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A biopsy of lymphoid tissue is currently required to diagnose Kaposi sarcoma-associated herpesvirus (KSHV)-associated multicentric Castleman disease (KSHV-MCD). Patients showing clinical manifestations of KSHV-MCD but no pathological changes of KSHV-MCD are diagnosed as KSHV inflammatory cytokine syndrome. However, a lymph node biopsy is not always feasible to make the distinction. A pathognomonic feature of lymph nodes in KSHV-MCD is the expansion of KSHV-infected, lambda-restricted but polyclonal plasmablasts. To investigate whether these cells also reside in extra-nodal sites, effusion from 11 patients with KSHV-MCD and 19 with KSHV inflammatory cytokine syndrome was analysed by multiparametric flow cytometry. A distinct, lambda-restricted plasmablastic population (LRP) with highly consistent immunophenotype was detected in effusions in 8/11 patients with KSHV-MCD. The same population was also observed in 7/19 patients with KSHV inflammatory cytokine syndrome. The detection of LRP stratified KSHV inflammatory cytokine syndrome into two clinically distinct subgroups; those with detectable LRP closely resembled KSHV-MCD, showing similar KSHV viral load, comparable severity of thrombocytopenia and hypoalbuminaemia, and similar incidences of hepatosplenomegaly. Collectively, the detection of LRP by flow cytometry can serve as a valuable tool in diagnosing KSHV-MCD. KSHV inflammatory cytokine syndrome with LRP in effusions may represent a liquid-form of KSHV-MCD.

Figure S3

Supplementary Tables show the findings for published studies describing the incidence of epithelial cytologic atypia (Table S1), ductal endoscopic abnormalities (Table S2), and MRI findings (Table S3) in women at high risk for breast cancer.

Purpose: Metastasis heterogeneity presents a significant obstacle to the development of targeted cancer therapeutics. In this study, we sought to establish from a large series of human melanoma metastases whether there exists a determined pattern in tumor cellular heterogeneity that may guide the development of future targeted immunotherapies.Experimental Design: From a cohort of 1,514 patients with metastatic melanoma, biopsies were procured over a 17-year period from 3,086 metastatic tumors involving various anatomic sites. To allow specific tumor cell profiling, we used established immunohistochemical methods to perform semiquantitative assessment for a panel of prototypic melanocyte differentiation antigens (MDA), including gp100, MART-1, and tyrosinase. To gain insight into the endogenous host immune response against these tumors, we further characterized tumor cell expression of MHC I and MHC II and, also, the concomitant CD4+ and CD8+ T-cell infiltrate.Results: Tumor cell profiling for MDA expression demonstrated an anatomic site-specific pattern of antigen expression that was highest in brain, intermediate in soft tissues/lymph nodes, and lowest in visceral metastases. Hierarchical clustering analysis supported that melanoma metastases have a phylogenetically determined, rather than a stochastic, pattern of antigen expression that varies by anatomic site. Furthermore, tyrosinase expression was more frequently lost in metastatic sites outside of the brain and was uniquely correlated with both endogenous CD8+ and CD4+ T-cell infiltrates.Conclusion: Site-specific antigen heterogeneity represents a novel attribute for human melanoma metastases that should be considered in future therapy development and when assessing the responsiveness to antigen-specific immunotherapies. Clin Cancer Res; 20(10); 2607–16. ©2014 AACR.

PDF file - 51K, Pairwise comparison of antigen expression by individual anatomic site cluster. Shown are p value results of Cochran-Armitage test for trend. p

PDF file - 47K, Individual dendrogram distance matrices for the melanoma differentiation antigens. Dendrogram distant matrices calculated from DendroUPGMA.

PDF file - 57K, Pairwise comparison of antigen expression by individual anatomic site. Shown are p value results of Cochran-Armitage test for trend. p

PDF file - 73K, MDA and MHC II expression demonstrates a site cluster-specific pattern. (A) The distribution of antigen expression for each anatomic site cluster. The p-values indicate the probability that no variability exists across the site clusters as determined by the Kruskal-Wallis test.

PDF file - 65K, TYR expression varies with the degree of CD8+ and CD4+ T cell infiltrate. (A) The distribution of antigen expression for given CD8 (blue) infiltrate score (0-3+, column). (B) The distribution of antigen expression for given CD4 (green) infiltrate score (0-3+, column).

PDF file - 50K, Dendrogram distance matrix for all melanoma differentiation antigens. Dendrogram distant matrix calculated from DendroUPGMA.

Context Epidemiologic studies have demonstrated that overweight/obese girls (OW/OB) undergo thelarche and menarche earlier than normal weight girls (NW). There have been no longitudinal studies to specifically investigate how body weight/fat affects both clinical and biochemical pubertal markers in girls. Objective To investigate the effect of total body fat on reproductive hormones and on the maturation of estrogen-sensitive tissues during puberty in girls. Methods Ninety girls (36 OW/OB, 54 NW), aged 8.2 to 14.7 years, completed 2.8 ± 1.7 study visits over 4 years. Visits included dual-energy x-ray absorptiometry to calculate total body fat (TBF), Tanner staging, breast ultrasound for morphological staging (BMORPH; A-E), pelvic ultrasound, hormone tests, and assessment of menarchal status. The effect of TBF on pubertal markers was determined using a mixed, multistate, or Cox proportional hazards model, controlling for baseline BMORPH. Results NW were older than OW/OB (11.3 vs 10.2 years, P Conclusion In late puberty, girls with higher TBF demonstrate differences in standard hormonal and clinical markers of puberty. Investigation of the underlying causes and clinical consequences of these differences in girls with higher TBF deserves further study.

