Manju L. Prasad

Somatic and germline CACNA1D calcium channel mutations in aldosterone-producing adenomas and primary aldosteronism

Adrenal aldosterone-producing adenomas (APAs) constitutively produce the salt-retaining hormone aldosterone and are a common cause of severe hypertension. Recurrent mutations in the potassium channel gene KCNJ5 that result in cell depolarization and Ca2+ influx cause ∼40% of these tumors. We identified 5 somatic mutations (4 altering Gly403 and 1 altering Ile770) in CACNA1D, encoding a voltage-gated calcium channel, among 43 APAs without mutated KCNJ5. The altered residues lie in the S6 segments that line the channel pore. Both alterations result in channel activation at less depolarized potentials; Gly403 alterations also impair channel inactivation. These effects are inferred to cause increased Ca2+ influx, which is a sufficient stimulus for aldosterone production and cell proliferation in adrenal glomerulosa. We also identified de novo germline mutations at identical positions in two children with a previously undescribed syndrome featuring primary aldosteronism and neuromuscular abnormalities. These findings implicate gain-of-function Ca2+ channel mutations in APAs and primary aldosteronism.

Recurrent activating mutation in PRKACA in cortisol-producing adrenal tumors

Adrenal tumors autonomously producing cortisol cause Cushings syndrome. We performed exome sequencing of 25 tumor-normal pairs and identified 2 subgroups. Eight tumors (including three carcinomas) had many somatic copy number variants (CNVs) with frequent deletion of CDC42 and CDKN2A, amplification of 5q31.2 and protein-altering mutations in TP53 and RB1. Seventeen tumors (all adenomas) had no somatic CNVs or TP53 or RB1 mutations. Six of these had known gain-of-function mutations in CTNNB1 (β-catenin) or GNAS (Gαs). Six others had somatic mutations in PRKACA (protein kinase A (PKA) catalytic subunit) resulting in a p.Leu206Arg substitution. Further sequencing identified this mutation in 13 of 63 tumors (35% of adenomas with overt Cushings syndrome). PRKACA, GNAS and CTNNB1 mutations were mutually exclusive. Leu206 directly interacts with the regulatory subunit of PKA, PRKAR1A. Leu206Arg PRKACA loses PRKAR1A binding, increasing the phosphorylation of downstream targets. PKA activity induces cortisol production and cell proliferation, providing a mechanism for tumor development. These findings define distinct mechanisms underlying adrenal cortisol-producing tumors.

Recurrent gain of function mutation in calcium channel CACNA1H causes early-onset hypertension with primary aldosteronism

Many Mendelian traits are likely unrecognized owing to absence of traditional segregation patterns in families due to causation by de novo mutations, incomplete penetrance, and/or variable expressivity. Genome-level sequencing can overcome these complications. Extreme childhood phenotypes are promising candidates for new Mendelian traits. One example is early onset hypertension, a rare form of a global cause of morbidity and mortality. We performed exome sequencing of 40 unrelated subjects with hypertension due to primary aldosteronism by age 10. Five subjects (12.5%) shared the identical, previously unidentified, heterozygous CACNA1HM1549V mutation. Two mutations were demonstrated to be de novo events, and all mutations occurred independently. CACNA1H encodes a voltage-gated calcium channel (CaV3.2) expressed in adrenal glomerulosa. CACNA1HM1549V showed drastically impaired channel inactivation and activation at more hyperpolarized potentials, producing increased intracellular Ca2+, the signal for aldosterone production. This mutation explains disease pathogenesis and provides new insight into mechanisms mediating aldosterone production and hypertension. DOI: http://dx.doi.org/10.7554/eLife.06315.001

Characterization of the mutational landscape of anaplastic thyroid cancer via whole-exome sequencing

