Allison Zemek

Identification of tumorigenic cells and therapeutic targets in pancreatic neuroendocrine tumors

Significance This is the first in-depth profiling of pancreatic neuroendocrine tumors (PanNETs), to our knowledge, that illuminates fundamental biological processes for this class of tumors. Beginning with the index case and confirmed with additional patient tumors, we showed the dependence on paracrine signaling through the hepatocyte growth factor (HGF)/receptor tyrosine kinase MET axis. We created a novel cell line derived from a well-differentiated PanNET with autocrine HGF/MET signaling. We also identified the cell-surface protein CD90 as a marker of the tumor-initiating cell population in PanNETs that allows for prospective isolation of this critical cell population. Finally, we demonstrated the efficacy of anti-CD47 therapy in PanNETs. These findings provide a foundation for developing therapeutic strategies that eliminate tumor-initiating cells in PanNETs and show how deep examination of individual cases can lead to potential therapies. Pancreatic neuroendocrine tumors (PanNETs) are a type of pancreatic cancer with limited therapeutic options. Consequently, most patients with advanced disease die from tumor progression. Current evidence indicates that a subset of cancer cells is responsible for tumor development, metastasis, and recurrence, and targeting these tumor-initiating cells is necessary to eradicate tumors. However, tumor-initiating cells and the biological processes that promote pathogenesis remain largely uncharacterized in PanNETs. Here we profile primary and metastatic tumors from an index patient and demonstrate that MET proto-oncogene activation is important for tumor growth in PanNET xenograft models. We identify a highly tumorigenic cell population within several independent surgically acquired PanNETs characterized by increased cell-surface protein CD90 expression and aldehyde dehydrogenase A1 (ALDHA1) activity, and provide in vitro and in vivo evidence for their stem-like properties. We performed proteomic profiling of 332 antigens in two cell lines and four primary tumors, and showed that CD47, a cell-surface protein that acts as a “don’t eat me” signal co-opted by cancers to evade innate immune surveillance, is ubiquitously expressed. Moreover, CD47 coexpresses with MET and is enriched in CD90hi cells. Furthermore, blocking CD47 signaling promotes engulfment of tumor cells by macrophages in vitro and inhibits xenograft tumor growth, prevents metastases, and prolongs survival in vivo.

Performance of Targeted Fungal Sequencing for Culture-Independent Diagnosis of Invasive Fungal Disease

Background Identification of fungi causing invasive fungal disease (IFD) is critical for guiding antifungal therapy. We describe the performance and clinical impact of a targeted panfungal polymerase chain reaction (PCR) amplicon sequencing assay for culture-independent diagnosis of IFD. Methods Between January 2009 and September 2016, 233 specimens, consisting of fresh and formalin-fixed, paraffin-embedded (FFPE) tissues and sterile body fluids with known diagnosis of IFD based on reference method results (n = 117), and specimens with negative fungal culture, but with microscopic and ancillary findings indicative of IFD (n = 116), were included. PCR amplicons from the internal transcribed spacer 2 and the D2 region of 28S ribosomal RNA gene were sequenced and fungi identified. Results Sensitivity and specificity of fungal sequencing in specimens with known diagnosis were 96.6% (95% confidence interval [CI], 87.4%-99.4%; 58/60) and 98.2% (95% CI, 89.4%-99.9%; 56/57). In patients with suspected IFD, the diagnostic yield of fungal sequencing was 62.9% (73/116) overall and 71.3% (57/80) in patients classified with proven IFD based on the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and Mycoses Study Group (EORTC/MSG) criteria. Samples obtained by open biopsy had a significantly higher diagnostic yield (71.5% [40/56]) compared with core-needle biopsy (50% [17/34] P = .04) and fine needle aspiration (0% [0/2]; P = .009). Additionally, D2 sequencing diagnosed 5 cases of invasive protozoal infections due to Toxoplasma gondii (n = 3), Trypanosoma cruzi, and Leishmania species. Sequencing results altered patient management in the majority of suspected cases. Conclusions The targeted fungal sequencing assay allowed accurate identification of fungi causing IFD and additionally provided partial-protozoal coverage. The diagnostic yield was dependent on the amount of tissue available for testing.

