BID expression determines the apoptotic fate of cancer cells after abrogation of the spindle assembly checkpoint by AURKB or TTK inhibitors.

BACKGROUND: Drugs targeting the spindle assembly checkpoint (SAC), such as inhibitors of Aurora kinase B (AURKB) and dual specific protein kinase TTK, are in different stages of clinical development. However, cell response to SAC abrogation is poorly understood and there are no markers for patient selection. METHODS: A panel of 53 tumor cell lines of different origins was used. The effects of drugs were analyzed by MTT and flow cytometry. Copy number status was determined by FISH and Q-PCR; mRNA expression by nCounter and RT-Q-PCR and protein expression by Western blotting. CRISPR-Cas9 technology was used for gene knock-out (KO) and a doxycycline-inducible pTRIPZ vector for ectopic expression. Finally, in vivo experiments were performed by implanting cultured cells or fragments of tumors into immunodeficient mice. RESULTS: Tumor cells and patient-derived xenografts (PDXs) sensitive to AURKB and TTK inhibitors consistently showed high expression levels of BH3-interacting domain death agonist (BID), while cell lines and PDXs with low BID were uniformly resistant. Gene silencing rendered BID-overexpressing cells insensitive to SAC abrogation while ectopic BID expression in BID-low cells significantly increased sensitivity. SAC abrogation induced activation of CASP-2, leading to cleavage of CASP-3 and extensive cell death only in presence of high levels of BID. Finally, a prevalence study revealed high BID mRNA in 6% of human solid tumors. CONCLUSIONS: The fate of tumor cells after SAC abrogation is driven by an AURKB/ CASP-2 signaling mechanism, regulated by BID levels. Our results pave the way to clinically explore SAC-targeting drugs in tumors with high BID expression.

Reference standards for gene fusion molecular assays on cytological samples: an international validation study.

AIMS: Gene fusions assays are key for personalised treatments of advanced human cancers. Their implementation on cytological material requires a preliminary validation that may make use of cell line slides mimicking cytological samples. In this international multi-institutional study, gene fusion reference standards were developed and validated. METHODS: Cell lines harbouring EML4(13)-ALK(20) and SLC34A2(4)-ROS1(32) gene fusions were adopted to prepare reference standards. Eight laboratories (five adopting amplicon-based and three hybridisation-based platforms) received, at different dilution points two sets of slides (slide A 50.0%, slide B 25.0%, slide C 12.5% and slide D wild type) stained by Papanicolaou (Pap) and May Grunwald Giemsa (MGG). Analysis was carried out on a total of 64 slides. RESULTS: Four (50.0%) out of eight laboratories reported results on all slides and dilution points. While 12 (37.5%) out of 32 MGG slides were inadequate, 27 (84.4%) out of 32 Pap slides produced libraries adequate for variant calling. The laboratories using hybridisation-based platforms showed the highest rate of inadequate results (13/24 slides, 54.2%). Conversely, only 10.0% (4/40 slides) of inadequate results were reported by laboratories adopting amplicon-based platforms. CONCLUSIONS: Reference standards in cytological format yield better results when Pap staining and processed by amplicon-based assays. Further investigation is required to optimise these standards for MGG stained cells and for hybridisation-based approaches.

Multiplex RNA-based detection of clinically relevant MET alterations in advanced non-small cell lung cancer.

MET inhibitors have shown activity in non-small-cell lung cancer patients (NSCLC) with MET amplification and exon 14 skipping (METΔex14). However, patient stratification is imperfect, and thus, response rates have varied widely. Here, we studied MET alterations in 474 advanced NSCLC patients by nCounter, an RNA-based technique, together with next-generation sequencing (NGS), fluorescence in situ hybridization (FISH), immunohistochemistry (IHC), and reverse transcriptase polymerase chain reaction (RT-PCR), exploring correlation with clinical benefit. Of the 474 samples analyzed, 422 (89%) yielded valid results by nCounter, which identified 13 patients (3%) with METΔex14 and 15 patients (3.5%) with very-high MET mRNA expression. These two subgroups were mutually exclusive, displayed distinct phenotypes and did not generally coexist with other drivers. For METΔex14, 3/8 (37.5%) samples positive by nCounter tested negative by NGS. Regarding patients with very-high MET mRNA, 92% had MET amplification by FISH and/or NGS. However, FISH failed to identify three patients (30%) with very-high MET RNA expression, among which one received MET tyrosine kinase inhibitor treatment deriving clinical benefit. Our results indicate that quantitative mRNA-based techniques can improve the selection of patients for MET-targeted therapies.

