some interesting issues that merit discussion. First, Dr. Surov argues that the frequency of skeletal muscle metastases (SMMs) in our case series is higher (2.6 %) than in previously reported data by other authors. The prevalence of SMMs in cancer patients has mainly been reported in postmortem studies, with highly variable estimates. In addition, apart from anecdotal case reports, this topic has not been adequately addressed in real-life clinical practice, at least in our opinion. Data from retrospective case series including different primary tumors also contribute to incomparable results due to differences in epidemiological and clinical/methodological settings. Dr. Surov [2] states that frequency of lung-cancer-related SMMs occurred only in the 0.84 % of cases in his retrospective evaluation of 5,170 patients suffering from a wide variety of primary malignancies, thus suggesting lung cancer to be a rare cause of SM involvement compared with other tumors. This, along with previous observations, is not really in contrast with our experience, which may help the scientific community further delineate this scenario. Indeed, while being a retrospective study, ours was reinforced by the strategy of analyzing a single primary tumor in a large cohort of patients within a given time, which is disease presentation at initial staging. We believe that taken together, these observations expose the need for additional efforts in larger, tailored prospective studies [3]. Dr. Surov [2] states that we described only two patterns of muscle metastases in lung cancer patients, visualized on multidetector computed tomography (MDCT) after i.v. injection of contrast medium, that were type I and II lesions, in agreement with SMMs classification provided in his retrospective analysis. To our knowledge, there are no literature data addressing the type of SM involvement in a given tumor setting for comparison. Therefore, data reported in our case series are complementary to those reported by Dr. Surov in his retrospective analysis. From a speculative point of view, it may be hypothesized that type III, IV, and V lesions, respectively, characterized by local bleeding, calcification, and infiltration, may be related to tumors of different origin and biology. However, this issue remains to be demonstrated. Unlike literature data, we also described a higher prevalence of type II lesions, but no definite comparison was feasible. To date, among the socalled target-like lesions, which are SMMs with ring-like enhancement and central hypoattenuation, we also reported the occurrence of large abscesses with a significant necrotic component in the 24 % of cases [3]. These findings may be classifiable as a subgroup of type II lesions. Again, the same consideration as given to SMM type should be done when looking at the frequency distribution of SM sites involved. Despite the fact that we described a higher prevalence of gluteal muscle involvement in comparison with data by Dr. Surov, overall, our observations are in line with previous literature reports. More evidence addressing This comment refers to the article available at doi:10.1007/s11604014-0299-8.

Do Angiotensin-Converting Enzyme Inhibitors Reduce the Risk of Symptomatic Radiation Pneumonitis in Patients With Non-Small Cell Lung Cancer After Definitive Radiation Therapy? Analysis of a Single-Institution Database

Preclinical studies have suggested that angiotensin-converting enzyme inhibitors (ACEIs) can mitigate radiation-induced lung injury. We sought here to investigate possible associations between ACEI use and the risk of symptomatic radiation pneumonitis (RP) among patients undergoing radiation therapy (RT) for non-small cell lung cancer (NSCLC). We retrospectively identified patients who received definitive radiation therapy for stages I to III NSCLC between 2004 and 2010 at a single tertiary cancer center. Patients must have received a radiation dose of at least 60 Gy for a single primary lung tumor and have had imaging and dosimetric data available for analysis. RP was quantified according to Common Terminology Criteria for Adverse Events, version 3.0. A Cox proportional hazard model was used to assess potential associations between ACEI use and risk of symptomatic RP. Of 413 patients analyzed, 65 were using ACEIs during RT. In univariate analysis, the rate of RP grade ≥2 seemed lower in ACEI users than in nonusers (34% vs 46%), but this apparent difference was not statistically significant (P=.06). In multivariate analysis of all patients, ACEI use was not associated with the risk of symptomatic RP (hazard ratio [HR] = 0.66; P=.07) after adjustment for sex, smoking status, mean lung dose (MLD), and concurrent carboplatin and paclitaxel chemotherapy. Subgroup analysis showed that ACEI use did have a protective effect from RP grade ≥2 among patients who received a low (≤20-Gy) MLD (P<.01) or were male (P=.04). A trend toward reduction in symptomatic RP among patients taking ACEIs during RT for NSCLC was not statistically significant on univariate or multivariate analyses, although certain subgroups may benefit from use (ie, male patients and those receiving low MLD). The evidence at this point is insufficient to establish whether the use of ACEIs does or does not reduce the risk of RP.

