Tumour-specific amplitude-modulated radiofrequency electromagnetic fields induce differentiation of hepatocellular carcinoma via targeting Cav3.2 T-type voltage-gated calcium channels and Ca2+ influx.

BACKGROUND: Administration of amplitude modulated 27·12 MHz radiofrequency electromagnetic fields (AM RF EMF) by means of a spoon-shaped applicator placed on the patient's tongue is a newly approved treatment for advanced hepatocellular carcinoma (HCC). The mechanism of action of tumour-specific AM RF EMF is largely unknown. METHODS: Whole body and organ-specific human dosimetry analyses were performed. Mice carrying human HCC xenografts were exposed to AM RF EMF using a small animal AM RF EMF exposure system replicating human dosimetry and exposure time. We performed histological analysis of tumours following exposure to AM RF EMF. Using an agnostic genomic approach, we characterized the mechanism of action of AM RF EMF. FINDINGS: Intrabuccal administration results in systemic delivery of athermal AM RF EMF from head to toe at levels lower than those generated by cell phones held close to the body. Tumour shrinkage results from differentiation of HCC cells into quiescent cells with spindle morphology. AM RF EMF targeted antiproliferative effects and cancer stem cell inhibiting effects are mediated by Ca2+ influx through Cav3·2 T-type voltage-gated calcium channels (CACNA1H) resulting in increased intracellular calcium concentration within HCC cells only. INTERPRETATION: Intrabuccally-administered AM RF EMF is a systemic therapy that selectively block the growth of HCC cells. AM RF EMF pronounced inhibitory effects on cancer stem cells may explain the exceptionally long responses observed in several patients with advanced HCC. FUND: Research reported in this publication was supported by the National Cancer Institute's Cancer Centre Support Grant award number P30CA012197 issued to the Wake Forest Baptist Comprehensive Cancer Centre (BP) and by funds from the Charles L. Spurr Professorship Fund (BP). DWG is supported by R01 AA016852 and P50 AA026117.

Differentiating Intrarenal Ectopic Adrenal Tissue From Renal Cell Carcinoma in the Kidney.

BACKGROUND: Adrenal rest (AR) is the presence of ectopic adrenal cortical tissue, often identified incidentally during autopsy (20% of postmortem examination). In the kidney, AR can be found in 6% of the general population. Ectopic adrenal tissue is of no functional significance but may in some cases, pose a diagnostic challenge for the pathologist, especially in the context of renal clear cell renal cell carcinoma (RCC) and small needle biopsies. AIM: To investigate the utility of immunohistochemical stains in distinguishing AR from RCC. METHODS: Archival cases of AR, in our institution, were reviewed and compared with a cohort of RCC cases using a panel of immunohistochemical stains, including PAX2, PAX8, calretinin, and inhibin. RESULTS: Nine of 10 (90%) cases of AR showed positive staining for inhibin and negative staining for calretinin, PAX2 and PAX8. One AR case was positive for PAX2 and PAX8 in addition to inhibin. All (100%) RCC cases were positive for PAX2 and PAX8, but negative for inhibin and calretinin. CONCLUSIONS: A panel of PAX2, PAX8 and inhibin may be useful markers for distinguishing AR from RCC. Calretinin was noncontributory in our study.

Mutational Landscapes of Smoking-Related Cancers in Caucasians and African Americans: Precision Oncology Perspectives at Wake Forest Baptist Comprehensive Cancer Center.