RNA-Based Multiplexing Assay for Routine Testing of Fusion and Splicing Variants in Cytological Samples of NSCLC Patients.

The detection of ALK receptor tyrosine kinase (ALK), ROS proto-oncogen1, receptor tyrosine kinase (ROS1), ret proto-oncogen (RET), and MET proto-oncogen exon 14 skipping (METΔex14) allows for the selection of specific kinase inhibitor treatment in patients with non-small cell lung cancer (NSCLC). Multiplex technologies are recommended in this setting. We used nCounter, a multiplexed technology based on RNA hybridization, to detect ALK, ROS1, RET, and METΔex14 in RNA purified from cytological specimens (n = 16) and biopsies (n = 132). Twelve of the 16 cytological samples (75.0%) were evaluable by nCounter compared to 120 out of 132 (90.9%) biopsies. The geometrical mean (geomean) of the housekeeping genes of the nCounter panel, but not the total amount of RNA purified, was significantly higher in biopsies vs. cytological samples. Among cytological samples, we detected ALK (n = 3), METΔex14 (n = 1) and very high MET expression (n = 1) positive cases. The patient with METΔex14 had a partial response to tepotinib, one of the patients with ALK fusions was treated with crizotinib with a complete response. Cell blocks and cytological extensions can be successfully used for the detection of fusions and splicing variants using RNA-based methods such as nCounter.

AURKB as a target in non-small cell lung cancer with acquired resistance to anti-EGFR therapy.

Non-small cell lung cancer (NSCLC) tumors harboring mutations in EGFR ultimately relapse to therapy with EGFR tyrosine kinase inhibitors (EGFR TKIs). Here, we show that resistant cells without the p.T790M or other acquired mutations are sensitive to the Aurora B (AURKB) inhibitors barasertib and S49076. Phospho-histone H3 (pH3), a major product of AURKB, is increased in most resistant cells and treatment with AURKB inhibitors reduces the levels of pH3, triggering G1/S arrest and polyploidy. Senescence is subsequently induced in cells with acquired mutations while, in their absence, polyploidy is followed by cell death. Finally, in NSCLC patients, pH3 levels are increased after progression on EGFR TKIs and high pH3 baseline correlates with shorter survival. Our results reveal that AURKB activation is associated with acquired resistance to EGFR TKIs, and that AURKB constitutes a potential target in NSCLC progressing to anti-EGFR therapy and not carrying resistance mutations.

Carcinoma seroso peritoneal y adenocarcinoma mucinoso apendicular sincrónicos. Estudio inmunohistoquímico y molecular / Synchronous serous peritoneal carcinoma and mucinous appendicular adenocarcinoma. Immunohistochemical and molecular study

Paciente mujer de 60 años, en seguimiento por historia familiar de cáncer de mama y ovario, en la que se detectaron implantes tumorales peritoneales y depósitos de mucina en serosa apendicular. Los implantes peritoneales fueron diagnosticados de carcinoma seroso tipo ovárico/peritoneal con inmunofenotipo característico, siendo la mucina acelular. Se realizaron estudios de mutación de KRAS y BRAF mediante PCR que demostró la presencia de mutación GD12 únicamente en el componente mucinoso, lo que permitió mostrar la existencia de una doble neoplasia. La cirugía posterior confirmó la existencia sincrónica de un adenocarcinoma mucinoso apendicular y un carcinoma seroso peritoneal. Creemos que este caso permite ilustrar que la incorporación de los estudios moleculares en la actividad asistencial del patólogo puede aportarnos información adicional con valor diagnóstico (AU)

A 60 year old female with a family history of ovarian and breast cancer underwent a follow-up laparoscopic exploration which revealed peritoneal implants and mucinous deposits on the appendicular surface. The peritoneal implants were diagnosed as serous carcinoma with a characteristic immunophenotype, while the mucinous material was acellular. KRAS and BRAF mutation studies with PCR showed a GD12 mutation in the mucinous component only, suggesting the presence of a synchronous carcinoma. Subsequent cytorreductive surgery confirmed the presence of an infiltrating appendicular mucinous adenocarcinoma, synchronous with the peritoneal infiltrating serous carcinoma. We believe that this case, where the different immunohistochemical and molecular profiles allowed a correct diagnosis of two independent neoplasms, emphasizes the value of molecular studies in routine diagnostic procedure (AU)

[Overlap syndrome. LEOPARD and neurofibromatosis. A case report]. / Síndrome de superposición. LEOPARD con neurofibromatosis. Reporte de caso.