Molecular and Histopathologic Characteristics of Multifocal Papillary Thyroid Carcinoma

Papillary thyroid carcinoma (PTC) is frequently multifocal, which can represent either intraglandular spread from a single primary tumor or multiple synchronous primary tumors (MSPTs). To distinguish and characterize these entities, we investigated whether multifocal PTCs contain genetically similar or different mutations and have particular histopathologic characteristics. In 60 cases of PTC with 2 to 4 discrete tumor foci, each focus was tested for BRAF, NRAS, HRAS, and KRAS point mutations and RET/PTC1 and RET/PTC3 rearrangements and analyzed for various histopathologic features. Overall, BRAF mutations were found in 43% of tumors, RAS in 27%, and RET/PTC in 2%. Four different patterns of mutation occurrence were identified: (i) 2 foci containing different mutations (30%); (ii) 1 tumor containing a mutation and another carrying no mutations (32%); (iii) both/all tumors containing the same mutation (25%); (iv) all tumors having no mutations (13%). The 30% of cases with 2 different mutations represent a group of tumors that are unequivocally MSPT. These tumors more commonly occurred in different lobes, although they could be located as close as 0.6 cm from each other. Moreover, MSPTs typically demonstrated distinct histologic variants/microscopic features, were encapsulated or had a smooth border, and showed no microscopic peritumoral dissemination. In conclusion, we demonstrate that at least 30% of multifocal PTCs represent unequivocal MSPTs that develop through distinct molecular alterations and that as many as 60% of multifocal PTCs are likely MSPTs. Histopathologically, MSPTs are typically located in different lobes, have distinct growth patterns, and do not show microscopic peritumoral dissemination.

Abstract 4569: Personalizing cancer therapy using dynamic BH3 profiling.

Abstract Personalizing cancer therapy using dynamic BH3 profiling. J. Montero, A. Letai. Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA. Background: There are many targeted therapies that lack effective predictive biomarkers. Many of these agents kill via the mitochondrial pathway of apoptosis. Apoptosis can be considered as a threshold, and we have designed a tool, BH3 profiling, that can measure mitochondrial ‘priming for death’, essentially the proximity of a cell to the this threshold of apoptosis. When effective death signaling is initiated by a targeted therapy, dynamic BH3 profiling can be used to measure the increase in mitochondrial priming caused by the agent within hours, without the requirement for prolonged ex vivo culture. This means that death signaling induced by any number of distinct agents can be simultaneously compared on a single cell line or primary tumor sample. Hypothesis : Early apoptotic signaling detected via dynamic BH3 profiling can predict later cytotoxic cellular response. Results: Our first test was whether dynamic BH3 profiling performed following only 16 hours of drug exposure could predict cytotoxicity at a much later (72-96 hours) time point. We initiated our studies on a diverse panel of cancer cell lines treated with a wide range of kinase inhibitors. These cell lines included: PC9 (NSCLC), K562 (CML), MCF7 (Breast Cancer), LP1 (Multiple Myeloma), DHL6 (Diffuse Large B cell lymphoma) and Sk5 mel (Melanoma) using nine different treatments (primary targets): gefitinib (EGFR inh), imatinib (BCR-ABL inh), lapatinib (HER2 inh), PD173074 (FGFR inh), TAE-684 (ALK inh), MK-2206 (Akt inh), PLX-4032 (BRaf V600E inh), AZD6244 (MEK inh) and BEZ235 (PI3K/mTOR inh). We observed a significant correlation between the increase in priming and cell death, demonstrating the predicting capacity of dynamic BH3 profiling in different cancer cells. We also tested the predictive ability of dynamic BH3 profiling in chronic myelogenous leukemia (CML). We tested samples from stable phase CML patients of known clinical outcome who were either sensitive (underwent at least complete cytogenetic response) or refractory to clinical imatinib treatment. We found that the induction of mitochondrial priming caused by short term (16 hr) ex vivo exposure to imatinib predicted clinical response. Conclusions: Our cell line and clinical experiments demonstrate the potential for dynamic BH3 profiling to be used as a powerful real time predictive tool across many cancers and many agents, including combinations of agents. This tool could be used to identify niche populations sensitive to an individual agent. Alternatively, it could be used to recognize the best agent from a list of possible therapies for an individual tumor and improve cancer therapy. Citation Format: Joan Montero, Anthony Letai. Personalizing cancer therapy using dynamic BH3 profiling. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4569. doi:10.1158/1538-7445.AM2013-4569