Background: Cancers related to tobacco use and African-American ancestry are under-characterized by genomics. This gap in precision oncology research represents a major challenge in the health disparities in the United States. Methods: The Precision Oncology trial at the Wake Forest Baptist Comprehensive Cancer Center enrolled 431 cancer patients from March 2015 to May 2016. The composition of these patients consists of a high representation of tobacco-related cancers (e.g., lung, colorectal, and bladder) and African-American ancestry (13.5%). Tumors were sequenced to identify mutations to gain insight into genetic alterations associated with smoking and/or African-American ancestry. Results: Tobacco-related cancers exhibit a high mutational load. These tumors are characterized by high-frequency mutations in TP53, DNA damage repair genes (BRCA2 and ATM), and chromatin remodeling genes (the lysine methyltransferases KMT2D or MLL2, and KMT2C or MLL3). These tobacco-related cancers also exhibit augmented tumor heterogeneities. Smoking related genetic mutations were validated by The Cancer Genome Atlas dataset that includes 2,821 cases with known smoking status. The Wake Forest and The Cancer Genome Atlas cohorts (431 and 7,991 cases, respectively) revealed a significantly increased mutation rate in the TP53 gene in the African-American subgroup studied. Both cohorts also revealed 5 genes (e.g. CDK8) significantly amplified in the African-American population. Conclusions: These results provide strong evidence that tobacco is a major cause of genomic instability and heterogeneity in cancer. TP53 mutations and key oncogene amplifications emerge as key factors contributing to cancer outcome disparities among different racial/ethnic groups.

Circulating mutational portrait of cancer: manifestation of aggressive clonal events in both early and late stages.

BACKGROUND: Solid tumors residing in tissues and organs leave footprints in circulation through circulating tumor cells (CTCs) and circulating tumor DNAs (ctDNA). Characterization of the ctDNA portraits and comparison with tumor DNA mutational portraits may reveal clinically actionable information on solid tumors that is traditionally achieved through more invasive approaches. METHODS: We isolated ctDNAs from plasma of patients of 103 lung cancer and 74 other solid tumors of different tissue origins. Deep sequencing using the Guardant360 test was performed to identify mutations in 73 clinically actionable genes, and the results were associated with clinical characteristics of the patient. The mutation profiles of 37 lung cancer cases with paired ctDNA and tumor genomic DNA sequencing were used to evaluate clonal representation of tumor in circulation. Five lung cancer cases with longitudinal ctDNA sampling were monitored for cancer progression or response to treatments. RESULTS: Mutations in TP53, EGFR, and KRAS genes are most prevalent in our cohort. Mutation rates of ctDNA are similar in early (I and II) and late stage (III and IV) cancers. Mutation in DNA repair genes BRCA1, BRCA2, and ATM are found in 18.1% (32/177) of cases. Patients with higher mutation rates had significantly higher mortality rates. Lung cancer of never smokers exhibited significantly higher ctDNA mutation rates as well as higher EGFR and ERBB2 mutations than ever smokers. Comparative analysis of ctDNA and tumor DNA mutation data from the same patients showed that key driver mutations could be detected in plasma even when they were present at a minor clonal population in the tumor. Mutations of key genes found in the tumor tissue could remain in circulation even after frontline radiotherapy and chemotherapy suggesting these mutations represented resistance mechanisms. Longitudinal sampling of five lung cancer cases showed distinct changes in ctDNA mutation portraits that are consistent with cancer progression or response to EGFR drug treatment. CONCLUSIONS: This study demonstrates that ctDNA mutation rates in the key tumor-associated genes are clinical parameters relevant to smoking status and mortality. Mutations in ctDNA may serve as an early detection tool for cancer. This study quantitatively confirms the hypothesis that ctDNAs in circulation is the result of dissemination of aggressive tumor clones and survival of resistant clones. This study supports the use of ctDNA profiling as a less-invasive approach to monitor cancer progression and selection of appropriate drugs during cancer evolution.

Diagnosing bone lesions: a practical non-pathology centric formulaic approach.

Bone lesions are perceived to be some of the most difficult lesions that pathologists encounter. The reasons for this are multiple and include lack of experience/familiarity, the need to rely heavily on non-pathology information and data, and the fact that many lesions are associated with either procedures or treatments with significant morbidity. However, in fact, the majority of bone lesions can be accurately assessed on the basis of data not directly related to traditional pathologic based assessment. In order to achieve this state, the pathologist must understand the consistent clinical parameters of most bone lesions, including their clinical presentation, the bone involved, particularly the anatomic site of the bone involved, and a fundamental, basic understanding of imaging studies, especially the plain radiograph. Once these principles are understood and mastered, the pathologist can easily diagnose most bone lesions, using traditional pathologic assessment to confirm the diagnosis.