We expose a clinical case of a 43-year-old patient who was attended at the Dermatology service in a general hospital of the Instituto Mexicano del Seguro Social, with a disseminated pattern of lentigines, psychomotor retardation and electrocardiographic abnormalities. Afterwards, we made an analysis of the literature.

Se presenta el caso de un paciente varón de 43 años de edad, que fue atendido en el servicio de Dermatología de un hospital general de zona del Instituto Mexicano del Seguro Social, con lesiones lentiginosas diseminadas, retraso psicomotor y alteraciones electrocardiográficas. Posteriormente, realizamos un análisis de la literatura.

EGFR mutations in lung cancer / Mutaciones de EGFR en cancer de pulmón

The study of epidermal growth factor receptor mutations has become essential for the treatment of lung cancer. The aim of this study was to find a correlation between morphological changes and EGFR mutational status using both immunohistochemistry and molecular techniques. We also analyzed the cross-reaction of the L858R mutation-specific monoclonal antibody in cases with HER2 amplification described previously in breast and gastric cancer. A series of 100 primary lung adenocarcinomas were examined. Exon 19 E746_A750del and exon 21 L858R mutations were studied using immunohistochemistry with two specific monoclonal antibodies. Gene mutational status was determined using real-time PCR or Sanger sequencing followed by real-time PCR when negative. EGFR mutations were detected in 22 cases (22%) by molecular techniques, being significantly more frequent in women, low grade carcinoma and lepidic subtype, (p-value <0.05 in all cases). In addition, in our series presence of tumoral necrosis correlated with absence of mutations. The anti-E746_A750del antibody achieved a 100% positive predictive value and a negative predictive value of 97.7% which could restrict the use of molecular techniques to the 7% of cases with an equivocal result. The antibody for L858R mutation showed inconsistent results compared to molecular techniques, giving false positive result in two adenocarcinomas with HER2 amplification. However, its negative predictive value was very high (98.9%). The use of real-time PCR identifies mutations not detected by the other two techniques. These new antibodies could be useful as a screening tool prior to EGFR molecular techniques (AU)

El estudio de las mutaciones de EGFR ha resultado ser esencial en el tratamiento del cáncer de pulmón. La finalidad de este trabajo ha sido estudiar la correlación entre los cambios morfológicos y el estado mutacional de EGFR utilizando técnicas de inmunohistoquímica y moleculares. Asimismo se analizó la reacción cruzada del anticuerpo anti-L858R en casos con amplificación de HER2 descrita previamente en cáncer de mama y gástrico. La serie consta de 100 adenocarcinomas pulmonares primarios. Las mutaciones E746_A750del exón-19 y L858R exón-21 se estudiaron por inmunohistoquímica con dos anticuerpos monoclonales específicos. El estado mutacional del gen se determinó mediante PCR en tiempo-real o secuenciación por Sanger seguida de PCR en tiempo-real cuando resultó negativa. Se detectaron mutaciones de EGFR en 22 casos (22%) por técnicas moleculares, siendo significativamente más frecuente en mujeres, carcinoma de bajo grado y subtipo lepídico, (p-valor<0,05 en todos los casos). Además, en nuestra serie la presencia de necrosis tumoral se correlaciona con ausencia de mutaciones. El anticuerpo anti-E746_A750del presentó un valor-predictivo-positivo del 100% y un valor-predictivo-negativo del 97,7%, lo que podría restringir el uso de técnicas moleculares al 7% de casos no-concluyentes. El anticuerpo anti-L858R mostró resultados inconsistentes en comparación con las técnicas moleculares, dando resultado falso-positivo en dos adenocarcinomas con amplificación de HER2. Sin embargo, su valor-predictivo-negativo es muy alto (98,9%). El uso de PCR en tiempo-real rescata mutaciones no detectadas por las otras dos técnicas. Estos nuevos anticuerpos podrían ser útiles como herramienta de cribado previo al estudio de EGFR mediante técnicas moleculares (AU)