Bilateral Appendicular Bone Tumors in Four Dogs

Bilateral synchronous appendicular bone tumors, occurring in the same bone and same anatomic site within the bone are very rare. This report describes the clinical presentation and oncologic outcome for four dogs with this rare presentation. All cases presented to the authors following a history of unilateral lameness for several weeks. On presentation, case 1 had pain elicited in the contralateral proximal humerus but all the other cases had no abnormalities detectable on physical examination of the contralateral limb. All dogs had technetium 99m ((99m)Tc) nuclear scintigraphy performed that identified bilateral lesions of the distal radii in two dogs, proximal humeri and distal tibiae in one dog each. Thoracic radiographs performed on all dogs showed no evidence of pulmonary metastases. Three dogs were treated with palliative radiation therapy (two dogs received concurrent bisphosphonates) resulting in survival times from initial presentation of 50 days, 193 days, and 523 days, respectively. One dog had stereotactic radiation therapy (SRT) and a surgical limb-salvage performed followed by carboplatin chemotherapy, resulting in a survival time of 926 days from initial presentation. Palliative and curative-intent treatments for the bilateral synchronous appendicular bone tumors resulted in survival times similar to those reported for treatment of a single primary appendicular bone tumor.

Radiofrequency ablation of hepatocellular carcinoma sized &gt; 3 and ≤ 5 cm: Is ablative margin of more than 1 cm justified?

To investigate whether an ablative margin (AM) > 1.0 cm might reduce chance of recurrence for patients with hepatocellular carcinoma (HCC) tumors 3.1 to 5.0 cm in size, compared with an AM of 0.5-1.0 cm. From October 2005 to December 2012, 936 consecutive patients with HCC who received radiofrequency ablation were screened. Of these, 281 patients, each with a single primary HCC tumor of 3.1 to 5.0 cm in size on its greatest diameter, were included in the study. Based on the AM width, we categorized patients into the 0.5-1.0 cm group and the > 1.0 cm group. Local tumor progression (LTP)-free survival, intrahepatic distant recurrence (IDR)-free survival and overall survival (OS) rates were obtained using the Kaplan-Meier method. The 1-, 2-, 3-, 4-, and 5-year LTP-free survival rates and IDR-free survival rates were significantly higher in the > 1.0 cm group compared with the 0.5-1.0 cm group (97.5%, 86.3%, 73.6%, 49.5% and 26.4% vs 91.3%, 78.4%, 49.5%, 27.8%, and 12.8%; 95.1%, 90.3%, 77.0%, 61.0% and 48.3% vs 95.2%, 85.9%, 62.6%, 47.2% and 28.5%; P < 0.05). The 1-, 2-, 3-, 4-, and 5-year OS rates were 98.6%, 91.5%, 69.2%, 56.0% and 42.2%, respectively, in the 0.5-1.0 cm group and 100%, 98.9%, 90.1%, 68.7% and 57.4%, respectively, in the > 1.0 cm group (P = 0.010). There were no significant differences in complication rates between the two groups. Both univariate and multivariate analyses identified AM as an independent prognostic factor linked to LTP, IDR, and OS. For HCC tumors > 3.0 cm and ≤ 5.0 cm, AM > 1.0 cm could reduce chances of recurrence compared with AM of 0.5-1.0 cm, emphasizing the need for a more defensive strategy using AMs > 1.0 cm for ablating HCC tumors of 3.1 to 5.0 cm.