Cartilage-forming tumors.

Cartilage-forming tumors as a group are the most common primary bone tumors; this is largely due to the common occurrence of asymptomatic benign lesions such as osteochondroma and enchondroma. The common feature of these tumors is the presence of chondrocytic cells and the formation of cartilaginous tumor matrix. Some of these tumors are true neoplasms while others are hamartomas or developmental abnormalities. The morphologic heterogeneity of these tumors may be explained by a common multipotent mesenchymal cell differentiating along the lines of fetal-adult cartilage maturation. Recently mutations in IDH1 and IDH2 have been detected in a variety of benign and malignant cartilaginous tumors.(1-4.)

Secondary chondrosarcoma of the pelvis arising from a solitary exostosis in an 11-year-old patient: a case report with 5-year follow-up.

Although conversion of an osteochondroma to chondrosarcoma is a well-described rare occurrence, it is usually associated with syndromes such as multiple hereditary exostoses and is much more common after maturity. We present here a rare case of secondary pelvic chondrosarcoma arising from a solitary exostosis in a pediatric patient. An 11-year-old, otherwise healthy, female was referred to our clinic for evaluation of a pelvic mass detected on a radiograph. The radiographs obtained by the referring physician demonstrated a large lesion arising from the right superior pubic ramus, which was visible but not identified on an abdominal radiograph several years prior. Histopathologic analysis showed chondrosarcoma which was supported by an additional opinion to rule out chondroblastic osteosarcoma. The patient was treated with wide resection without adjuvant therapy and is doing well with no evidence of recurrence five years post-operatively. There have been only a few small case series describing chondrosarcoma in the pediatric patient. Even rarer are descriptions of secondary chondrosarcoma with only occasional cases reported as part of larger case series. Chondrosarcoma is a rare and difficult diagnosis in the pediatric patient. There is often considerable debate between chondrosarcoma and chondroblastic osteosarcoma, and the treatment implications of differentiating these diagnoses are of paramount importance.

Relationship between HER-2 overexpression and brain metastasis in esophageal cancer patients.

AIM: To study if HER-2 overexpression by locally advanced esophageal cancers increase the chance of brain metastasis following esophagectomy. METHODS: We retrospectively reviewed the medical records of esophageal cancer patients who underwent esophagectomy at University of Iowa Hospitals and Clinics between 2000 and 2010. Data analyzed consisted of demographic and clinical variables. The brain metastasis tissue was assayed for HER-2 overexpression utilizing the FDA approved DAKO Hercept Test(®). RESULTS: One hundred and forty two patients were reviewed. Median age was 64 years (36-86 years). Eighty eight patients (62%) received neoadjuvant chemoradiotherapy. Pathological complete and partial responses were achieved in 17 (19%) and 71 (81%) patients. Cancer relapsed in 43/142 (30%) patients. The brain was the first site of relapse in 9/43 patients (21%, 95% CI: 10%-36%). HER-2 immunohistochemistry testing of the brain metastasis tissue showed that 5/9 (56%) cases overexpressed HER-2 (3+ staining). CONCLUSION: HER-2 overexpression might be associated with increased risk of brain metastasis in esophageal cancer patients following esophagectomy. Further studies will be required to validate this observation.

Putative multifunctional signature of lung metastases in dedifferentiated chondrosarcoma.

Chondrosarcomas are among the most malignant skeletal tumors. Dedifferentiated chondrosarcoma is a highly aggressive subtype of chondrosarcoma, with lung metastases developing within a few months of diagnosis in 90% of patients. In this paper we performed comparative analyses of the transcriptomes of five individual metastatic lung lesions that were surgically resected from a patient with dedifferentiated chondrosarcoma. We document for the first time a high heterogeneity of gene expression profiles among the individual lung metastases. Moreover, we reveal a signature of "multifunctional" genes that are expressed in all metastatic lung lesions. Also, for the first time, we document the occurrence of massive macrophage infiltration in dedifferentiated chondrosarcoma lung metastases.