Breast ductal cytologic atypia is an important risk factor for sporadic breast cancer. Characterization of the associated normal breast tissue is needed to develop additional methods of risk assessment and new targets for breast cancer prevention. We conducted a prospective clinical trial evaluating women at normal-risk or at high-risk for sporadic breast cancer. Breast ductal cells were collected and studied cytologically and by gene expression profiling, and breast ductal architectural changes were studied by breast ductal endoscopy (BDE) and breast MRI. One hundred and forty subjects were studied, 70 at high risk (RR, 2.0–4.6) and 70 at normal risk. Cytologic atypia was present in 22.9% of high-risk and 25.7% of normal-risk subjects. Ductal endoscopy was performed in 89 subjects and revealed benign intraductal abnormalities, primarily intraductal fibrous webbing suggesting chronic inflammation, in 40.4% of high-risk and 5.4% of normal-risk subjects, respectively (P2 = 0.0002). Two high-risk subjects with atypia and no normal-risk subjects with atypia developed invasive breast cancer. Gene expression profiling of ductal cells showed comparable gene expression profiles without enriched expression of previously defined oncogenic signatures in subjects with cellular atypia compared with those without atypia, and in high-risk subjects compared with normal-risk subjects (FDR > 0.5). Cytologic ductal atypia in normal-risk subjects does not appear to be of clinical significance. Atypia in women at high risk may be associated with benign and malignant breast ductal abnormalities; these characteristics of high-risk ductal cells may not be reflected in gene expression profiles.

OBJECTIVE To assess the cytokine and viral profiles of effusions and peripheral blood among patients diagnosed with HIV and Kaposi sarcoma herpesvirus [KSHV, also known as human herpesvirus 8 (HHV-8)]-associated conditions. DESIGN Retrospective comparative study evaluating clinicopathologic findings in patients with HIV and KSHV-associated conditions presenting with an effusion between 2010 and 2018. METHODS Paired peripheral blood and effusion samples collected at the time of pathological diagnosis of KSHV-associated conditions [Kaposi sarcoma, KSHV-associated multicentric Castleman disease (KSHV-MCD), primary effusion lymphoma (PEL), or KSHV-associated inflammatory cytokine syndrome (KICS)] were evaluated for disease-specific and compartment-specific (effusion vs. blood) characteristics. We assessed 12 cytokines, KSHV viral DNA (KSHV-VL), and Epstein--Barr virus (EBV) viral DNA (EBV-VL). RESULTS Nine patients had PEL, five patients had KSHV-MCD, and eight patients met criteria for KICS; all but one patient had concurrent Kaposi sarcoma in addition to these conditions. PEL effusions had substantially higher levels of IL-13 (median 16.9 pg/ml; interquartile range 9.7--26.9 pg/ml) compared with KSHV-MCD (median

Molecular testing for genetic alterations in thyroid neoplasms, including BRAF V600E (BRAF) mutation, are often applied to thyroid aspirates falling into the Bethesda System for Reporting Thyroid Cytopathology indeterminate categories. Current methods typically use dedicated aspirated material, without morphological determination of containing the cells of interest and may be of elevated cost. We describe our experience with BRAF mutation analysis on material obtained from Papanicolaou (PAP)-stained ThinPrepEighty-three cases collected between 2012 and 2019 with more than 100 cells were selected. An electronic record of a whole slide scan was made for each case before testing. The coverslips were removed, and DNA was extracted from material scraped from each slide using the Qiagen QIAamp DNA FFPE Tissue Kit. BRAF testing was performed using a highly sensitive mutation detection assay, either COLD-PCR, castPCR, or droplet digital PCR.Fourteen out of 83 cases had a BRAF mutation. Of these, 8 were classified as atypia of undetermined significance or suspicious for malignancy in which follow-up showed conventional papillary thyroid carcinoma in 5 out of 6 cases. The specificity and positive predictive value were 97% and 91%, respectively.BRAF mutation analysis can be performed on material obtained from routine clinical PAP-stained TP slides. As a first step, this unconventional effective approach may reduce costs related to the molecular evaluation of thyroid nodule aspirates and provides the opportunity for cytomorphological confirmation that the cells of interest are present in material submitted for BRAF mutation analysis.

Background Girls who are overweight/obese (OB) develop breast tissue but do not undergo menarche (the first menstrual period) significantly earlier than girls of normal weight (NW). It has been proposed that estrogen synthesized by adipose tissue may be contributory, yet OB do not have higher serum estrogen levels than NW matched on breast stage. We hypothesized that estrogen synthesized locally, in mammary fat, may contribute to breast development. This hypothesis would predict that breast development would be more advanced than other estrogen-sensitive tissues as a function of obesity and body fat. Methods 80 pre-menarchal girls (26 OB, 54 NW), aged 8.2–14.7 yrs, underwent dual-energy x-ray absorptiometry to calculate percent body fat (%BF), Tanner staging of the breast, breast ultrasound for morphological staging, trans-abdominal pelvic ultrasound, hand x-ray (bone age), a blood test for reproductive hormones, and urine collection to determine the vaginal maturation index (VMI), an index of estrogen exposure in urogenital epithelial cells. Results When controlling for breast morphological stage determined by ultrasound, %BF was not associated with serum estrogen or gonadotropin (LH and FSH) levels or on indices of systemic estrogen action (uterine volume, endometrial thickness, bone age advancement, and VMI). Tanner breast stage did not correlate with breast morphological stage and led to misclassification of chest fatty tissue as breast tissue in some OB. Conclusions These studies do not support the hypothesis that estrogen derived from total body fat or local (mammary) fat contributes to breast development in overweight/obese girls.

Background GATA2 deficiency is a genetic disorder of hematopoiesis, lymphatics, and immunity caused by autosomal dominant or sporadic mutations in GATA2. The disease has a broad phenotype encompassing immunodeficiency, myelodysplasia, leukemia, and vascular or lymphatic dysfunction as well as prominent pulmonary manifestations. Research Question What are the pulmonary manifestations of GATA2 deficiency? Study Design and Methods A retrospective review was conducted of clinical medical records, diagnostic imaging, pulmonary pathologic specimens, and tests of pulmonary function. Results Of 124 patients (95 probands and 29 ascertained), the lung was affected in 56%. In addition to chronic infections, pulmonary alveolar proteinosis (11 probands) and pulmonary arterial hypertension (nine probands) were present. Thoracic CT imaging found small nodules in 54% (54 probands and 12 relatives), reticular infiltrates in 40% (45 probands and four relatives), paraseptal emphysema in 25% (30 probands and one relative), ground-glass opacities in 35% (41 probands and two relatives), consolidation in 21% (23 probands and two relatives), and a typical crazy-paving pattern in 7% (eight probands and no relatives). Nontuberculous mycobacteria were the most frequent organisms associated with chronic infection. Allogeneic hematopoietic stem cell transplantation successfully reversed myelodysplasia and immune deficiency and also improved pulmonary hypertension and pulmonary alveolar proteinosis in most patients. Interpretation GATA2 deficiency has prominent pulmonary manifestations. These clinical observations confirm the essential role of hematopoietic cells in many aspects of pulmonary function, including infections, alveolar proteinosis, and pulmonary hypertension, many of which precede the formal diagnosis, and many of which respond to stem cell transplantation.