Primary Peritoneal Serous Papillary Carcinoma Presenting as a Single Colonic Mass without Peritoneal Dissemination

Peritoneal serous papillary carcinoma (PSPC), a rare primary malignancy arising from the abdominal and pelvic peritoneum, is characterized by peritoneal carcinomatosis with ascites and a histological pattern similar to that of papillary serous ovarian carcinoma. We herein describe a rare case of PSPC with unusual clinical presentations involving a single primary tumor originating from the peritoneal lining of the sigmoid colon with no evidence of intraperitoneal spread, pelvic lymph node involvement or distant metastasis. Awareness of such unusual presentations of PSPC should assist in the diagnosis of this disease, thereby improving the management of patients with this condition.

Abstract A50: Is there a reverse racial/ethnic disparity in chemotherapy treatment among breast cancer patients?

Abstract Background: There is evidence that minority women are more likely than White women to receive suboptimal breast cancer care, however this may not be the case with regards to chemotherapy for which White women may be less likely than Black or Hispanic women to receive chemotherapy. In order to better understand this apparent reverse disparity, this study seeks to examine how changes in the treatment guidelines may affect the disparity observed in chemotherapy treatment. Methods: Interview and medical record data came from a population-based study of 989 breast cancer patients (397 non-Hispanic White, 411 non-Hispanic Black, 181 Hispanic) diagnosed between 2005-2008. Of these, 87% (N=849) consented to medical record abstraction, including a linkage with the Illinois State Cancer Registry (ISCR). Patients who consented to the medical record abstraction and had single primary tumors were considered for this study. Eligibility for chemotherapy (CT) was defined according to the National Comprehensive Cancer Network (NCCN) guidelines in two ways. First, CT eligibility was defined according to the 2005 guidelines and then also according to the 2006-2007 guidelines. The outcome variables included treatment recommendation, acceptance, and initiation which were derived from the interview, medical record, and ISCR data. Age-adjusted risk differences (RDs) were estimated using logistic regression (with marginal standardization) in order to report percentages. Results: The medical record consent rate was 87%. Among those with a medical record consent and single primary tumors (n=824), 460 were CT-eligible per the 2005 NCCN guidelines and 284 were CT-eligible per the 2006-2007 guidelines (eligibility could not be determined for 107 node-negative, hormone receptor-positive women because their HER2 status was unavailable). Using the 2005 NCCN guidelines, chemotherapy was recommended to 74% of CT-eligible patients and non-Hispanic White patients were less likely than minority patients to receive a recommendation (0.62 vs. 0.75, RD=13%p=0.001). However, if the 2006-2007 guidelines were used, 94% of eligible patients received a recommendation and there were no racial/ethnic differences. Of these women who received a recommendation, 98% accepted it and a resulting 100% initiated treatment. However, the overall treatment initiation was 92% and in multivariate analysis, patients with lower income (p=0.03), breast conserving surgery (p=0.01), and low grade tumors (p=0.05) were less likely to initiate chemotherapy. Conclusions: The apparent reverse racial/ethnic disparity in chemotherapy treatment may appear or disappear depending on which guideline is used to determine treatment eligibility. It is reasonable that clinicians change treatment practices ahead of published guidelines as to provide their patients with the best care. Future studies on treatment quality should consider this when defining treatment eligibility. The vast majority of CT-eligible patients are receiving chemotherapy, although patients with less aggressive appearing tumors are less likely to receive CT. Citation Format: Abigail Silva, Garth H. Rauscher, Kent Hoskins, Ruta D. Rao. Is there a reverse racial/ethnic disparity in chemotherapy treatment among breast cancer patients? [abstract]. In: Proceedings of the Fifth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2012 Oct 27-30; San Diego, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2012;21(10 Suppl):Abstract nr A50.

Abstract A49: Mediators of racial/ethnic disparities in radiation treatment among breast cancer patients.