Consensus statement on effective communication of urgent diagnoses and significant, unexpected diagnoses in surgical pathology and cytopathology from the College of American Pathologists and Association of Directors of Anatomic and Surgical Pathology.

CONTEXT: Recognizing the difficulty in applying the concept of critical values to anatomic pathology diagnoses, the College of American Pathologists and the Association of Directors of Anatomic and Surgical Pathology have chosen to reevaluate the concept of critical diagnoses. OBJECTIVE: To promote effective communication of urgent and significant, unexpected diagnoses in surgical pathology and cytology. DESIGN: A comprehensive literature search was conducted and reviewed by an expert panel. RESULTS: A policy of effective communication of important results in surgical pathology and cytology is desirable to enhance patient safety and to address multiple regulatory requirements. CONCLUSIONS: Each institution should create its own policy regarding urgent diagnoses and significant, unexpected diagnoses in anatomic pathology. This policy should be separate from critical results or panic-value policies in clinical pathology, with the expectation of a different time frame for communication. Urgent diagnosis is defined as a medical condition that, in most cases, should be addressed as soon as possible. Significant, unexpected diagnosis is defined as a medical condition that is clinically unusual or unforeseen and should be addressed at some point in the patient's course. Further details of this statement are provided.

Leiomyosarcoma of the head and neck: a population-based analysis.

OBJECTIVES: To describe the characteristics of head and neck leiomyosarcoma and to identify factors associated with survival. DESIGN: Retrospective population-based study. PATIENTS: The 17-registry Surveillance, Epidemiology, and End Results database was used to identify 578 patients with leiomyosarcoma of the head and neck. INTERVENTIONS: Surgery and primary and adjuvant radiotherapy. MAIN OUTCOME MEASURES: Patient demographics and tumor characteristics were examined. Treatment modalities were compared, and survival was assessed using the log-rank test. RESULTS: The mean age at diagnosis was 64 years. Most tumors were smaller than 5 cm in greatest dimension (87%) and high grade (44% were moderately differentiated and 39% were poorly differentiated). The primary tumor demonstrated deep extension in 39% of cases, and 2% had lymph node metastases. The most common primary site was the skin and soft tissue of the head and neck (83%). Surgical treatment was provided to 89% of patients, 14% received adjuvant radiotherapy, and 4% received radiotherapy alone. The median observed survival was 84.7 months. The 5-year disease-specific survival rate was 87.6% in patients with well-differentiated tumors, 85.7% in patients with moderately differentiated tumors, and 52.7% in patients with poorly differentiated tumors (P < .001). Survival was better for patients who received surgery alone (median survival, 100.1 months [n = 413]) than for those who received radiotherapy alone (median survival, 16 months [n = 16]) or adjuvant radiotherapy (median survival, 64.2 months [n = 80]) (P < .001). The latter group was more likely to have poorly differentiated, large, locally invasive tumors. CONCLUSIONS: Leiomyosarcoma typically presents in older patients; it is often poorly differentiated; and improved survival is associated with surgical treatment.

Micro-recanalization in a biodegradable graft for reconstruction of the vas deferens is enhanced by sildenafil citrate.

