Prognostic Value of ACVRL1 Expression in Metastatic Colorectal Cancer Patients Receiving First-line Chemotherapy With Bevacizumab: Results From the Triplet Plus Bevacizumab (TRIBE) Study.

BACKGROUND: No biomarkers exist to predict benefit from antiangiogenic therapy in metastatic colorectal cancer patients. ACVRL1 (activin receptor like-protein 1) encodes for ALK1, a member of the transforming growth factor-ß receptor family, which directs pathologic angiogenesis. We examined the intratumoral expression of ACVRL1 and other angiogenesis pathway-related genes to identify molecular markers in the TRIBE study. MATERIALS AND METHODS: Of 503 randomized patients, 228 had sufficient tissue for analysis. Formalin-fixed paraffin-embedded specimens were examined for expression of VEGF-A, VEGF-B, VEGF-C, VEGFR1, VEGFR2, ACVRL1, EphB4, and EGFL7 using reverse transcription polymerase chain reaction. A maximal χ2 approach was used to determine the messenger RNA levels associated with progression-free survival (PFS), overall survival (OS), response rate, early tumor shrinkage, and depth of response. Recursive partitioning trees were constructed to identify composite prognostic biomarker profiles. External validation was conducted in silico using the Oncomine database. RESULTS: High ACVRL1 expression was associated with superior OS in both treatment arms (FOLFOXIRI [5-fluorouracil, leucovorin, oxaliplatin, irinotecan]-bevacizumab, 32.7 vs. 13.5 months, hazard ratio [HR], 0.38, P = .023; FOLFIRI [5-fluorouracil, leucovorin, irinotecan]-bevacizumab, 35.1 vs. 22.0 months, HR, 0.36, P = .006) and prolonged PFS (11.7 vs. 5.9 months, multivariate HR, 0.17; P = .001) for patients receiving FOLFOXIRI-bevacizumab on univariate and multivariate analyses. In recursive partitioning analysis, ACVRL1 was the strongest discriminator of the response rate, PFS, and OS in patients receiving FOLFOXIRI-bevacizumab and of OS in patients receiving FOLFIRI-bevacizumab. In silico validation revealed significant associations between ACVRL1 expression, disease recurrence, and 1-year survival (P < .05) among all colorectal cancer stages. CONCLUSION: ACVRL1 expression could serve as a prognostic biomarker in metastatic colorectal cancer patients receiving chemotherapy and bevacizumab and warrants further evaluation in prospective studies.

Impact of a transfusion-free program on non-Jehovah's Witness patients undergoing liver transplantation.

BACKGROUND: Orthotopic liver transplantation (OLT) is associated with a large amount of blood loss. This article examines the impact of the initiation of a transfusion-free program in January 2000 for Jehovah's Witnesses (JWs) on the overall use of blood products in non-JW patients undergoing OLT. DESIGN: Retrospective review of OLT from January 1997 through December 2004. SETTING: University of Southern California University Hospital. PATIENTS: A total of 272 OLTs were performed on non-JW adults. This number includes 216 (79.4%) deceased donor and 56 (20.6%) living donor liver transplantations. Thirty-three OLTs were performed before January 2000 (ie, before the initiation of a transfusion-free program) (group 1), and 239 OLTs were performed after January 2000 (group 2). In group 2, all patients underwent OLT using cell-scavenging techniques and acute normovolemic hemodilution whenever feasible. Demographic, laboratory, and clinical data were collected and matched for severity of disease (model of end-stage liver disease [MELD] score). Transfusion records of packed red blood cells (PRBCs), platelets, and fresh frozen plasma (FFP) were obtained from the University of Southern California blood bank. RESULTS: In comparing group 2 with group 1, the mean MELD score was statistically significantly higher (P < .001), whereas the mean number of intraoperative PRBC and FFP transfusions was significantly lower (P = .03 and P = .004, respectively). The number of preoperative and postoperative PRBC, FFP, and platelet transfusions between the 2 groups was not statistically different. CONCLUSION: The development of a transfusion-free surgical program for JW patients has had a positive impact on reducing the overall blood use in non-JW patients undergoing OLT, despite the increase in MELD score.

Recombinant human coagulation factor VIIa in Jehovah's Witness patients undergoing liver transplantation.