Big data modeling to predict platelet usage and minimize wastage in a tertiary care system

Significance In modern hospital systems where complicated, severely ill patient populations are the norm, there is currently no reliable way to forecast the use of perishable medical resources to enable a smart and economic way to deliver optimal patient care. We here demonstrate a statistical model using hospital patient data to quantitatively forecast, days in advance, the need for platelet transfusions. This approach can be leveraged to significantly decrease platelet wastage, and, if adopted nationwide, would save approximately 80 million dollars per year. We believe our approach can be generalized to all other aspects of patient care involving timely delivery of perishable medical resources. Maintaining a robust blood product supply is an essential requirement to guarantee optimal patient care in modern health care systems. However, daily blood product use is difficult to anticipate. Platelet products are the most variable in daily usage, have short shelf lives, and are also the most expensive to produce, test, and store. Due to the combination of absolute need, uncertain daily demand, and short shelf life, platelet products are frequently wasted due to expiration. Our aim is to build and validate a statistical model to forecast future platelet demand and thereby reduce wastage. We have investigated platelet usage patterns at our institution, and specifically interrogated the relationship between platelet usage and aggregated hospital-wide patient data over a recent consecutive 29-mo period. Using a convex statistical formulation, we have found that platelet usage is highly dependent on weekday/weekend pattern, number of patients with various abnormal complete blood count measurements, and location-specific hospital census data. We incorporated these relationships in a mathematical model to guide collection and ordering strategy. This model minimizes waste due to expiration while avoiding shortages; the number of remaining platelet units at the end of any day stays above 10 in our model during the same period. Compared with historical expiration rates during the same period, our model reduces the expiration rate from 10.5 to 3.2%. Extrapolating our results to the ∼2 million units of platelets transfused annually within the United States, if implemented successfully, our model can potentially save ∼80 million dollars in health care costs.

Sa1141 Clinical and Immunohistochemical Features of High Grade Neuroendocrine Neoplasia of the Gastrointestinal Tract

Background: Neuroendocrine neoplasms of the gastrointestinal tract and pancreas are classified in the WHO 2010 system into three grades, using mitotic figure counts and Ki-67 indices. There is increasing evidence that neuroendocrine carcinomas, WHO grade 3, have a broad range of clinical behavior. Morphology and immunohistochemistry may aid in predicting outcome and distinguishing between poorly-differentiated neuroendocrine tumors and poorly-differentiated neuroendocrine carcinoma.

Perianal Extramammary Paget’s Disease: More Than Meets the Eye

A 75-year-old man was initially evaluated for a 1-year history of perianal pruritus that was refractory to multiple topical therapies. He had a sharply demarcated, erythematous and scaly rash involving the left perianal skin, originating from the left hemi-circumference of the anal verge and extending anteriorly, laterally and posteriorly to involve a 6 cm × 4 cm area of skin (Fig. 1). A punch biopsy of the lesion confirmed extramammary Paget’s disease (EMPD). Immunostaining of the biopsy was positive for cytokeratin (CK)7 and GATA3, weakly positive for cytokeratin 20, and negative for CDX2 and prostate-specific antigen (PSA), all consistent with likely primary EMPD. Patient’s past history was notable for coronary artery disease and prostate cancer, for which he had undergone prostatectomy. His recent serum PSA had been unremarkable at 0.02 ng/mL. PSA immunostaining of the punch biopsy was negative, as was urine cytology, excluding underlying urothelial intraepithelial neoplasia. Further evaluation to exclude an underlying malignancy included computed tomography (CT) of the abdomen and pelvis, which was unremarkable. Colonoscopy from several months prior to initial presentation was likewise negative. He was referred for colorectal surgery consultation to discuss management, at which time wide local excision was recommended. Given the size of his lesion, the patient was also evaluated by plastic surgery for staged reconstruction after confirming negative microscopic margins. The patient then underwent wide local excision of the perianal EMPD with a 5 mm margin of grossly normal perianal skin, which included excision of an external hemorrhoid, since it was abutted by the grossly involved medial margin. Additional biopsies of grossly normal skin around the main excision site were taken to direct potential re-excisions. Pathology of the main specimen showed malignant cells infiltrating the superficial dermis without angiolymphatic invasion (Fig. 2a) and positive posterolateral margins. The additional posterolateral biopsy sites were also positive for EMPD. Due to these findings, re-excision was performed with additional posterolateral margins from the first excision site and additional biopsies around the re-excision site taken for further mapping (Fig. 3a). Pathology of the re-excision specimen confirmed EMPD infiltrating the superficial dermis, with negative margins. Nevertheless, rare EMPD cells within the superficial dermis were present in one of the lateral biopsy sites (Fig. 2b). The patient underwent a third excision, which resulted in negative margins. The wound bed after this excision was now approximately 10 cm at the greatest diameter (Fig. 3b, c). The patient subsequently underwent a left gluteal fasciocutaneous V–Y advancement flap (Fig. 3d) and was discharged after an uneventful hospital course. Unfortunately, the patient was evaluated at a local emergency department the following week with increased perianal pain and flap erythema. He was febrile to 101.2 °F, had leukocytosis to 20.2 K/μL, with pelvic CT revealing a 4 × 5 cm abscess under his flap, which eventually broke down. He was treated with intravenous antibiotics and underwent a diverting colostomy. Though split thickness skin graft was considered, he was found to have several sinus tracts within the wound bed, which did not communicate with the anal canal (Fig. 3e). The patient was able to go home with a course of oral antibiotics and local wound care involving wet-to-dry dressing changes. * Cindy Kin cindykin@stanford.edu