PURPOSE: To evaluate an integrated liver biopsy platform that combined CT image fusion, electromagnetic (EM) tracking and optical molecular imaging (OMI) of indocyanine green (ICG) to target hepatocellular carcinoma (HCC) lesions and a point-of-care (POC) OMI to assess biopsy cores, all based on tumor retention of ICG compared to normal liver, in phantom and animal model. MATERIAL: A custom CT image fusion and EM-tracked guidance platform was modified to integrate the measurement of ICG fluorescence intensity signals in targeted liver tissue with an OMI stylet or a POC-OMI system. Accuracy was evaluated in phantom and a woodchuck with HCC, 1 day after administration of ICG. Fresh biopsy cores and paraffin embedded formalin fixed liver tissue blocks were evaluated with the OMI stylet or POC system to identify ICG fluorescence signal and ICG peak intensity. RESULTS: The mean distance between the initial guided needle delivery location and the peak ICG signal was 5.0 ± 4.7mm in the phantom. There was complete agreement between the reviewers of the POC acquired ICG images, cytology, and histopathology in differentiating HCC positive from HCC negative biopsy cores. The peak ICG fluorescence intensity signal in the ex vivo liver blocks was 39 ± 12 and 281 ± 150 for HCC negative and HCC positive, respectively. CONCLUSION: Biopsy guidance with fused CT imaging, EM tracking, and ICG tracking with an OMI stylet to detect HCC is feasible. Immediate assessment of ICG uptake in biopsy cores with the POC-OMI system is feasible and correlates with presence of HCC in the tissue.

Most primary thyroid tumours are of epithelial origin. Primary thyroid mesenchymal tumours are rare but are being increasingly detected. A vast majority of thyroid mesenchymal tumours occur between the fourth and seventh decades of life, presenting as progressively enlarging thyroid nodules that often yield non-diagnostic results or spindle cells on fine needle aspiration biopsy. Surgery is the preferred mode of treatment, with adjuvant chemoradiotherapy used for malignant thyroid mesenchymal tumours. Benign thyroid mesenchymal tumours have excellent prognosis, whereas the outcome of malignant thyroid mesenchymal tumours is variable. Each thyroid mesenchymal tumour is characterised by its unique histopathology and immunohistochemistry. Because of the rarity and aggressive nature of malignant thyroid mesenchymal tumours, a multidisciplinary team-based approach should ideally be used in the management of these tumours. Comprehensive guidelines on the management of thyroid mesenchymal tumours are currently lacking. In this Review, we provide a detailed description of thyroid mesenchymal tumours, their clinical characteristics and tumour behaviour, and provide recommendations for the optimal management of these tumours.

Background Adenoid cystic carcinoma (AdCC) is an uncommon malignancy of the salivary gland characterized by slow growth, increased risk of recurrence and poor prognosis. The annual incidence in the United States is approximately 1200 cases per year and rarely involves the body cavities. Case presentation We present a case of a 48-year-old male diagnosed with AdCC of the left submandibular gland. He received his last chemotherapy in 2006 and presented with pleural metastasis. After undergoing pleurectomy and decortication procedure, pericardial fluid and biopsies from the chest wall, sixth rib, diaphragm, pleural cavity and pericardium were sent for pathologic evaluation. A diagnosis of metastatic adenoid cystic carcinoma was confirmed, including in the pericardium, pericardial fluid and diaphragm. Conclusion AdCC of the submandibular gland is a malignant tumor with a high mortality rate. It is very rare for AdCC to metastasize to the pericardium and diaphragm. Metastasis to uncommon sites such as seen in our case with metastases to the pericardium and diaphragm shows the aggressive and unpredictable nature of this tumor, requiring close follow up, and indicating the need for molecular profile analysis and biomarker-stratified clinical trials.

Key Points Question Is alkaptonuria associated with thyroid dysfunction? Findings In this cohort study, 125 adults with alkaptonuria followed up for a median of 93 months were found to have a 16.0% prevalence of primary hypothyroidism. This prevalence was significantly higher than the 3.7% prevalence in the general population. Meaning Results of this study suggest that screening for hypothyroidism should be considered in patients with alkaptonuria., Importance Alkaptonuria is an autosomal recessive disorder caused by pathogenic variants in the HGD gene. Deficiency of the HGD enzyme leads to tissue deposition of homogentisic acid (HGA), causing severe osteoarthropathies and cardiac valve degeneration. Although HGD is vital for the catabolism of tyrosine, which provides the basis for thyroid hormone synthesis, the prevalence of thyroid dysfunction in alkaptonuria is unknown. Objective To assess thyroid structure and function in patients with alkaptonuria. Design, Setting, and Participants A single-center cohort study was conducted in a tertiary referral center including patients with alkaptonuria followed up for a median of 93 (interquartile range, 48-150) months between February 1, 2000, and December 31, 2018. The alkaptonuria diagnosis was based on clinical presentation and elevated urine HGA levels. A total of 130 patients were considered for participation. Main Outcomes and Measures Prevalence of thyroid dysfunction in adults with alkaptonuria compared with the general population. Thyrotropin and free thyroxine levels were measured by immunoassay and repeated in each patient a median of 3 (interquartile range, 2-22) times. Neck ultrasonographic scans were analyzed in a subset of participants. Logistic regression was used to test the association of thyroid dysfunction with age, sex, thyroid peroxidase (TPO) antibodies, serum tyrosine levels, and urine HGA levels. Results Of the 130 patients, 5 were excluded owing to thyroidectomy as the cause of hypothyroidism. The study cohort consisted of 125 patients; the median age was 45 (interquartile range, 35-51) years. Most of the patients were men (72 [57.6%]). The prevalence of primary hyperthyroidism was 0.8% (1 of 125 patients), similar to 0.5% observed in the general population (difference, 0.003; 95% CI, −0.001 to 0.04; P = .88). The prevalence of primary hypothyroidism was 16.0% (20 of 125 patients), which is significantly higher than 3.7% reported in the general population (difference, 0.12; 95% CI, 0.10-0.24; P, This cohort study examines thyroid function and structure in patients with alkaptonuria.