Expresión inmunohistoquímica de CK19 en variantes comunes del carcinoma de mama y en subtipos histológicos infrecuentes / CK19 immunoexpression in common breast carcinomas and in special histological subtypes

El estudio del ganglio centinela es una parte esencial de la estadificación en cáncer de mama. El One-Step Nucleic Acid Amplification (OSNA) es un ensayo molecular intraoperatorio que mide la cantidad de ARNm de citoqueratina 19 (CK19) presente en la totalidad del ganglio. Debido a su mayor sensibilidad y especificidad en comparación con el examen histológico convencional se está incorporando progresivamente para la detección de metástasis en ganglio centinela, con la recomendación de demostrar la expresión inmunohistoquímica de CK19 en una biopsia previa para evitar falsos negativos. El objetivo de nuestro estudio fue evaluar la expresión de CK19 en carcinomas de mama con especial énfasis en los subtipos especiales o poco frecuentes. Se estudiaron un total de 337 carcinomas de mama distribuidos en tres series: una retrospectiva de casos no seleccionados estudiados sobre «tissue microarrays», otra retrospectiva de secciones completas, enriquecida con subtipos histológicos especiales y/o perfil molecular triple-negativo y una serie prospectiva de biopsias, sobre secciones completas, previas al estudio OSNA. Considerando los resultados de nuestras tres series, la gran mayoría de los carcinomas de mama presentan positividad difusa de CK19; por tanto, sería muy poco probable, especialmente en variantes comunes, obtener un falso negativo de OSNA debido a la falta de expresión de CK19 en el tumor primario. Se demuestra expresión heterogénea únicamente en casos de subtipos especiales (carcinoma apocrino, adenoide quístico y medular) y/o perfil TN (AU)

Sentinel lymph node assessment is an essential part of the staging procedure in breast cancer. Increasingly, One-Step Nucleic Acid Amplification (OSNA), an intraoperative molecular assay that measures the amount of cytokeratin 19 (CK19) mRNA present in the whole of the sentinel lymph node, is being used in the detection of sentinel lymph node metastasis, due to its superior sensitivity and equal specificity compared with conventional histological examination. CK19 positive immunostaining in a previous biopsy is recommended to avoid false negative results with OSNA analysis. The aim of our study was to assess the degree of CK19 positivity in series of breast carcinomas with particular emphasis on special histological subtypes. The total of 337 breast carcinomas studied were distributed in three series as follows: a retrospective unselected series studied on tissue microarray, a series of whole sections enriched for special histological subtypes and triple negative carcinomas, and a prospective series of biopsies studied in whole sections, prior to a routine OSNA assay. The results of our three series revealed that the great majority of breast carcinomas exhibits a diffuse CK19 positivity. Therefore it would be highly unlikely, especially in frequent variants, to have a false negative OSNA result due to lack of CK19 expression in the primary tumour. Heterogeneous expression was only observed in patients with infrequent variants (apocrine, adenoid cystic and medullary carcinomas) and/or TN profile (AU)

Cross-reactivity of EGFR L858R mutation-specific antibody in HER2 positive gastric adenocarcinomas / Reacción cruzada del anticuerpo específico para la mutación EGFR L858R en los adenocarcinomas gástricos HER2 positivos

Recently, the study of the EGFR mutations in lung cancer has become mandatory due to their usefulness in target therapy. Lately, the use of specific antibodies to study the most prevalent of these mutations has been introduced. The newly published discovery of the existence of a cross-reaction between the HER2 protein and the EGFR L858R-specific antibody in breast cancer led us to corroborate this cross-reactivity in other tumors; specifically, we have chosen gastric carcinomas where HER2 is routinely evaluated as a molecular therapy driver. Our series consists of 15 primary gastric carcinomas, 7 HER2 cases positive for overexpression/amplification and 8 negative cases for both techniques. EGFR L858R mutation was studied with immunohistochemistry and complemented with real time PCR when positive. Immunohistochemistry assay with EGFR L858R was positive in 5 of the HER2 positive carcinomas (71%), none of which was confirmed by PCR, and negative in all HER2 negative carcinomas. Conclusions: The EGFR L858R antibody gives false positive results in most gastric carcinomas with HER2 overexpression/amplification, confirming the results described previously in breast cancer. It is also important to bear in mind that HER2 has also been described in other carcinomas, including lung cancer (AU)