Abstract Background: Radiation after surgery can reduce recurrence and breast cancer mortality yet there is some evidence that not all women receive guideline-concordant radiation treatment. Indeed, studies that have examined the receipt of radiation among women who underwent breast-conserving surgery have found that Black and Hispanic women were less likely than White women to complete their locoregional treatment. However, little is known about the factors that may facilitate or impede treatment. In order to better understand the causes of disparities in radiation treatment, this study seeks to: 1) determine the extent to which there is a racial/ethnic disparity in radiation treatment initiation, and 2) examine patient factors and hospital characteristics that may help explain the variation. Methods: Interview and medical record data came from a population-based study of 989 breast cancer patients (397 non-Hispanic White, 411 non-Hispanic Black, 181 Hispanic) diagnosed between 2005-2008. Of these, 87% (N=849) consented to medical record abstraction, including a linkage with the Illinois State Cancer Registry (ISCR). Patients who consented to the medical record abstraction and had single invasive primary tumors were considered for this study. Radiation treatment eligibility was defined according to the 2005-2007 National Comprehensive Cancer Network (NCCN) guidelines. The outcome variables included treatment recommendation, acceptance, and initiation which were derived from the interview, medical record, and ISCR data. Risk differences (RDs) were estimated using logistic regression (with marginal standardization). Potential mediators related to radiation initiation were identified, and then assessed by rescaling model coefficients using the method of Karlson, Holm, and Breen. All models were adjusted for age and time from diagnosis to interview. Results: Among patients with single invasive primary tumors (n=614), 443 patients (72%) were eligible for radiation treatment (RT) per the NCCN guidelines. Radiation treatment was recommended to 88% of eligible patients of which 93% accepted it. Among those who accepted treatment, 97% received radiation. This translated into an overall treatment initiation of 79%. Minority patients were less likely than non-Hispanic (nH) White patients to initiate radiation (0.75 vs. 0.85, RD=10%p=0.000). Minorities were more likely to have moderate-high grade tumors and symptomatically detected tumors which in turn were less likely to receive radiation (all p-values &amp;lt;0.01). Minority women were also more likely than nH White women (p&amp;lt;0.0001) to receive chemotherapy, which in turn was associated with lower receipt of RT (p&amp;lt;0.05). Together these factors explained 46% of the disparity (p=0.002). Conclusions: Patients who are eligible for radiation and have more aggressive appearing tumors at diagnosis are more likely to receive chemotherapy but at the expense of completing their locoregional (radiation) therapy. This disproportionately affects minority patients and results in underuse of radiation in these women. Greater diffusion of gene expression profiling (e.g. Oncotype) may improve cancer care not only by reducing overuse of chemotherapy but by eliminating chemotherapy as a potential barrier to receipt of RT. Citation Format: Abigail Silva, Garth H. Rauscher, Rao D. Ruta, Kent Hoskins. Mediators of racial/ethnic disparities in radiation treatment among breast cancer patients. [abstract]. In: Proceedings of the Fifth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2012 Oct 27-30; San Diego, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2012;21(10 Suppl):Abstract nr A49.

Abstract 1702: A novel diagnostic approach using genome-wide single nucleotide polymorphism arrays for ovarian metastases in endometrial cancers

Abstract [Introduction] Synchronous cancers involving ovaries as well as uterine corpus are well-known events in gynecologic malignancies. These tumors may be independently derived, non-metastatic tumors (Dual Primary tumors; DP) or a tumor from one organ with metastasis to the other (Single Primary tumor with Metastasis; SPM). In the majority of these synchronous endometrial and ovarian cancers, both tumors are histologically endometrioid adenocarcinomas, which may cause a diagnostic difficulty to distinguish DP and SPM. By genome-wide genotyping, single nucleotide polymorphism (SNP) arrays, we can obtain chromosomal copy number alterations (CNA) throughout the genome in a single assay. We hypothesized that the copy number profiles by SNP arrays might be highly informative for genetic diagnosis in the synchronous endometrioid adenocarcinomas. [Material and Methods] We genetically diagnosed ten tumors from five patients with synchronous endometrial and ovarian carcinomas, using 250K SNP typing arrays and mutational analysis of PIK3CA, PTEN, K-Ras and CTNNB1 (beta-catenin). We evaluated whether conventional pathological diagnosis is compatible with the genetic diagnosis. [Results] Three of the five patients show identical copy number alterations (CNA), including type, loci and degree of each alteration, between the endometrial and the ovarian carcinomas. The other two show CNA only in either endometrial or ovarian carcinoma. All the five tumors possess one or more genetic mutations in the genes examined. One patient showed mutations both in PIK3CA and PTEN at discordant sites between endometrial and ovarian carcinomas, whereas the other four showed concordant mutations. Together, four of the five were genetically diagnosed as SPM and the remaining one was as DP. The pathological diagnosis was not in agreement with the molecular diagnosis in four of the five cases. [Conclusion] Our data suggest that most of the copy number alterations might occur before metastases and that genome-wide genotyping may represent a useful approach to distinguish between SPM and DP in synchronous endometrial and ovarian carcinomas. As chromosomal instability is commonly observed in various types of tumors, SNP array genotyping might be applicable to synchronous tumors in other organs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1702. doi:1538-7445.AM2012-1702

Synchronous and metachronous multiple gastrointestinal stromal tumors.