Introduction BRCA1-associated protein-1 (BAP1), a nuclear deubiquitinase thought to be involved in DNA double-strand break repair, is frequently mutated in mesothelioma. Because poly(adenosine diphosphate-ribose) polymerase inhibitors (PARPIs) induce synthetic lethality in BRCA1/2 mutant cancers, we evaluated whether BAP1 inactivating mutations confer sensitivity to PARPIs in mesothelioma and if combination therapy with temozolomide (TMZ) would be beneficial. Methods A total of 10 patient-derived mesothelioma cell lines were generated and characterized for BAP1 mutation status, protein expression, nuclear localization, and sensitivity to the PARPIs, olaparib, and talazoparib, alone or in combination with TMZ. BAP1 deubiquitinase (DUB) activity was evaluated by ubiquitin with 7-amido-4-methylcoumarin assay. BAP1 knockout mesothelioma cell lines were generated by CRISPR-Cas9. Because Schlafen 11 (SLFN11) and O6-methylguanine-DNA methyltransferase also drive response to TMZ and PARPIs, we tested their expression and relationship with drug response. Results BAP1 mutations or copy-number alterations, or both were present in all 10 cell lines. Nonetheless, four cell lines exhibited intact DUB activity and two had nuclear BAP1 localization. Half maximal-inhibitory concentrations of olaparib and talazoparib ranged from 4.8 μM to greater than 50 μM and 0.039 μM to greater than 5 μM, respectively, classifying them into sensitive (two) or resistant (seven) cells, independent of their BAP1 status. Cell lines with BAP1 knockout resulted in the loss of BAP1 DUB activity but did not increase sensitivity to talazoparib. Response to PARPI tended to be associated with high SLFN11 expression, and combination with temozolomide increased sensitivity of cells with low or no MGMT expression. Conclusions BAP1 status does not determine sensitivity to PARPIs in patient-derived mesothelioma cell lines. Combination of PARPI with TMZ may be beneficial for patients whose tumors have high SLFN11 and low or no MGMT expression.

Background Accurate disease detection is integral to risk stratification in B-cell acute lymphoblastic leukemia (ALL). The gold standard used to evaluate response in the United States includes morphologic evaluation and minimal residual disease (MRD) testing of aspirated bone marrow (BM) by flow cytometry (FC). This MRD assessment is usually made on a single aspirate sample that is subject to variability in collection techniques and sampling error. Additionally, central nervous system (CNS) assessments for ALL include evaluations of cytopathology and cell counts, which can miss subclinical involvement. Procedure We retrospectively compared BM biopsy, aspirate, and FC samples obtained from children and young adults with relapsed/refractory ALL to identify the frequency and degree of disease discrepancies in this population. We also compared CNS FC and cytopathology techniques. Results Sixty of 410 (14.6%) BM samples had discrepant results, 41 (10%) of which were clinically relevant as they resulted in a change in the assignment of marrow status. Discrepant BM results were found in 28 of 89 (31.5%) patients evaluated. Additionally, cerebrospinal fluid (CSF) FC identified disease in 9.7% of cases where cytopathology was negative. Conclusions These results support further investigation of the role of concurrent BM biopsy, with aspirate and FC evaluations, and the addition of FC to CSF evaluations, to fully assess disease status and response, particularly in patients with relapsed/refractory ALL. Prospective studies incorporating more comprehensive analysis to evaluate the impact on clinical outcomes are warranted.

Background: Studies comparing the timing and pace of puberty in overweight/obese girls (OW/OB) vs normal weight girls (NW) have produced conflicting results; some suggest earlier activation of the central components of the reproductive axis in OB while others are more consistent with a peripheral source of estrogen (e.g. adipose tissue) driving puberty in OB. Importantly, there have been no longitudinal assessments of both clinical and biochemical pubertal markers in OB vs. NW. Methods: 90 healthy pre-menarchal girls (26 OW/OB, 54 NW) from the community, aged 8.2-14.7 years, completed 2.8 ± 1.7 (mean, SD) study visits over the course of 4 years. Visits included dual-energy x-ray absorptiometry to calculate percent total body fat (TBF), Tanner staging, breast ultrasound for morphological staging (BMORPH; stages A-E), pelvic ultrasound, hand x-ray (bone age, BA), blood tests for reproductive hormones, and urine collection to determine a vaginal maturation index (VMI), an index of estrogen exposure in urogenital epithelial cells. Menarchal status was determined at each visit and via follow-up questionnaires. The effect of TBF on hormones and markers of estrogen action, the pace of breast maturation, and age at menarche were determined using a mixed, multi-state, or Cox proportional hazards model, respectively. Mixed and Cox models controlled for BMORPH at visit 1 (V1) and race. Results: NW girls were older than OW/OB (11.3 vs. 10.2 yrs, p