En los últimos años, el estudio de las mutaciones de EGFR en cáncer de pulmón se ha convertido en esencial por su utilidad como diana terapéutica. Recientemente se ha introducido el uso de anticuerpos para la detección de las mutaciones más prevalentes. El descubrimiento de la existencia de una reacción cruzada entre la proteína HER2 y el anticuerpo específico EGFFR L858R, recientemente publicado en cáncer de mama, nos condujo a explorar la existencia de dicha reacción cruzada en otros tumores. Seleccionamos en primer lugar el cáncer gástrico, en el que se evalúa HER2 de forma rutinaria para la identificación de candidatos a tratamiento con terapia dirigida. La serie incluyó 15 carcinomas gástricos primarios, 7 casos positivos para sobreexpresión/amplificación de HER2 y 8 casos negativos para ambas técnicas. La presencia de la mutación EGFR L858R se estudió por inmunohistoquímica, y en caso de resultado positivo se complementó con el estudio molecular por PCR a tiempo real para su confirmación. El estudio inmunohistoquímico de EGFR L858R resultó positivo en 5 de los carcinomas HER2 positivos (71%), sin que ninguno se confirmara por PCR, y fue negativo para todos los carcinomas HER2 negativos. Conclusiones: El anticuerpo EGFR L858R presenta resultado falso positivo en la mayoría de carcinomas con sobreexpresión/amplificación de HER2, confirmando los resultados descritos previamente en cáncer de mama. Es importante tener presente que la presencia de HER2 ha sido descrita en otros carcinomas, incluido el cáncer de pulmón (AU)

Prospective validation of the microsatellite instability prediction models: RERtest6 and RERtest8

El estudio de la inestabilidad de microsatélites (MSI) es altamente recomendable en todos los carcinomas colorrectales, tanto como una primera aproximación para identificar posibles pacientes con síndrome de Lynch, como debido a su valor pronóstico y a su asociación con una mala respuesta a los regímenes basados en 5-fluorouracilo. A pesar de estas evidencias, la aplicación del estudio de MSI en la práctica general es bastante limitada. Este hecho pone de relieve la necesidad de herramientas de cribado para facilitar la implementación del estudio de MSI. Este trabajo presenta la validación de dos modelos de predicción previamente publicados, RERtest6 y RERtest8, basados en parámetros clínico-patológicos y dirigidos a una población no seleccionada por edad. La serie incluye 206 tumores colorrectales primarios de 199 pacientes, a los que se les aplicaron los modelos de predicción que contienen 6 ó 8 parámetros, respectivamente, antes de la evaluación del estado de MSI con el panel NCI consenso o el kit de MSI Promega. Se detectó alta inestabilidad de microsatélites (MSI-H) en 21 casos (10,1%). Ambos modelos han confirmado su robustez y fueron capaces de mantener valores predictivos negativos cercanos al 95%, lo que permite la reducción del número de casos que requieren un estudio molecular al 10%. Asimismo, la naturaleza de los parámetros incluidos en los modelos, que en su mayoría ya forman parte de un examen histopatológico de rutina, los convierten en una herramienta útil y de fácil aplicación en la práctica clínica (AU)

Determining microsatellite instability status (MSI) is now highly recommended for all diagnosed colorectal carcinomas, both as a first approach to identify putative Lynch syndrome patients, and as a valuable prognostic indicator as it is associated with a poor response to 5-fluorouracil-based regimes. However, generally the implementation of MSI testing is still quite limited, suggesting the need for screening tools which would simplify MSI testing. This study presents the validation of two previously published prediction models, RERtest6 and RERtest8, based on clinicopathological features and without age restriction. The series includes 206 primary colorectal tumors from 199 patients, to which the models containing 6 or 8 parameters were applied before the assessment of MSI status, using the consensus NCI panel or the MSI Promega kit. High-level microsatellite instability (MSI-H) was detected in 21 cases (10.1%). Both models confirmed their reliability and were able to maintain negative predictive values close to 95%, reducing the number of cases to be tested by molecular methods to 10%. Furthermore, the nature of the parameters included in the models, mostly already part of a routine histopathological examination, makes them a useful tool that can be easily implemented into routine practice (AU)

Mechanical properties and defect sensitivity of diamond nanothreads.