Sporadic multiple gastrointestinal stromal tumors (GISTs) are rare events especially those developed metachronously. This study aimed to investigate the clinico-pathologic and genetic features defining multiple GISTs. 624 cases of GISTs were retrieved for retrospective review. 15 cases were identified as multiple GISTs including 13 synchronous and 2 metachronous ones. 32 tumors and 15 normal tissues were obtained from these cases each containing 2-3 tumor nodules and the genomic DNA was extracted for mutational analysis of KIT and PDGFRA genes. The associated patients were recruited for clinical follow-up studies, including 5 males and 10 females at 49 to 84 years of age. Multiple GISTs comprised of 2.4% of GIST cases in our consecutive series. Twenty-six tumors showed mutations at KIT gene in exon 11 and one at PDGFRA gene in exon 18. In seven synchronous cases, different tumors from the same patients displayed different genotypes of KIT or PDGFRA, suggesting their polyclonal origin. In the two multiple GISTs occurring metachronously, the tumors from each patient showed different KIT mutations, suggesting that the second tumors were not the relapse or metastasis of the primary GISTs. Based on types of KIT or PDGFRA mutations and other pathological features, multiple primary GISTs can be differentiated from multiple GISTs resulting from recurrence or metastasis of a single primary tumor. Unlike recurrence or metastasis of GISTs that are malignant, most multiple GISTs are mostly benign and do not require aggressive adjuvant therapy. Therefore, correct diagnosis is critical for proper treatment.

Expression of Sodium–Iodide Symporter and TSH Receptor in Subclinical Metastatic Lymph Nodes of Papillary Thyroid Microcarcinoma

The clinical significance of the subclinical lymph node (LN) metastasis in clinically node-negative (cN0) papillary thyroid microcarcinoma (PTMC) has been debated. We investigated the expression of sodium-iodide symporter (NIS) and thyroid-stimulating hormone receptor (TSHR) in the subclinical metastatic LNs of PTMC, which are crucial prerequisites for the response to radioactive iodine treatment. Among 149 consecutive patients who received total thyroidectomy in conjunction with prophylactic central neck dissection for cN0 PTMC from October 2005 to December 2007, 20 who had single PTMC and subclinical LN metastasis (cN0, pN1, single PTMC) were included. Immunohistochemical staining was performed with anti-human NIS antibody and anti-human TSHR antibody in 20 primary tumors and 52 metastatic LNs. NIS and TSHR expression was detected in 19 (95%) and 18 (90%) of 20 PTMCs, respectively. NIS and TSHR expression were also detected in 50 (96.2%) and 39 (75%) of 52 metastatic LNs, respectively. In 85% of patients, the presence of NIS expression in primary PTMCs was concordant with that in corresponding metastatic LNs. Intensities of NIS and TSHR expression were diverse. In 6 of 12 cases of multiple metastatic LNs, the metastatic LNs showed heterogeneous intensities of NIS expression. The presence of NIS and TSHR expression was observed with high frequency in both PTMCs and corresponding subclinical metastatic LNs. However, the intensity of NIS and TSHR expression was diverse. Multiple metastatic LNs from single primary tumor focus could have heterogeneous intensity of NIS expression.