Background: Mesenchymal tumors represent around 1% of all thyroid neoplasms. Schwannoma is one such tumor that arises from the peripheral nerve sheath within the thyroid gland. Case: A 40-year-old lady was referred to our center for evaluation of a thyroid mass that was initially noticed 2 years prior and had been rapidly enlarging over the past several months. The patient experienced occasional dysphagia, but no shortness of breath nor change in voice. There was no family history of thyroid cancer or exposure to head and neck radiation. On physical exam, a firm, non-tender, 3 cm nodule was palpated in the right thyroid lobe. There was no cervical lymphadenopathy. Pemberton sign was negative. There was no evidence of café-au-lait spots or cutaneous neurofibromas on skin exam. The thyroid function tests were normal (NL), with serum TSH of 2.06 µIU/mL (NL: 0.27 - 4.2) and free thyroxine of 1.2 ng/dL (NL: 0.9 - 1.7), anti-TPO antibody was elevated (483 IU/mL; NL: 0 - 34.9), serum calcitonin was undetectable (

Overweight/obese girls (OB) undergo thelarche but not menarche significantly earlier than normal weight girls (NW). It has been proposed that estrogen synthesized by adipose tissue may be contributory, yet OB do not have higher serum estrogen levels than NW matched on breast stage. We hypothesized that in OB, adipose tissue overlying the breast bud may be a ready source of estrogen that is sufficient to initiate thelarche because the primitive breast bud is intimately associated with the mammary fat pad during development (Anbazhagan Am J Anat ’91, Neville J Mammary Gland Biol Neoplasia ’98). Estrogen derived from mammary fat could act locally to induce thelarche but would not be detected in the circulation and therefore would not act systemically to induce uterine maturation/menses. To address this hypothesis, we measured reproductive hormones and imaged the breast, uterus, and ovaries in 80 pubertal, pre-menarchal girls (26 OB, 54 NW) aged 8.2-14.7 yrs. We used total percent body fat (%BF), determined by DXA, as a proxy for mammary fat, taking advantage of the positive correlation between these two measures (Novotny Am J Hum Biol ’11, Schautz Eur J Clin Nutr ’11, Zhu Eur Radiol ’16). We predicted that if estrogen derived from mammary fat contributes to breast maturation, then girls with higher %BF (and thus more mammary fat) would demonstrate immaturity of other estrogen-sensitive tissues (e.g. uterus, endometrial lining, vaginal epithelium) compared with girls of the same breast morphological stage (stages A-E based on Bruni et al. Adolesc Pediatr Gynecol ‘90) with lower %BF. OB were younger than NW (10.1 ± 1.1 vs. 11.3 ± 1.3 yrs, p

Background: Alkaptonuria (AKU) is a rare autosomal recessive disorder caused by pathogenic variants in the gene encoding homogentisate-1,2-deoxygenase (HGD). Deficiency of HGD leads to tissue deposition of homogentisic acid (HGA), causing severe osteoarthropathies, aortic and mitral valve degeneration and kidney stones. HGD is vital for the catabolism of tyrosine, which provides the backbone of iodination into thyroxine (T4) within the thyroid gland. The goal of this study was to assess thyroid function (TFT) in patients (pts) with AKU. Methods: We performed a cohort study including pts with AKU followed at our center between 2000 and 2018. All pts had thyroid stimulation hormone (TSH), freeT4 (fT4), anti-thyroid peroxidase antibodies (anti-TPO-abs) measured repeatedly by immunoassay. In addition, a subset of patients underwent a thyroid ultrasound (US). Hypothyroidism (HYPO) was defined as TSH above the reference range (0.27-4.2 mIU/ml) with fT4 below the reference range (0.9-1.7 ng/dl) on two repeated tests or normalization of TSH on levothyroxine (LT4) replacement therapy. Subclinical HYPO (S-HYPO) was defined as TSH above the reference range with normal fT4 on two repeated tests. Results:The study cohort consisted of 130 pts with AKU, majority men (55.4%), aged 59.1±15 years and followed for 36 (range 1-63) months, during which median 10 (range 2-35) TFTs were performed. Twenty-four pts were diagnosed with HYPO, but 3 pts were excluded due to post-thyroidectomy etiology of HYPO, giving a prevalence of HYPO of 16.5% (21/127) - significantly higher than reported 5% of the general population (p=0.006). S-HYPO was diagnosed in 4.7% (6/127) and overt HYPO in 11.8% (15/127) pts, who were treated with a median LT4 dose of 100 mcg daily (1.4 mcg/kg). The median thyroid volume in HYPO pts was 7 cc (range 3.9-13). The prevalence of elevated anti-TPO-abs in the whole cohort was 14% (18/127), while its prevalence in HYPO group was 43% (9/21) - significantly lower than reported 89.8% for HYPO pts in the general population (p

Background Thyroid ultrasound (US) and US-guided fine needle aspiration biopsy (FNAB) have been widely used to stratify the risk of malignancy in thyroid nodules (TN). Cytology material characterized as category III and IV by the Bethesda System for Reporting Thyroid Cytopathology is associated with thyroid cancer (TC) risk ranging between 5-30%. The use of molecular markers may help defining the management strategy in these indeterminate nodules. The goal of this study was to create a risk stratification system based on US features and the results of molecular profiling of cytologically indeterminate TN and to assess its diagnostic accuracy. Methods We performed a cohort study including patients with TN who underwent thyroid US and FNAB revealing indeterminate cytology (Bethesda III, IV). Final pathology diagnosis was available for all patients. DNA and RNA were extracted from pathology material and subjected to Ion Torrent™ Oncomine™ Comprehensive Assay v3 (OCAv3) next-generation sequencing, analyzing single nucleotide variants (SNV), small insertions and deletions (INDEL), copy number variants (CNV) and gene fusions (GF) from 161 cancer driver genes. Each genetic alteration was annotated (X) based on its strength of association with TC per TCGA and COSMIC database. US features of TN were similarly categorized (Xus) by two independent clinicians, blinded to pathology report. The total risk score (TRS) was estimated based on the following formula TRS= (XSNV/INDEL)*n + XGF + XCNV + XUS , where X is the annotated score and n is the number of SNVs/INDELs. ROC curve was performed to assess the diagnostic accuracy of US alone, OCAv3 alone and TRS. Results We analyzed 84 TN in 50 patients (39 females and 11 males), age 47.2 ± 14.8 years. Thirty-four patients (68%) were diagnosed with TC: 21 papillary (PTC), 6 follicular variant of PTC (FVPTC), 1 noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP), 2 Hürthle cell (HTC), 1 poorly differentiated carcinoma (PD) and 3 medullary TC (MTC). The molecular signature revealed the BRAF V600E mutation as the most common in PTC, while KRAS and NRAS mutations were associated with FVPTC. A NIFTP showed a PAX8-PPARG fusion. HTC had pathogenic variants of SLX4, ATM and NRAS genes, PD was associated with HRAS mutation, and MTC with RET pathogenic variants. Benign lesions were characterized by either no mutations or the presence of GNAS, HRAS, NRAS and ESR1 variants. The diagnostic accuracy of US alone was 68% (sensitivity 41.2%, specificity 93.8%), while for OCAv3 alone it was 79% (sensitivity 65%, specificity 93.8%). TRS was associated with the highest diagnostic accuracy of 83.5% (sensitivity 79.4%, specificity 88%). Conclusions Combining risk stratification based on thyroid US and molecular signature improves diagnostic accuracy of TC in patients with indeterminate TN. Our pilot study requires validation in independent larger cohorts.