One of the newest carbon allotropes synthesized are diamond nanothreads. Using molecular dynamics, we determine the stiffness (850 GPa), strength (26.4 nN), extension (14.9%), and bending rigidity (5.35 × 10(-28) N·m(2)). The 1D nature of the nanothread results in a tenacity of 4.1 × 10(7) N·m/kg, exceeding nanotubes and graphene. As the thread consists of repeating Stone-Wales defects, through steered molecular dynamics (SMD), we explore the effect of defect density on the strength, stiffness, and extension of the system.

Cross-reactivity of EGFR mutation-specific immunohistochemistry assay in HER2-positive tumors.

The coexpression of HER2 and EGFR L858R in a solitary nodule removed from the lung, whose mutation was not confirmed by molecular techniques, made us think about the possible existence of a cross-reaction between HER2 and the EGFR L858R-specific antibody. Our study was designed to further analyze the existence of this cross-reaction and stress the need to exclude a metastatic breast cancer when dealing with EGFR L858R-positive cases. The series consists of 42 primary breast carcinomas, 22 HER2 positive for overexpression and amplification, and 20 negative for both. EGFR mutations were studied by immunohistochemistry and confirmed using real-time PCR when positive. Immunohistochemistry assay with EGFR L858R was positive in 19 (86%) of the HER2-positive breast carcinomas and negative in all HER2-negative carcinomas. The EGFR L858R antibody gives false-positive results in most of the breast carcinomas with HER2 overexpression/amplification. As a consequence, it is essential to confirm any EGFR L858R-positive cases by molecular methods or at least discard the presence of HER2 overexpression/amplification before rendering a diagnosis. It is also important to consider that HER2 has been described in other carcinomas such as urothelial, gastric or ovarian, as well as lung, although infrequently.

Differences in prevalence of inflammatory bowel disease in Puerto Rico between commercial and government-sponsored managed health care insured individuals.

BACKGROUND: Evaluation of ethnic and racial patterns of Inflammatory Bowel Disease (IBD) has demonstrated a higher incidence of IBD in Jews, and lower rates in blacks and Hispanics when compared to whites. There is limited data describing incidence and prevalence among Hispanics, the fastest growing minority in the United States. METHODS: To estimate the prevalence of IBD computerized records of all physicians billing and hospital discharges classified with ICD-9-CM IBD related codes were searched. Prevalence was estimated by age group, sex, and type of insurance (commercial versus government-sponsored managed care). RESULTS: Of 1,248,993 insured individuals in 2005, 186 had a diagnosis of Crohn's disease and 291 of ulcerative colitis. The estimated prevalence per 100,000 was 14.9 for Crohn's disease, 23.3 for ulcerative colitis, and 38.2 cases for IBD. The most significant difference was found when comparing insurance type, with a total IBD prevalence rate of 61.75 cases among commercial versus 14.36 cases among government-sponsored insured. CONCLUSIONS: The prevalence of IBD in this insured population in Puerto Rico places it among the highest described in a Hispanic population. Given the continued rise in prevalence of IBD and the limited studies describing the epidemiology of IBD in Hispanics, further studies which may provide important clues to the etiology of the disease as well as valuable information for appropriate health care planning are important.

ERG expression and prostatic adenocarcinoma.

ERG gene rearrangement has been identified as a highly specific alteration that is present in 40-50 % of prostate carcinomas. The standardization of an immunohistochemical assay with a novel anti-ERG antibody recently described would have significant diagnostic value. The aims of this study were to identify the incidence of this rearrangement in a Spanish population and to test the specificity of immunohistochemical ERG evaluation for prostate carcinomas. Three prostate tissue microarrays were constructed using radical prostatectomy specimens and related to grade, local invasion, and regional invasion. In addition to samples from malignant cases (160), specimens of prostatic hyperplasia (26) and high-grade prostatic intraepithelial neoplasia (10) were included. Tissue microarrays of 270 samples from most common malignant tumors (breast, colon, lung, and bladder) were also tested. All were analyzed by immunohistochemistry. Seventy-five out of 154 evaluable cases (49 %) of prostate carcinoma showed ERG expression; 52/75 showed strong staining. No ERG expression was observed in any of the high-grade prostatic intraepithelial neoplasia. ERG expression was independent of Gleason score (p = 0.160), extent of invasion (p = 0.517), and regional lymph node involvement (p = 0.816). No ERG expression was found in any other type of tumor, with the exception of one bladder cancer sample that showed focal and weak expression. The frequency of ERG detected in our study correlated with the results published for other Caucasian populations. Strong ERG protein expression was exclusively detected in prostate carcinomas, corroborating the specificity of ERG rearrangements for these tumors. Thus, detecting ERG using immunohistochemistry may be useful in routine practice in pathology departments.

