PIK3CA Mutations are Common in Many Tumor Types and are Often Associated With Other Driver Mutations.

OBJECTIVES: Genotyping clinical cancer specimens determines a fuller spectrum of mutations that an individual tumor harbors, and thus provides better insight into its molecular pathogenesis. Using genotyping data collected during routine clinical care our objective was to better determine the genomic landscape associated with PIK3CA mutations since much interest has been placed on PIK3CA targeted therapy. METHODS: We performed multiplexed tumor genotyping within our CLIA-certified clinical laboratory on all consenting cancer patients who presented to the Brigham and Women's Hospital/Dana-Farber Cancer Center, regardless of histologic subtype. A total of 3252 cancers were genotyped for 471 mutations in 41 cancer-associated genes (including 23 mutations in PIK3CA), using a PCR-mass spectrometry assay. RESULTS: A total of 288 (9%) samples contained a mutation in PIK3CA, involving 25 different primary sites. In 117 (41%) cases, the PIK3CA mutation was found with at least 1 other mutation, many known oncogenic drivers, while only 7% of the non-PIK3CA mutated cases, when comparing like tumor types, had >1 mutation (P<0.0001). Breast cancers had the highest rate of PIK3CA mutations (34%), which correlated with estrogen receptor + status (P=0.0002). CONCLUSIONS: These findings suggest that PIK3CA mutations may be a relatively late event and may function primarily in a supporting/modifying role, and not as a primary driver of oncogenesis. Although further studies are needed, our observations during clinical tumor genotyping suggest that when other pro-oncogenic pathways are mutated along with PIK3CA, then, PIK3CA inhibition alone may not be effective and combination therapy may be warranted.

A Cluster of CNS Infections Due to B. cereus in the Setting of Acute Myeloid Leukemia: Neuropathology in 5 Patients.

Bacillus cereus typically causes a self-limited foodborne gastrointestinal (GI) illness. Severe invasive infection occurs rarely, mainly among immunocompromised hosts. We describe a cluster of B. cereus infections among 5 patients with acute myeloid leukemia and chemotherapy-induced neutropenia. The initial case presented with occipital lobe abscess and was found on biopsy to have organisms consistent with Bacillus species. Within 1 week, a second patient died of fulminant brain swelling and hemorrhage. Neuropathologic autopsy and culture revealed B. cereus; hospital infection control and public health officials were notified. Three more patients died within the subsequent 9 months (2 patients had rapid massive hemorrhage and many bacilli reminiscent of Bacillus anthracis infection, and 1 patient had sparse bacilli, petechial hemorrhages, and border zone infarcts). Blood cultures yielded positive results in 3 of 5 cases. A possible route of infection was hematogenous dissemination via GI mucosal breaches (GI symptoms occurred in 3 of 5 cases, and postmortem GI ulceration was found in 3 of 4 cases). Bacilli were seen in 2 of 3 GI ulcerations. Epidemiologic work-up, including a site visit conducted by the Centers for Disease Control and Prevention, did not identify a clear common source but suggested the possibility of bananas as a food source. Bacillus cereus causes a rapidly progressive, hemorrhagic meningoencephalitis with high mortality among patients with neutropenia. Neuropathologists can play a key role in the detection of outbreaks.

Novel molecular insights from routine genotyping of colorectal carcinomas.

Routine tumor genotyping enables identification of concurrent mutations in tumors and reveals low-frequency mutations that may be associated with a particular tumor phenotype. We genotyped 311 colorectal carcinomas (CRCs) for 471 mutation hot spots in 41 cancer-associated genes. At least 1 mutation was present in 239 (77%) of 311 tumors. Two concurrent mutations were identified in 89 (29%) tumors, 3 mutations in 24 (8%), 4 mutations in 6 (2%), and 5 mutations in 1 tumor. KRAS mutations were most frequent and identified in 132 (42%) tumors, followed by APC in 79 (25%) and TP53 in 64 (21%) tumors. Mutations in PIK3CA, BRAF, CTNNB1, and NRAS were identified in 41, 27, 11, and 9 cases, respectively. Rare mutations not typically associated with CRC included AKT1 (4), AKT2 (1), IDH1 (1), KIT (1), MAP2K1 (1), PTEN (2), and GNAS (6). GNAS mutations in CRC correlated with a mucinous phenotype and were present in 20% of all mucinous adenocarcinomas evaluated in this study. Among CRCs with a PIK3CA mutation, 77% showed concurrent mutations in other cancer-associated genes, and 4% of CRC did not neatly fit into either the chromosomal instability pathway or CpG island methylator phenotype/microsatellite instability pathway, suggesting overlapping mutational profile in some tumors. Our findings indicate that routine tumor genotyping is helpful in identifying low-frequency mutations, such as GNAS, that may correlate with a specific morphological phenotype and also reveal multiplicity of concurrent mutations in a significant proportion of CRC that may have significant implications for clinical trial design and personalized therapy.

Predictive factors in patients with advanced and metastatic squamous cell carcinoma of the head and neck: a study based on SWOG protocol S0420.