Primary effusion lymphoma (PEL) is an aggressive HIV-associated lymphoma with a relatively poor prognosis in the era of effective HIV therapy. Kaposi sarcoma herpesvirus (KSHV) is the etiologic agent, and ∼80% of tumors are coinfected with Epstein-Barr virus (EBV). A better understanding of how KSHV-related immune dysregulation contributes to the natural history of PEL will improve outcomes. Twenty patients with PEL diagnosed between 2000 and 2013, including 19 treated with modified infusional etoposide, vincristine, and doxorubicin with cyclophosphamide and prednisone (EPOCH), were identified. We compared their clinical, virologic, and immunologic features vs 20 patients with HIV-associated diffuse large B-cell lymphoma and 19 patients with symptomatic interleukin (IL)-6 related KSHV-associated multicentric Castleman disease. Survival analyses of treated patients with PEL were then performed to identify prognostic factors and cancer-specific mortality. Compared with HIV-associated diffuse large B-cell lymphoma, PEL was associated with significant hypoalbuminemia (P < .0027), thrombocytopenia (P = .0045), and elevated IL-10 levels (P < .0001). There were no significant differences in these parameters between PEL and KSHV-associated multicentric Castleman disease. Median overall survival in treated patients with PEL was 22 months, with a plateau in survival noted after 2 years. Three-year cancer-specific survival was 47%. EBV-positive tumor status was associated with improved survival (hazard ratio, 0.27; P = .038), and elevated IL-6 level was associated with inferior survival (hazard ratio, 6.1; P = .024). Our analysis shows that IL-6 and IL-10 levels contribute to the natural history of PEL. Inflammatory cytokines and tumor EBV status are the strongest prognostic factors. Pathogenesis-directed first-line regimens are needed to improve overall survival in PEL.

The PPAR-γ agonist, pioglitazone, was associated with significant side effects and did not improve the primary outcome measure of the Juniper Asthma Quality of Life Questionnaire (AQLQ) score in severe asthmatics. We conclude that no further studies should be performed with pioglitazone for severe asthma.

Purpose: Metastasis heterogeneity presents a significant obstacle to the development of targeted cancer therapeutics. In this study, we sought to establish from a large series of human melanoma metastases whether there exists a determined pattern in tumor cellular heterogeneity that may guide the development of future targeted immunotherapies. Experimental Design: From a cohort of 1,514 patients with metastatic melanoma, biopsies were procured over a 17-year period from 3,086 metastatic tumors involving various anatomic sites. To allow specific tumor cell profiling, we used established immunohistochemical methods to perform semiquantitative assessment for a panel of prototypic melanocyte differentiation antigens (MDA), including gp100, MART-1, and tyrosinase. To gain insight into the endogenous host immune response against these tumors, we further characterized tumor cell expression of MHC I and MHC II and, also, the concomitant CD4+ and CD8+ T-cell infiltrate. Results: Tumor cell profiling for MDA expression demonstrated an anatomic site-specific pattern of antigen expression that was highest in brain, intermediate in soft tissues/lymph nodes, and lowest in visceral metastases. Hierarchical clustering analysis supported that melanoma metastases have a phylogenetically determined, rather than a stochastic, pattern of antigen expression that varies by anatomic site. Furthermore, tyrosinase expression was more frequently lost in metastatic sites outside of the brain and was uniquely correlated with both endogenous CD8+ and CD4+ T-cell infiltrates. Conclusion: Site-specific antigen heterogeneity represents a novel attribute for human melanoma metastases that should be considered in future therapy development and when assessing the responsiveness to antigen-specific immunotherapies. Clin Cancer Res; 20(10); 2607–16. ©2014 AACR.

The Papanicolaou Society of Cytopathology has developed a set of guidelines for pancreatobiliary cytology including indications for endoscopic ultrasound-guided fine-needle aspiration, terminology and nomenclature of pancreatobiliary disease, ancillary testing, and post-biopsy management. All documents are based on the expertise of the authors, a review of the literature, discussions of the draft document at several national and international meetings, and synthesis of selected online comments of the draft document. This document presents the results of these discussions regarding the use of ancillary testing in the cytologic diagnosis of biliary and pancreatic lesions. Currently, fluorescence in situ hybridization (FISH) appears to be the most clinically relevant ancillary technique for cytology of bile duct strictures. The addition of FISH analysis to routine cytologic evaluation appears to yield the highest sensitivity without loss in specificity. Loss of immunohistochemical staining for the protein product of the SMAD4 gene and positive staining for mesothelin support a diagnosis of ductal adenocarcinoma. Immunohistochemical markers for endocrine and exocrine differentiation are sufficient for a diagnosis of endocrine and acinar tumors. Nuclear staining for beta-catenin supports a diagnosis of solid-pseudopapilary neoplasm. Cyst fluid analysis for amylase and carcinoembryonic antigen aids in the preoperative classification of pancreatic cysts. Many gene mutations (KRAS, GNAS, VHL, RNF43, and CTNNB1) may be of aid in the diagnosis of cystic neoplasms. Other ancillary techniques do not appear to improve diagnostic sensitivity sufficiently to justify their increased costs.