This study investigated the effect of sildenafil citrate on micro-recanalization and neovascularization, which were previously demonstrated in a rat model using biodegradable grafts (BGs) for vas deferens reconstruction. A total of 24 male rats underwent bilateral vasectomy with removal of a 0.5-cm vasal segment and were randomly assigned to four groups. Groups 1 and 2 underwent immediate vasovasostomy. Groups 3 and 4 underwent interposition of a 0.5-cm BG in the vasal gap. Groups 1 and 3 were given 5 mg kg(-1) day(-1) oral sildenafil. Other groups were given placebo. Rats were housed with females 12 weeks postoperatively. Reconstructed vasal segments were harvested 16 weeks postoperatively and analyzed histologically. Fluid from the distal vasal stump was analyzed for motile sperm. Urine samples obtained 16 weeks postoperatively were analyzed for cGMP levels. cGMP levels in rats treated with sildenafil were significantly higher than in control rats. No pregnancies were sired by grafted groups. In all, 5/6 rats in group 1 and 3/6 rats in group 2 sired litters. No motile sperm were noted in the vasal fluid of the grafted groups. Motile sperm were noted in all rats in group 1 and in 5/6 rats in group 2. In addition, 29 and 4 microcanals were detected in the sildenafil and placebo groups, respectively (P = 0.023). No microcanal exceeded 3 mm in length. An average of 12 and 28 blood vessels per graft were noted in the placebo and sildenafil groups, respectively (P < 0.0001). In conclusion, sildenafil enhances micro-recanalization and neovascularization in BG used for vas deferens reconstruction, but does not increase the microcanal length after 16 weeks.

Antibody expiration in the context of resource limitation: what is the evidence basis?

The implementation and enforcement of the College of American Pathologists Survey Checklist ANP 22432 has renewed attention on the issue of outdating of antibodies used for immunohistochemical analysis. The current study examined the staining patterns of 26 recently acquired primary antibodies and their expired counterparts. Two reviewers examined sequential sections of formalin-fixed, paraffin-embedded tissue samples for staining intensity and percentage of positivity. Appropriate positive and negative control studies were performed. Of the 26 antibodies, 20 exhibited no difference in percentage of positivity or staining intensity. Of the remaining 6, 3 showed better performance with the expired cohort and 3 with nonexpired antibodies. However, no antibody staining characteristics varied by more than 1 step, and in no case was positive staining lost after antibody expiration. Negligible differences exist in immunostaining between outdated and current antibodies. Thus, exemption for primary antibodies from existing regulations would conserve resources without adversely impacting patient care.

Frequent attenuation of the WWOX tumor suppressor in osteosarcoma is associated with increased tumorigenicity and aberrant RUNX2 expression.

The WW domain-containing oxidoreductase (WWOX) is a tumor suppressor that is deleted or attenuated in most human tumors. Wwox-deficient mice develop osteosarcoma (OS), an aggressive bone tumor with poor prognosis that often metastasizes to lung. On the basis of these observations, we examined the status of WWOX in human OS specimens and cell lines. In human OS clinical samples, WWOX expression was absent or reduced in 58% of tumors examined (P < 0.0001). Compared with the primary tumors, WWOX levels frequently increased in tumors resected following chemotherapy. In contrast, tumor metastases to lung often exhibited reduced WWOX levels relative to the primary tumor. In human OS cell lines having reduced WWOX expression, ectopic expression of WWOX inhibited proliferation and attenuated invasion in vitro, and suppressed tumorigenicity in nude mice. Expression of WWOX was associated with reduced RUNX2 expression in OS cell lines, whereas RUNX2 levels were elevated in femurs of Wwox-deficient mice. Furthermore, WWOX reconstitution in HOS cells was associated with downregulation of RUNX2 levels and RUNX2 target genes, consistent with the ability of WWOX to suppress RUNX2 transactivation activity. In clinical samples, RUNX2 was expressed in the majority of primary tumors and undetectable in most tumors resected following chemotherapy, whereas most metastases were RUNX2 positive. Our results deepen the evidence of a tumor suppressor role for WWOX in OS, furthering its prognostic and therapeutic significance in this disease.

Pathology education: quo vadis?

The teaching of pathology and the face of pathology as a career are changing rapidly. A short questionnaire was constructed covering key areas impacted by curricular change to collect data for a workshop to be held at the Annual Meeting of the West and Mid-West Region of the Association of Pathology Chairs. Questions included the ongoing existence of pathology as a separate discipline, the degree of 'control' exercised by pathology faculty, the length of the course (hours), the use of microscopes, and selected outcomes data, specifically National Board of Medical Examiners graphs of comparative performance of disciplines and US Medical Licensing Examination Step 1 data. The written responses and discussion by attendees at the meeting are presented with preliminary analysis and commentary. It was concluded that 'evidence-based education' represents something of a novelty, worthy of further study.