To evaluate the prognostic values of different protein expression in the progression of squamous cell carcinoma of the head and neck (SCCHN) patients, we conducted immunohistochemical (IHC) analysis in tissue samples of different patients enrolled on SWOG protocol S0420. S0420 was a phase II trial to evaluate the efficacy and safety of single-agent sorafenib in chemotherapy-naïve patients with metastatic or recurrent SCCHN. The primary end point was response probability, i.e., confirmed complete (CR) and partial response (PR). Sorafenib was administered orally at 400 mg twice daily on a continuous basis in 28-day cycles to eligible patients. Responses were evaluated according to RECIST (Response Evaluation Criteria in Solid Tumors) criteria. IHC analysis was performed for various markers and data were analyzed statistically. IHC data were obtained from 19 patients enrolled on S0420. There was a high frequency of cases with expression of the angiogenesis markers SMA, HIF-1α, Raf-1, VEGF and VEGF-R. None of the markers were significantly associated with response. Negative HER-2 status was associated with longer progression-free survival (PFS), P=0.04. Negative NRP-1 status was associated with longer overall survival (OS), P=0.04. There were no other significant associations. An almost universal overexpression of angiogenesis markers in the samples analyzed supports the evaluation of angiogenesis inhibition as a potential target for therapy. High levels of NRP-1 and HER-2 in SCCHN samples appear to be associated with decreased survival and earlier progression of disease, respectively, in SCCHN patients and may represent targets for therapy.

Expression of Vascular Endothelial Growth Factor correlates with the advance of clinical osteosarcoma.

PURPOSE: Osteosarcoma is the most common primary malignancy in orthopaedic surgery. Studies suggest that expression of VEGF and high vascularity within osteosarcoma may correlate with poor prognosis. The purpose of this study was to determine whether there was a correlation of VEGF expression with clinical tumour stage and metastasis. METHODS: This retrospective case series examined 54 cases of osteosarcoma patients who were treated during a ten-year period. Relevant clinical information included age, gender, tumour location, stage, adjuvant therapy, morbidity, mortality, and tumour subtypes. The clinical information was analysed for correlation of VEGF expression and tumour prognosis. Tumour sections were examined by routine H&E and by immunohistochemistry for VEGF, CD31, and the oncogenes c-myc and c-fos. RESULTS: There was a significantly positive correlation between VEGF expression and tumour stages among these cases (p < 0.01). The data also suggested a higher cancer recurrence and more frequent cases of remote metastasis in the high-VEGF group compared to the low-VEGF group. VEGF expression also positively associated with c-fos and c-myc expressions in the primary tumour sections. CONCLUSION: The results of this study highlight the role of VEGF in angiogenesis and tumour burden. Data also suggest the influence of VEGF may associate with the elevations of c-fos and c-myc expression. The development of novel therapies to target the VEGF pathway in osteosarcoma may lead to improved survival.

Simultaneous measurement of several cytokines using small volumes of biospecimens.

The role of host immunity in the development of virus-induced cancers has been difficult to elucidate, in part because of our inability to effectively measure multiple immune parameters using available amounts of biological material. The objective of the present study was to validate the use of a newly developed multiplex assay, the LINCOplex assay, for the simultaneous measurement of multiple cytokines [interleukin/(IL)-1beta, IL-2, IL-4, IL-6, IL-8, IL-10, IFN-gamma, and tumor necrosis factor-alpha]. Supernatants obtained from peripheral blood mononuclear cell cultures stimulated with various different mitogens and antigens (including phytohemagglutinin, influenza, tetanus, HPV16 E6 and E7 peptides, and media alone) were selected for study. In total, 750 specimens obtained from 26 participants were tested in replicate using the 8-plex LINCOplex assay (25 micro l of specimen required per well). Every specimen was included in duplicate in a blinded fashion. For some specimens, multiple 2-fold dilutions of the same specimen were included to evaluate the linearity of results. The availability of independently obtained IL-2 and IFN-gamma results from standard single cytokine (simplex) assays allowed for a direct comparison between the LINCOplex results and those obtained from the simplex assays. Spearman correlation coefficients for continuous results, and exact agreement rates and weighted kappa statistics for quartiled variables, were computed to evaluate intra- and interassay agreement. IL-4 levels were consistently below the detectable level of the assay (3 pg/ml) whereas IL-6 and IL-8 levels were consistently above the highest detectable level of the assay (10,000 pg/ml), and these three cytokines were, therefore, not evaluated further. For the remaining five cytokines, excellent intra-assay reproducibility was observed, with Spearman correlation coefficients consistently above 0.90 for all five cytokines. Exact agreement rates ranging from 77.6-90.3% and weighted kappas ranging from 0.81-0.92 were observed. Similar results were observed when analysis was restricted to supernatants obtained from cultures that had been stimulated with HPV16 peptides and when analysis was restricted to supernatants obtained from cultures containing no antigen or mitogen, suggesting that the LINCOplex assay is applicable under conditions where moderate or weak cytokine responses/levels are expected. Linearity of results was observed when dilutions of a single specimen were blindly tested, with the exception of IL-2 and IL-10, where deviations from linearity were sometimes observed. For IL-2 and IFN-gamma, where results from simplex assays were available for comparison, the LINCOplex assay and the simplex assay results agreed well. Spearman correlation coefficients were 0.86 and 0.93 for IL-2 and IFN-gamma, respectively. Exact agreement and weighted kappa values were 68.5% and 0.72 (95% confidence interval, 0.65-0.79), respectively, for IL-2 and 67.3% and 0.73 (95% confidence interval, 0.67-0.80), respectively, for IFN-gamma. These results indicate the applicability of the LINCOplex assay for the simultaneous measurement of multiple cytokines using small amounts of biological material.