Background: Several lines of evidence suggest a dichotomy between immune active and quiescent cancers, with the former associated with a good prognostic phenotype and better responsiveness to immunotherapy. Central to such dichotomy is the master regulator of the acute inflammatory process interferon regulatory factor (IRF)-1. However, it remains unknown whether the responsiveness of IRF-1 to cytokines is able to differentiate cancer immune phenotypes. Methods: IRF-1 activation was measured in 15 melanoma cell lines at basal level and after treatment with IFN-γ, TNF-α and a combination of both. Microarray analysis was used to compare transcriptional patterns between cell lines characterised by high or low IRF-1 activation. Results: We observed a strong positive correlation between IRF-1 activation at basal level and after IFN-γ and TNF-α treatment. Microarray demonstrated that three cell lines with low and three with high IRF-1 inducible translocation scores differed in the expression of 597 transcripts. Functional interpretation analysis showed mTOR and Wnt/β-cathenin as the top downregulated pathways in the cell lines with low inducible IRF-1 activation, suggesting that a low IRF-1 inducibility recapitulates a cancer phenotype already described in literature characterised by poor prognosis. Conclusion: Our findings support the central role of IRF-1 in influencing different tumour phenotypes.

Precision medicine promises to enhance patient treatment through the use of emerging molecular technologies, including genomics, transcriptomics, and proteomics. However, current tools in surgical pathology lack the capability to efficiently isolate specific cell populations in complex tissues/tumors, which can confound molecular results. Expression microdissection (xMD) is an immuno-based cell/subcellular isolation tool that procures targets of interest from a cytological or histological specimen. In this study, we demonstrate the accuracy and precision of xMD by rapidly isolating immunostained targets, including cytokeratin AE1/AE3, p53, and estrogen receptor (ER) positive cells and nuclei from tissue sections. Other targets procured included green fluorescent protein (GFP) expressing fibroblasts, in situ hybridization positive Epstein-Barr virus nuclei, and silver stained fungi. In order to assess the effect on molecular data, xMD was utilized to isolate specific targets from a mixed population of cells where the targets constituted only 5% of the sample. Target enrichment from this admixed cell population prior to next-generation sequencing (NGS) produced a minimum 13-fold increase in mutation allele frequency detection. These data suggest a role for xMD in a wide range of molecular pathology studies, as well as in the clinical workflow for samples where tumor cell enrichment is needed, or for those with a relative paucity of target cells.

Objective: In the past decade molecular diagnostics has changed the clinical management of lung adenocarcinoma patients. Molecular diagnostics, however, is largely dependent on the quantity and quality of the tumor DNA that is retrieved from the tissue or cytology samples. Frequently, patients are diagnosed on cytology specimens where the tumor cells are scattered within the cell block, making selecting for tumor enrichment difficult. In the past we have used laser capture microdissection (LCM) to select for pure populations of tumor cells to increase the sensitivity of molecular assays. This study explores several methods for semiautomated computer-guided LCM. Study Design: Hematoxylin and eosin- or TTF-1-immunostained slides from a pleural effusion cell block with metastatic lung adenocarcinoma were used for LCM with either AutoScan or a recently described pattern-matching algorithm, spatially invariant vector quantization (SIVQ), to define morphologic predicates (vectors) to select cells of interest. Results: We retrieved pure populations of tumor cells using both algorithm-guided LCM approaches with slight variations in cellular retrievals. Both methods were semiautomated, requiring minimum technical supervision. Conclusion: In this study we demonstrate the first semiautomated, computer-guided LCM of a cytology specimen using SIVQ and AutoScan, a first step towards the long-term goal of integrating LCM into the clinical cytology-molecular workflow.

BACKGROUND. There is an unmet clinical need for economic, minimally invasive procedures that use a limited number of cells for the molecular profiling of tumors in individual patients. Reverse-phase protein microarray (RPPM) technology has been applied successfully to the quantitative analysis of breast, ovarian, prostate, and colorectal cancers using frozen surgical specimens. METHODS. For this report, the authors investigated the novel use of RPPM technology for the analysis of both archival cytology aspirate smears and frozen fine-needle aspiration (FNA) samples. RPPMs were printed with 63 breast FNA samples that were obtained before, during, and after treatment from 21 patients who were enrolled in a Phase II trial of neoadjuvant capecitabine and docetaxel therapy for breast cancer. RESULTS. Based on an MCF7 cell line model of breast adenocarcinoma, the sensitivity of the RPPM detection method was in the femtomolar range with a coefficient of variance

Breast implant-associated anaplastic large-cell lymphoma (BIA-ALCL) is a rare form of ALK-negative ALCL, usually presenting as an accumulation of seroma fluid between the implant itself and the surrounding fibrous capsule.1 It was first described in 1997.2 From 1997 to 2012, the United States Food and Drug Administration reported a total of 60 registered instances of BIA-ALCL with a mean interval to lymphoma diagnosis of 10.9 years.3-5 It is estimated that 1 of 500,000 women receiving breast implants will develop BIA-ALCL.5, 6 Recent studies have suggested that patients can be managed conservatively, by drainage of the fluid and removal of the implant and capsule, if no invasion of the capsule or breast tissue is identified. 3, 7 However, some patients manifest invasion of the capsule, and involvement of breast, lymph nodes, or more rarely distant sites. The risk factors for progression are not fully delineated.