Indisputably, liver transplantation is among the most technically challenging operations in current practice and is compounded by significant coagulopathy and portal hypertension. Recombinant human coagulation factor VIIa (rFVIIa) is a new product that was initially described to treat bleeding in hemophilia patients. We present in this paper 10 liver transplants in Jehovah's Witness patients using this novel product at University of Southern California-University Hospital. The subject population included nine males and one female with an average age of 50 years. Six patients underwent cadaveric and four live donor liver transplantation. Surgeries were conducted following our established protocol for transfusion-free liver transplantation, which includes preoperative blood augmentation, intraoperative blood salvage, acute normovolemic hemodilution, and postoperative blood conservation. Factor rFVIIa was used at a dose of 80 microg/kg intravenously just prior to the incision in all patients, and a second intraoperative dose was used in 3 patients. All living donor liver transplantation (LDLT) recipients did well and were discharged uneventfully with normal liver functions. Two of the six cadaveric recipients died. One patient died intraoperatively from acute primary graft nonfunction, and the other died 38 hours postoperatively from severe anemia. This report suggests factor rFVIIa might have a much broader application in surgery in the control of bleeding associated with coagulopathy.

Fetal-Maternal Hemorrhage Detected by Sudden Disappearance of Rh Immune Globulin-Related Anti-D.

BACKGROUND: Fetal-maternal hemorrhage is usually spontaneous and goes undetected but can be associated with adverse perinatal outcomes. CASE: We describe the detection of a fetal-maternal hemorrhage by abrupt disappearance of prophylactic anti-D on antibody screen in an Rh-negative mother with dichorionic twins admitted for atrial flutter of one twin. Both rosette and Kleihauer-Betke tests were positive. The diagnosis was confirmed by anemia in one twin at birth. CONCLUSION: Fetal-maternal hemorrhage requires a high index of suspicion for diagnosis. An unexpected sudden decline in Rh immune globulin-related anti-D may be an indication of fetal-maternal hemorrhage.

Large-scale screening and molecular characterization of EML4-ALK fusion variants in archival non-small-cell lung cancer tumor specimens using quantitative reverse transcription polymerase chain reaction assays.

INTRODUCTION: The objective of this study was to identify and characterize echinoderm microtubule-associated protein-like 4 anaplastic lymphoma kinase fusion (EML4-ALK+) cancers by variant-specific, quantitative reverse transcription polymerase chain reaction (RT-PCR) assays in a large cohort of North American non-small-cell lung cancer (NSCLC) patients. METHODS: We developed a panel of single and multiplex RT-PCR assays suitable for rapid and accurate detection of the eight most common EML4-ALK+ variants and ALK gene expression in archival formalin-fixed, paraffin-embedded NSCLC specimens. EGFR and KRAS genotyping and thymidylate synthase RNA level by RT-PCR assays were available in a subset of patients. RESULTS: Between December 2009 and September 2012, 7344 NSCLC specimens were tested. An EML4-ALK+ transcript was detected in 200 cases (2.7%), including 109 V1 (54.5%), 20 V2 (10.0%), 68 V3 (34.0%), and three V5a (1.5%) variants. Median age was 54.5 years (range, 23-89), and 104 patients (52.0%) were women. The great majority (n=188, 94.0%) of EML4-ALK+ NSCLC tumors had adenocarcinoma histology. ALK expression level varied significantly among different EML4-ALK+ variants and individual tumors. Only one case each of concurrent EGFR or KRAS mutation was detected. The median thymidylate synthase RNA level from 85 EML4-ALK+ cancers was significantly lower compared with that of EML4-ALK-negative lung adenocarcinomas (2.02 versus 3.29, respectively, p<0.001). CONCLUSIONS: This panel of variant-specific, quantitative RT-PCR assays detects common EML4-ALK+ variants as well as ALK gene expression level in archival formalin-fixed paraffin-embedded NSCLC specimens. These RT-PCR assays may be useful as an adjunct to the standard fluorescence in situ hybridization assay to better understand biologic variability and response patterns to anaplastic lymphoma kinase inhibitors.

Histology-related associations of ERCC1, RRM1, and TS biomarkers in patients with non-small-cell lung cancer: implications for therapy.