ORAOV1 is amplified in oral squamous cell carcinoma

Oral cancer overexpressed 1 (ORAOV1) was found as a candidate oncogene in the 11q13 chromosomal region, based on its amplification and overexpression in oral cancer cell lines. Because gene amplification often leads to increased levels of gene expression, we aimed to verify the relationship between ORAOV1 gene status and mRNA expression primarily in oral squamous cell carcinoma (OSCC) by quantitative assay, correlating with clinical and pathological characteristics in patients. Levels of ORAOV1 amplification and expression were evaluated by qPCR and RT-qPCR in OSCC cell lines and in tumor and non-tumoral surgical margins from 33 patients with OSCC. All subjects were smokers and habitual alcohol drinkers, mostly men above 40 years of age and with a single primary tumor. ORAOV1 exhibited increased gene expression levels as well as higher copy number in three OSCC cell lines with 11q13 amplified chromosomal region when compared with the OSCC cell line without the amplification (one-way ANOVA, P < 0.05). Weak correlation between ORAOV1 mRNA levels and DNA copy number was seen in tumor samples (Spearman, P = 0.07). Although ORAOV1 was amplified in tumor (Wilcoxon, P < 0.01), high levels of transcripts in margin did not reveal differences in comparison with tumor (Wilcoxon, P = 0.85). Aggressiveness and survival rate did not demonstrate statistical difference for both events in OSCC. The overexpression of ORAOV1 in non-tumoral margin samples can occur in the absence of amplification. The weak correlation between ORAOV1 amplification and expression in OSSC suggests that ORAOV1 expression can be regulated by mechanisms other than gene amplification.

Genome-wide single nucleotide polymorphism (SNP) arrays as a novel diagnostic tool in synchronous carcinomas.

5105 Background: Synchronous cancers involving ovaries as well as uterine corpus are well-known events in gynecologic malignancies. These tumors may be independently derived, non-metastatic tumors (dual primary tumors; DP) or a tumor from one organ with metastasis to the other (single primary tumor with Metastasis; SPM). In the majority of these synchronous cancers, both tumors are histologically endometrioid adenocarcinomas, which may cause a diagnostic difficulty to distinguish DP and SPM. By genome-wide single nucleotide polymorphism (SNP) arrays, we can obtain chromosomal copy number alterations (CNA) throughout the genome in a single assay. We hypothesized the CNA by SNP arrays might be highly informative for genetic diagnosis in the synchronous endometrioid adenocarcinomas. Methods: We genetically diagnosed ten tumors from five patients with synchronous endometrial and ovarian carcinomas, using 250K SNP typing arrays and mutational analysis of PIK3CA, PTEN, K-Ras and CTNNB1under informed consent and institutional reveiw board approval. We evaluated whether conventional pathological diagnosis is compatible with the genetic diagnosis. Results: Three of the five patients show identical CNA, including type, loci and degree of each alteration, between the endometrial and the ovarian carcinomas. The other two show CNA only in either endometrial or ovarian carcinoma. All the five tumors possess one or more genetic mutations in the genes examined. One patient showed mutations both in PIK3CA and PTEN at discordant sites between endometrial and ovarian carcinomas, whereas the other four showed concordant mutations. Together, four of the five were genetically diagnosed as SPM and the remaining one was as DP. The pathological diagnosis was not in agreement with the molecular diagnosis in four of the five cases. Conclusions: Our data suggest that most of the copy number alterations might occur before metastases and that genome-wide genotyping may represent a useful approach to distinguish between SPM and DP in synchronous endometrial and and ovarian carcinomas. As chromosomal instability is commonly observed, SNP array genotyping might be applicable to synchronous tumors in other organs.

Predicting Clinical Outcome in Patients Diagnosed with Synchronous Ovarian and Endometrial Cancer

Patients with synchronous ovarian and endometrial cancers may represent cases of a single primary tumor with metastasis (SPM) or dual primary tumors (DP). The diagnosis given will influence the patient's treatment and prognosis. Currently, a diagnosis of SPM or DP is made using histologic criteria, which are frequently unable to make a definitive diagnosis. In this study, we used genetic profiling to make a genetic diagnosis of SPM or DP in 90 patients with synchronous ovarian/endometrial cancers. We compared genetic diagnoses in these patients with the original histologic diagnoses and evaluated the clinical outcome in this series of patients based on their diagnoses. Combining genetic and histologic approaches, we were able make a diagnosis in 88 of 90 cases, whereas histology alone was able to make a diagnosis in only 64 cases. Patients diagnosed with SPM had a significantly worse survival than patients with DP (P = 0.002). Patients in which both tumors were of endometrioid histology survived longer than patients of other histologic subtypes (P = 0.025), and patients diagnosed with SPM had a worse survival if the mode of spread was from ovary to endometrium rather than from endometrium to ovary (P = 0.019). Genetic analysis may represent a powerful tool for use in clinical practice for distinguishing between SPM and DP in patients with synchronous ovarian/endometrial cancer and predicting disease outcome. The data also suggest a hitherto uncharacterized level of heterogeneity in these cases, which, if accurately defined, could lead to improved treatment and survival.