Background There is a strong need to define the molecular changes in normal at-risk breast epithelium to identify biomarkers and new targets for breast cancer prevention and to develop a molecular signature for risk assessment. Improved methods of breast epithelial sampling are needed to promote whole-genome molecular profiling, increase ductal epithelial cell yield, and reduce sample cell heterogeneity. Methods We developed an improved method of breast ductal sampling with ductal lavage through a 22-gauge catheter and collection of ductal samples with a microaspirator. Women at normal risk or increased risk for breast cancer were studied. Ductal epithelial samples were analyzed for cytopathologic changes, cellular yield, epithelial cell purity, quality and quantity of DNA and RNA, and use in multiple downstream molecular applications. Results We studied 50 subjects, including 40 subjects at normal risk for breast cancer and 37 subjects with non-nipple aspirate fluid-yielding ducts. This method provided multiple 1.0 mL samples of high ductal epithelial cell content (median ≥8 samples per subject of ≥5,000 cells per sample) with 80%–100% epithelial cell purity. Extraction of a single intact ductal sample (fluid and cells) or the separate frozen cellular component provided DNA and RNA for multiple downstream studies, including quantitative reverse transcription-polymerase chain reaction (PCR) for microRNA, quantitative PCR for the human telomerase reverse transcriptase gene, whole-genome DNA amplification, and array comparative genomic hybridization analysis. Conclusion An improved breast epithelial sampling method has been developed, which should significantly expand the acquisition and biomarker analysis of breast ductal epithelium in women at risk for breast cancer.

We assessed the cerebrospinal fluid (CSF) by flow cytometry and cytology in 51 newly diagnosed and 9 treated aggressive B-cell lymphomas at risk for central nervous system (CNS) involvement to examine the utility of flow cytometry, incidence of CSF disease, and clinical surrogates of CNS spread. Multicolor flow cytometry using multiple antibody panels for light chains and B- and T-cell antigens identified neoplastic clones that constituted as little as 0.2% of total CSF lymphocytes. Among 51 newly diagnosed patients, 11 (22%) had occult CSF involvement. All 11 were detected by flow cytometry but only 1 by cytology (P = .002). Among 9 treated patients, CSF involvement was detected by flow cytometry alone in 2 and also by cytology in 1 case. CSF chemistry and cell counts were similar in patients with and without CSF lymphoma. Only the number of extranodal sites was associated with occult CSF lymphoma in newly diagnosed patients by univariate (P = .006) or logistic regression analysis (P = .012). We hypothesize that the biologic phenotype associated with colonization of extranodal sites leads to CNS spread, possibly related to the microenvironment. Patients at risk for CNS spread should undergo staging CSF evaluation by flow cytometry.

A 9-year-old girl with autosomal recessive chronic granulomatous disease (CGD) presented with asymptomatic bilateral pulmonary infiltrates on routine computed tomography. Fine-needle aspirate of the infiltrates was obtained and showed fungal cells resembling Trichosporon inkin . The specimen grew in culture, and testing by means of both API 20C and PCR amplification confirmed the diagnosis of T inkin . The infiltrates increased in size, despite sequential therapy with voriconazole, liposomal amphotericin B, caspofungin, and posaconazole. The patient required resection of the infected lung tissue, after which she recovered completely. While she was undergoing therapy, her 13-year-old brother, also with CGD, was given a diagnosis of bilateral T inkin –induced pulmonary infection. He also required bilateral pulmonary resection for cure. These cases demonstrate the predisposition of patients with CGD to have invasive infections with unusual fungal organisms, such as T inkin . They also illustrate the difficulty of treating invasive T inkin infections with antifungal agents alone. There are 9 previously reported cases of invasive infections caused by T inkin , 3 of which are in patients with CGD. All patients required removal of infected prosthetic devices or surgical resection of infected tissue for cure.

Use of the polymerase chain reaction (PCR) for the detection of B- and T-cell clonality, Epstein-Barr virus (EBV) and Human Herpes Virus 8 (HHV 8) infection is gaining increasing importance as a diagnostic modality. These tests are usually performed on fresh specimens. There are instances when fresh material is not available and there is a clinical utility for the performance of PCR on archival material via slide scrape lysates (SSL). However, the suitability of archival material may be questioned. Records were searched for all archival cytology cases submitted for SSL molecular diagnostics tests since 1998. Results for each case were analyzed for PCR amplification status and individual test results. A randomly chosen control group of equivalent cytologic samples submitted fresh was evaluated for comparison of amplification status. In all, 241 PCR runs were performed on SSL of archival material from 112 cytologic samples (89 cerebrospinal fluids (CSFs), 13 fine-needle aspirates (FNAs), 10 effusions). Out of these samples, 95 (85%) had amplifiable DNA, as assessed by a positive reaction for glyceraldehyde phosphate dehydrogenase (GAPDH). For the control group, 320 PCR runs were performed on 112 fresh cytologic samples (89 CSFs, 13 FNAs, 10 effusions). In total, 102 samples (91%) had amplifiable DNA. There was no statistical difference in the amplification yield between the two groups (P = 0.2177). A morphologic review of 16 of the 17 SSL archival cytologic cases that did not show amplification revealed 11/16 to be of sparse cellularity. Molecular diagnostic tests are performed routinely on fresh cytologic samples with excellent results. At times critical decisions on patient care may need to be made when fresh tissue is not available for molecular diagnostic tests. SSL of archival cytologic material can be used with excellent results for molecular diagnostic tests when fresh material is not available or when the cytologic diagnosis needs further clarification.

The development of effective autologous cell transfer therapies for the treatment of patients with cancer has been difficult, in part because the cells used to treat each patient are different, as are the patient’s tumor and immune status. Much can thus be learned by sequential treatments of the same patient with the same cells, making single modifications in the treatments to determine which factors are critical. The authors have treated a single patient with five sequential administrations of the same cells with minor modifications in the mode of administration and the immune status of the patient. The treatment of this patient strongly suggested that 1) the highly avid recognition of tumor antigens in vitro by a transferred lymphocyte population does not necessarily predict in vivo antitumor activity; 2) the administration of highly avid antitumor autologous lymphocyte populations can be far more active in mediating tumor regression in vivo when administered after nonmyeloablative chemotherapy than when administered without this prior chemotherapy; 3) intra-arterial administration of highly avid antitumor autologous lymphocytes into the blood supply of the tumor can be more effective in mediating tumor regression than the intravenous administration of these same tumor infiltrating lymphocytes; 4) one mechanism of tumor escape from immunotherapy is loss of class I MHC antigen expression by the tumor due to mutation of the beta-2 microglobulin gene.