TB or Not TB: Crohn’s Disease, Peritoneal Tuberculosis, or Both?

A 25-year-old woman with a history of Crohn’s disease (CD) was evaluated in the Emergency Department for fevers, a persistent cough for several months, and abdominal cramps. Chest radiography and laboratory testing confirmed active tuberculosis (TB). She had been diagnosed with CD by colonoscopy and biopsy 3 years prior, due to complaints of abdominal pain, cramping, and diarrhea combined with a family history of CD. Her gastroenterologist treated her with adalimumab following screening for TB, which improved her gastrointestinal symptoms. One year later, disease progression, evidenced by extensive rectosigmoid ulcerations, prompted a switch to infliximab infusions. Due to prior travel to two countries with endemic TB 4 years previously, she was suspected to have latent TB that had been reactivated by anti-tumor necrosis factor (TNF) alpha therapy. Given such a diagnostic possibility, antiTNF-α therapy was discontinued and she was started on the anti-TB regimen consisting of rifampin, isoniazid, pyrazinamide, and ethambutol (RIPE). Three months after starting RIPE therapy, her respiratory symptoms of persistent cough and fevers resolved, but her diarrhea, abdominal pain, nausea, and vomiting returned. Because of ongoing failure to thrive and the possibility of relapsing CD or tuberculous enteritis, she was hospitalized; colonoscopy revealed an ileocecal stricture and moderate inflammation of the sigmoid and ascending colon (Fig. 1a). She was treated with intravenous methylprednisolone. Magnetic resonance enterography and computed tomographic enterography demonstrated irregular bowel wall thickening, mucosal hyper-enhancement, and phlegmonous inflammation in the right lower quadrant, compatible with severe CD (Fig. 2a, b). The imaging also showed severe peritoneal thickening and omental nodularities with hyper-enhancement concerning for tuberculosis peritonitis (Fig. 2c). The scans also showed that she had miliary tuberculosis of the lungs despite the resolution of her respiratory symptoms (Fig. 2d). The infectious disease specialists recommended continuing her RIPE therapy for a prolonged period while continuing steroid therapy for the Crohn’srelated ileocecal stricture. Microscopic examination of the ileocecal stricture biopsies showed severe chronic active enterocolitis although stains were negative for acid-fast bacteria. Continued steroid therapy as an outpatient was unsuccessful, as she had ongoing abdominal symptoms and significant weight loss of 30 lbs. Nutritional support with total parenteral nutrition was initiated and maintained for several weeks prior to surgical intervention. Intraoperatively during ileocolic resection, she was found to have dense seeding of the peritoneal cavity and visceral surfaces by TB (Fig. 3). Other findings included a chronic perforation of the terminal ileum with a chronic abscess cavity in the mesentery and fistulous connections to several other loops of small bowel and transverse colon. Final surgical pathology demonstrated florid granulomatous inflammation (necrotizing and non-necrotizing) with rare acid-fast bacilli (Fig. 4). Following surgery, the patient remained hospitalized and had an unremarkable recovery. She is continuing treatment for TB, but her steroids were discontinued given that she was unresponsive to therapy. Several months after her ileocolic resection, she has gained weight and continues to recover well with resolution of abdominal symptoms. There is still an ongoing discussion among her providers as to the future management of her diagnosis of CD and what medication to start or to resume should her symptoms return. * Miquell Miller miquellm@stanford.edu