INTRODUCTION: On the basis of the results of recent clinical trials, histology-based decision-making for therapy of non-small-cell lung cancer has been advocated. We hypothesized associations of the biomarkers excision repair cross-complementing 1 (ERCC1), ribonucleotide reductase M1 (RRM1), and thymidylate synthase (TS) with histology as a contributing factor to reported differences in chemotherapy outcomes between squamous cell carcinoma (SCCA) and adenocarcinoma (AC) subtypes. Here, we report analysis of the Response Genetics Inc., database and implications for histology-based therapy. METHODS: RNA from microdissected formalin-fixed paraffin-embedded tumors was extracted and analyzed as previously described. Specimens from 2540 individual non-small-cell lung cancer patients were analyzed for one or more biomarkers, of which 1457 were categorized as AC or SCCA. RESULTS: For each biomarker, gene expression was lower in AC compared with SCCA (<0.001), although there was a wide range between individual patients. Gene expression was higher in men versus women: ERCC1: 2.51 versus 2.22 (p = 0.005); RRM1: 1.41 versus 1.24 (p = 0.004); TS: 3.23 versus 2.83 (p < 0.001). However, SCCA was more frequent in men versus women (30%/19%; p < 0.001). When AC and SCCA were assessed separately, the statistical significance between gene expression and sex was lost (in SCCA: ERCC1, p = 0.14; RRM1, p = 0.26; TS, p = 0.11). CONCLUSIONS: This analysis represents the largest data set for gene expression of these biomarkers reported so far. Significant histology-related associations for ERCC1, RRM1, and TS are seen. However, marked heterogeneity exists in individual patient tumor expression levels. Randomized phase III trials assessing the predictive value of these chemotherapy-related biomarkers are warranted.

Identification of Novel Variant of EML4-ALK Fusion Gene in NSCLC: Potential Benefits of the RT-PCR Method.

BACKGROUND: The discovery of the transforming fusion gene of the anaplastic lymphoma kinase (ALK) with the echinoderm microtubule-associated protein like 4 (EML4) as an oncogene in 2007 has led to its validation as a clinical target in NSCLC patients in a short period of time. The inhibition of the anaplastic lymphoma receptor tyrosine kinase has demonstrated to prolong progression-free survival compared to the standard of care chemotherapy in patients with advanced NSCLC that are ALK positive. However, the clinical implications of the 15 different variants of the EML4-ALK transforming gene described so far are currently not defined. Here we present a novel variant of the EML4-ALK fusion gene which we named variant 3c. METHODS: RNA extracted from formalin fixed paraffin embedded (FFPE) specimens from patients with advanced and metastatic NSCLC was amplified, using primers and probes designed to detect specific EML4-ALK fusion gene fragments. Gel electrophoresis showed a different band for the new variant 3c compared to the known bands of positive cell lines for variant 3a and 3b. These findings were further investigated by dye-terminator Sequencing and FISH. RESULTS: The novel variant, detected in two NSCLC specimens, is longer than v3a and shorter than v3b, representing an 18 base pair insertion of intron 19 of ALK between exon 6 of EML4 and exon 20 of ALK. All of the two samples showed exactly the same sequencing result. One of the samples was negative for FISH break apart testing and the other one showed a positive result, defined by ≥15% split nuclei as indicative of an ALK rearrangement. CONCLUSIONS: Compared to FISH technology, RT-PCR enables the detection of different isoforms of the EML4-ALK transforming gene, which can be validated by sequencing. Only one out of two samples that were positive for the new variant by RT-PCR could be confirmed by FISH. The clinical significance of the different variants, notably to resistance and response to ALK-Inhibitors and the concordance and sensitivity of FISH and RT-PCR should be subject to further investigations.

Squamous cell carcinoma arising in a second branchial cleft cyst.

The existence of primary branchiogenic carcinoma is controversial. In 1950, Martin et al. established four criteria for the diagnosis of primary branchiogenic carcinoma. In 1989, Khafif et al. proposed new criteria, which are currently most recognized in the literature. A pathologic description of the surgical specimen is analyzed and compared with a critical review of the literature. A case is presented in which imaging established the diagnosis of a benign second branchial cleft cyst 2 years prior to the diagnosis of a branchiogenic carcinoma in the same cyst. This cyst was resected and was found to have squamous cell carcinoma arising from the benign epithelium. This case satisfies the histologic criteria established by Martin and Khafif for a primary branchiogenic carcinoma arising in a previously benign second branchial cleft cyst.