Her-2/neu expression in salivary duct carcinoma: an immunohistochemical and chromogenic in situ hybridization study.

Salivary duct carcinoma (SDC) shares significant morphologic and immunophenotypic overlap with ductal carcinoma of the breast, including HER-2/neu expression. Previous studies have detected HER-2/neu at the protein level in SDCs; however, no study, to date, has assayed whether this expression is related to gene amplification detected by chromogenic in situ hybridization (CISH). Formalin-fixed, paraffin-embedded tissue sections from 12 previously diagnosed SDCs were evaluated by immunohistochemistry (IHC) and CISH for HER-2/neu status. Result concordance was seen in all 12 cases. A total of 4 SDCs were positive by IHC; all 4 cases showed amplification with CISH. The remaining 8 cases were negative by IHC and showed no gene amplification with CISH. SDCs in this study show HER-2/neu overexpression on both the protein and gene levels in approximately 30% of cases. These findings suggest a role may exist for Herceptin (trastuzumab) based therapy in some SDC patients.

RDP58 inhibits T cell-mediated bladder inflammation in an autoimmune cystitis model.

Interstitial cystitis (IC) is a chronic inflammatory condition of the urinary bladder with a strong autoimmune component. Currently, the major challenge in IC treatment is the development of effective therapies. RDP58 is a novel d-amino acid decapeptide with potent immunosuppressive activity. In this study, we investigated whether RDP58 was effective as an intravesical agent for treating bladder autoimmune inflammation in a transgenic mouse model (URO-OVA mice). URO-OVA mice were adoptively transferred with syngeneic activated splenocytes of OT-I mice transgenic for the OVA-specific CD8(+) TCR for cystitis induction and treated intravesically with RDP58 at days 0 and 3. Compared with controls, the RDP58-treated bladders showed markedly reduced histopathology and expressions of mRNAs and proteins of TNF-alpha, NGF and substance P. To determine whether the inhibition of bladder inflammation by RDP58 was due to the interference with effector T cells, we treated the cells with RDP58 in vitro. Cells treated with RDP58 showed reduced production of TNF-alpha and IFN-gamma as well as apoptotic death. Collectively, these results indicate that RDP58 is effective for treating T cell-mediated experimental autoimmune cystitis and may serve as a useful intravesical agent for the treatment of autoimmune-associated bladder inflammation such as IC.

Somatostatin receptor scintigraphy in surveillance of pediatric brain malignancies.

BACKGROUND: Somatostatin receptor scintigraphy (SRS) has been successfully used in imaging PBTs and, as a functional imaging modality, may be better able to differentiate tumor from scar/necrosis. This retrospective study evaluates the role of SRS in post-treatment surveillance of PBTs. PROCEDURE: Twenty children (age range: 7 months to 24 years, mean: 9 years) with known brain malignancies underwent serial SRS and MRI. The sensitivity and specificity of SRS and MRI were compared for surveillance scanning using patient outcome as the reference standard. Somatostatin receptors (sstrs) expression was determined by immunohistochemistry (IHC) of available tumor specimens. RESULTS: SRS was true positive (TP) in 15 of 16 patients with proven disease found post-resection (n = 5) or during follow-up (n = 11). In contrast, MRI was positive in 12 of these 16 patients and equivocal in another two patients where it could not distinguish between radiation necrosis and tumor recurrence. The two patients with false negative (FN) MRI and proven disease were positive by SRS. SRS was negative in all four patients with no evidence of disease by follow-up (mean follow-up = 58 months). The only patient with a FN SRS (TP by MRI) was one without IHC evidence of sstrs. SRS was TP in 7/7 tumors with IHC documented sstrs. CONCLUSIONS: SRS is a useful adjunct to MRI for post-treatment surveillance of sstr-positive PBTs, particularly when MRI is equivocal.