Immunologic correlates of the abscopal effect in a patient with melanoma.

The abscopal effect is a phenomenon in which local radiotherapy is associated with the regression of metastatic cancer at a distance from the irradiated site. The abscopal effect may be mediated by activation of the immune system. Ipilimumab is a monoclonal antibody that inhibits an immunologic checkpoint on T cells, cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4). We report a case of the abscopal effect in a patient with melanoma treated with ipilimumab and radiotherapy. Temporal associations were noted: tumor shrinkage with antibody responses to the cancer-testis antigen NY-ESO-1, changes in peripheral-blood immune cells, and increases in antibody responses to other antigens after radiotherapy. (Funded by the National Institutes of Health and others.).

Immunotherapy for advanced melanoma.

The anticytotoxic T-lymphocyte antigen-4 (CTLA-4) monoclonal antibody ipilimumab was approved recently by the U.S. Food and Drug Administration for the treatment of patients with unresectable or metastatic melanoma. Anti-CTLA-4 treatment yields tumor responses or stable disease that may last months or years. Antitumor responses can occur within the first few weeks or even months after initiation of treatment, even as the disease appears to be progressing or new lesions are detected. Most side effects are immune related, consistent with the immune-based mechanism of action, and generally manageable with supportive measures and steroids. With anti-CTLA-4 therapy, patient response differs (both clinically and psychologically) to that generally observed with chemotherapy or other agents used to treat advanced cancer. Patients and caregivers require education about the likely patterns of response to treatment to help them understand why beneficial clinical outcomes may require weeks or months to occur and why continued treatment may be advisable, even when the disease may appear to be progressing. At the author's institution, the staff have noted that patients also need increased psychological support as a result of these clinical features and decisions. Patients and caregivers require instruction on recognition of potential side effects, the importance of reporting them in a timely manner, and their management.

KIT as a therapeutic target in metastatic melanoma.

CONTEXT: Some melanomas arising from acral, mucosal, and chronically sun-damaged sites harbor activating mutations and amplification of the type III transmembrane receptor tyrosine kinase KIT. We explored the effects of KIT inhibition using imatinib mesylate in this molecular subset of disease. OBJECTIVE: To assess clinical effects of imatinib mesylate in patients with melanoma harboring KIT alterations. DESIGN, SETTING, AND PATIENTS: A single-group, open-label, phase 2 trial at 1 community and 5 academic oncology centers in the United States of 295 patients with melanoma screened for the presence of KIT mutations and amplification between April 23, 2007, and April 16, 2010. A total of 51 cases with such alterations were identified and 28 of these patients were treated who had advanced unresectable melanoma arising from acral, mucosal, and chronically sun-damaged sites. INTERVENTION: Imatinib mesylate, 400 mg orally twice daily. MAIN OUTCOME MEASURES: Radiographic response, with secondary end points including time to progression, overall survival, and correlation of molecular alterations and clinical response. RESULTS: Two complete responses lasting 94 (ongoing) and 95 weeks, 2 durable partial responses lasting 53 and 89 (ongoing) weeks, and 2 transient partial responses lasting 12 and 18 weeks among the 25 evaluable patients were observed. The overall durable response rate was 16% (95% confidence interval [CI], 2%-30%), with a median time to progression of 12 weeks (interquartile range [IQR], 6-18 weeks; 95% CI, 11-18 weeks), and a median overall survival of 46.3 weeks (IQR, 28 weeks-not achieved; 95% CI, 28 weeks-not achieved). Response rate was better in cases with mutations affecting recurrent hotspots or with a mutant to wild-type allelic ratio of more than 1 (40% vs 0%, P = .05), indicating positive selection for the mutated allele. CONCLUSIONS: Among patients with advanced melanoma harboring KIT alterations, treatment with imatinib mesylate results in significant clinical responses in a subset of patients. Responses may be limited to tumors harboring KIT alterations of proven functional relevance. Trial Registration clinicaltrials.gov Identifier: NCT00470470.