P53 mutation in histologically normal mucosa of the aero-digestive tract is not a marker of increased risk for second primary carcinoma in head and neck cancer patients

Head and neck cancer patients are at high risk for developing second primary tumors. This is known as field cancerization of the aero-digestive tract. In a previous study, we showed that patients with multiple primary tumors were more likely to have p53 mutations in histologically normal mucosae than patients presenting with an isolated tumor. Based on this observation, we postulated that p53 mutations in normal tissue samples of patients bearing a single primary tumor could have a clinical value as a biomarker for the risk of developing second primary tumors. Thirty-five patients presenting with a single primary tumor were followed-up for a median of 51 months (range 1 month to 10.9 years) after biopsies of histologically normal squamous cell mucosa had been analyzed for p53 mutations with a yeast functional assay at the time of the primary tumor. During this follow-up, recurrences and non-sterilization of the primary tumor, occurrence of lymph node metastases, and of second primary tumors were evaluated. Sixteen (45.7%) patients were found to have p53 mutations in their normal squamous cell mucosa, and 19 (54.3%) patients showed no mutation. No relationship was found between p53 mutations and the occurrence of evaluated events during follow-up. Notably, the rate of second primary tumors was not associated with p53 mutations in the normal squamous mucosa. The correlation between p53 mutations in histologically normal mucosae and the incidence of second primary tumors is generally low. The benefit of analyzing p53 mutations in samples of normal squamous cell mucosa in every patient with a primary tumor of the head and neck is doubtful.

Cytopathologic features of olfactory neuroblastoma

Olfactory neuroblastoma (ONB) is an uncommon neoplasm arising from the olfactory mucosa. Because its cytopathology is largely limited to case reports, the goal was to evaluate a series of ONB cases, compare them with previously reported cases, and with a control group of pulmonary and cutaneous small cell neuroendocrine carcinoma (NEC). Six fine-needle aspiration (FNA) biopsies of metastatic ONB and one case with imprint smears of primary ONB were recovered from files. Aspirates from seven FNA cases of metastatic pulmonary small cell NEC and four cases of metastatic Merkel cell carcinoma to the head and neck functioned as a control group and were compared with those of ONB. Seven cases from 4 patients included 3 males (ages 33-39 yrs) and 1 female (age 58 yrs). Aspirates were acquired from soft tissues of the neck (three cases), cervical lymph nodes (two cases), parietal scalp (one case), and imprint of a nasal mass (one case). A correct cytologic diagnosis of metastatic ONB (five cases) or malignant small round cell tumor (one case) was made in six cases. One aspirate was misdiagnosed as a reactive lymph node. The single primary tumor and five of six metastatic tumors were histologically confirmed. Cytologic features were similar in all cases. These included high cellularity (seven of seven cases), distribution as single forms and cell clusters (seven of seven cases), a two-cell population of intact and apoptotic nuclei (seven of seven cases), nuclear molding (seven of seven cases), paranuclear 'blue bodies' (five of seven cases), necrosis (five of seven cases), and absence of lymphoglandular bodies (seven of seven cases). Unlike prior reports, no case exhibited rosettes or fibrillar neuropil on smears. All examples from the control group displayed nearly identical cytomorphologic features to those of the study group. The cytopathology of metastatic ONB is nonspecific unless fibrillar neuropil is identified. Nonetheless, a cytopathologic diagnosis of metastatic ONB can be made with confidence in nearly all patients if a well documented history of ONB exists. Minus such a clinical context, aspirates of metastatic ONB may be mistaken for metastatic pulmonary small cell NEC, cutaneous neuroendocrine (Merkel cell) carcinoma, and even small cell lymphoma.