Infectious rash after riding elephants

-old American male presented with a 2-week history of a persistent itchy rash on his A 17-year bilateral legs after riding elephants in Laos. He reported direct skin contact with the animal’s hide. Other family members who rode elephants also developed a similar rash. The physical examination revealed nonfluctuant pustules and erythematous papules with collarettes of scale on the medial and anterior aspects of his legs (Fig 1). Cultures from a pustule grew dry and crusty yellowish colonies on blood agar (Fig 2). Gram staining revealed filamentous branching, Gram-positive rods with clusters of coccoid forms (Fig 3).

Falsely low plasma human chorionic gonadotropin (hCG) concentrations: Corrective actions implemented.

Dear Editor: The human chorionic gonadotropin (hCG) is an important diagnostic and prognosticmarker in pregnancy andneoplastic disease [1–3]. An intact hCG is a dimer of an α and β subunit, and assays detect the intact hCG and free subunits either separately or together [1–3]. Virtually all commercially available hCG assays are two-site immunoassays based on the sandwich principle, where 1 antibody coupled to a solid phase captures the hCG from the sample, and another antibody labeled with an enzyme, dye, fluorophor or chemiluminescent agent is used to measure the concentration of bound hCG [1,4]. The two-site sandwich assays are susceptible to interference by heterophile antibodies and high concentrations of free subunits, which may produce false results and have harmful effects on the patient [1,4]. Gestational trophoblastic disease and germ cell tumors can elevate an intact hCG and free hCGβ [1–3]. The elevation of free hCGβ and/or hCGβ core fragments is also associated with non-trophoblastic malignancies and other carcinomas [1–3]. There are several different commercially available immunoassays used for measuring hCG in serum and urine specimens. These assays have antibodies that bind to varying epitopes of hCG, resulting in the detection of various combinations of hCG-related molecules such as intact and free subunits. HCG assays are FDA-approved solely for the diagnosis andmonitoring of early pregnancy, but not for ectopic and molar pregnancies, gestational trophoblastic diseases and germ cell tumors. These factors complicate the utility of hCG as a marker for trophoblastic and non-trophoblastic diseases. We recently encountered a case of falsely low hCG test results that were due to a high free hCGβ concentration.We report herein our strategies forworking up the discrepant hCG test results and suggestways of preventing recurrences. The patient is a 22-y-old female with no prior medical history, who presented with a positive home pregnancy test with reportedly no prior sexual activity for seven to eight months. Her serum hCG was 140 mIU/ml, 151 mIU/ml, 279 mIU/ml, 1015 mIU/ml and 5343 mIU/ml between December 2015 and March 2016 (performed at LabCorp on Roche CobasTM platform). She was referred to the gynecologic oncology clinic at Stanford Healthcare for further workup in February of 2016. Her plasma hCG concentrations were either 1 mIU/ml or b1 mIU/ml from February 2016 to March 2016 performed on a Siemens Dimension RxLTM intact hCG (reference value: b5 mIU/ml). During this time, her liver MRI showed numerous suspicious densities and a pelvic MRI indicated a bowel thickening. The diagnosis of the colon and liver biopsies revealed a poorly differentiated adenocarcinoma of uncertain primary site. There was no morphologic or immunohistochemical support for either a germ cell tumor or gestational trophoblastic disease. She had progressive liver failure and elected for palliative care due to the extent of her metastatic disease.