Clinicopathological and molecular characterization of colorectal micropapillary carcinoma.

Invasive micropapillary carcinoma is associated with frequent lymph node metastasis and adverse clinical outcome. Initially described as a variant of breast and ovarian carcinoma, it has subsequently been found in other organs, most recently the colon. Reports of colorectal micropapillary carcinoma to date are limited in number, and their molecular profile has not been established. The aims of the present study were to analyze their clinicopathological features and molecular profile, and compare them with those of conventional adenocarcinoma. Clinicopathological features of a cohort of 379 patients with primary colorectal cancer were retrospectively reviewed for the presence of the pattern characteristic of micropapillary carcinoma. We also assessed the expression of KRT7, KRT20, CEACAM5, MUC1 (EMA, clone E29), MUC1 (clone MA695), MLH1, MSH2, MSH6 and TP53 by immunohistochemistry. Genetic assessments of microsatellite instability, chromosomes 17p and 18q, and mutations in TP53, BRAF and KRAS were performed using DNA extracted from formalin-fixed, paraffin-embedded sections. In all, 60 of the reviewed cases (16%) had a micropapillary component that ranged from 5 to 95% of the tumor, characterized by a higher frequency of an infiltrative pattern, lymphovascular and perineural invasion, a higher depth of invasion and more positive lymph nodes than conventional adenocarcinoma. Immunohistochemistry for MUC1 (clone MA695) and MUC1 (EMA, clone E29) enhanced the characteristic inside-out staining pattern of the micropapillary carcinoma component, whereas the rest of the tumor showed luminal staining patterns. KRT7 expression was slightly increased in micropapillary carcinoma, but did not reach significance (17-3%, P=0.1967). The molecular parameters showed a higher frequency of TP53 alterations and a low incidence of microsatellite instability and RER phenotype (loss of mismatch repair protein) in micropapillary carcinoma. With regard to the histological parameters, micropapillary carcinoma appears to be more aggressive than conventional colorectal adenocarcinoma. The molecular profile supports the hypothesis that micropapillary carcinoma carcinogenesis develops through the classical chromosomal instability pathway.

Immunologic response to xenogeneic gp100 DNA in melanoma patients: comparison of particle-mediated epidermal delivery with intramuscular injection.

PURPOSE: Prior studies show that i.m. injection of xenogeneic orthologues of melanosomal antigens (tyrosinase, gp100) induces CD8(+) T-cell responses to the syngeneic protein. To further define the optimal vaccination strategy, we conducted a pilot clinical trial comparing i.m. injection with particle-mediated epidermal delivery (PMED). EXPERIMENTAL DESIGN: Human leukocyte antigen (HLA)-A*0201(+) disease-free melanoma patients were randomized to the PMED or i.m. arm, receiving eight vaccinations over 4 months. Patients received 4 microg or 2,000 microg per injection, respectively, of mouse gp100 DNA. Peripheral blood mononuclear cells were collected, cultured with gp100 peptides, and analyzed by tetramer and intracellular cytokine staining for responses to HLA-A*0201-restricted gp100 epitopes [gp100(209-217) (ITDQVPFSV) and gp100(280-288) (YLEPGPVTA)]. RESULTS: Twenty-seven patients with stage IIB-IV melanoma were analyzable for immune response. The only common toxicity was grade 1 injection site reaction in nine patients with no intergroup difference, and one dose-limiting toxicity of acute hypersensitivity occurred in a PMED patient with undiagnosed gold allergy. Four of 27 patients produced gp100 tetramer(+)CD8(+) T cells, all carrying the CCR7(lo)CD45RA(lo) effector-memory phenotype. Five of 27 patients generated IFN-gamma(+)CD8(+) T cells, one who was also tetramer-positive. Overall, vaccination induced a response in 30% of patients, which was not significantly associated with study arm or clinical outcome. However, the PMED group showed a trend toward increased IFN-gamma(+)CD8(+) T-cell generation (P = 0.07). CONCLUSION: A comparable efficacy and safety profile was shown between the i.m. and PMED arms, despite a significantly decreased dose of DNA used for PMED injection.