Anaplastic thyroid carcinoma (ATC) is a frequently lethal malignancy that is often unresponsive to available therapeutic strategies. The tumorigenesis of ATC and its relationship to the widely prevalent well-differentiated thyroid carcinomas are unclear. We have analyzed 22 cases of ATC as well as 4 established ATC cell lines using whole-exome sequencing. A total of 2674 somatic mutations (121/sample) were detected. Ontology analysis revealed that the majority of variants aggregated in the MAPK, ErbB and RAS signaling pathways. Mutations in genes related to malignancy not previously associated with thyroid tumorigenesis were observed, including mTOR, NF1, NF2, MLH1, MLH3, MSH5, MSH6, ERBB2, EIF1AX and USH2A; some of which were recurrent and were investigated in 24 additional ATC cases and 8 ATC cell lines. Somatic mutations in established thyroid cancer genes were detected in 14 of 22 (64%) tumors and included recurrent mutations in BRAF, TP53 and RAS-family genes (6 cases each), as well as PIK3CA (2 cases) and single cases of CDKN1B, CDKN2C, CTNNB1 and RET mutations. BRAF V600E and RAS mutations were mutually exclusive; all ATC cell lines exhibited a combination of mutations in either BRAF and TP53 or NRAS and TP53. A hypermutator phenotype in two cases with >8 times higher mutational burden than the remaining mean was identified; both cases harbored unique somatic mutations in MLH mismatch-repair genes. This first comprehensive exome-wide analysis of the mutational landscape of ATC identifies novel genes potentially associated with ATC tumorigenesis, some of which may be targets for future therapeutic intervention.

Expression of melanocytic differentiation markers in malignant melanomas of the oral and sinonasal mucosa.

Malignant melanomas of the oral and sinonasal mucosa are rare tumors. Amelanotic variants can, on occasion, be difficult to recognize by routine light microscopy. Immunohistochemical studies may be needed for a final diagnosis. A number of new monoclonal antibodies to melanocytic differentiation antigens have been studied recently on primary cutaneous and metastatic melanoma. However, little is known about these antibodies for the diagnosis of mucosal melanomas. In this study the authors analyzed 79 oral and sinonasal mucosal melanomas of 65 patients. A total of 35 tumors originated from the oral mucosa (21 primary tumors, eight local recurrences, and six metastases) and 44 melanomas were from the sinonasal tract (27 primary tumors, nine local recurrences, and eight metastases). Immunohistochemical studies were performed on paraffin-embedded tissues, using the following antibodies: anti-S-100 protein, T311 (anti-tyrosinase), A103 (anti-Mart-1/Melan-A), D5 (antimicrophthalmia-associated transcription factor), and HMB-45 (anti-gp100). Of 35 oral mucosal tumors, 34 (97%) were positive with anti-S-100 protein, 33 (94%) with T311, 30 (85%) with A103, 26 (74%) with D5, and 25 (71%) with HMB-45. All five desmoplastic melanomas of the oral mucosa were positive for S-100 protein, four for tyrosinase, and one each for HMB-45 and A103. No desmoplastic melanoma was positive with D5. All 44 sinonasal melanomas were positive for tyrosinase and Mart-1/Melan-A (100%). Forty-three (98%) were positive with HMB-45, 42 (95%) with anti-S-100 protein, and 40 (91%) with D5. These results reveal that T311 is the most sensitive marker for sinonasal melanomas and closely approaches the sensitivity of anti-S-100 protein for oral mucosal melanomas. For desmoplastic mucosal tumors, anti-S-100 protein remains the most sensitive marker.

Clinicopathologic Differences in Malignant Melanoma Arising in Oral Squamous and Sinonasal Respiratory Mucosa of the Upper Aerodigestive Tract

OBJECTIVE Primary mucosal melanomas are rare tumors. We compare melanomas arising in 2 histologically different mucosa, the stratified oral squamous mucosa and pseudostratified sinonasal respiratory mucosa, to investigate the clinicopathologic influence of native mucosal histology on the tumor. METHODS Clinicopathologic features of 36 melanomas arising in the squamous mucosa of the oral cavity were compared with 59 melanomas arising in the sinonasal respiratory mucosa. RESULTS The median age of patients was 61 and 63 years for oral and sinonasal melanomas, respectively, with the squamous and respiratory mucosa covering the maxilla being most frequently involved (68.7% and 66%, respectively). The former had a remarkable male predilection (28 men, 8 women), while the latter affected both sexes equally (29 men, 30 women). The oral melanomas were more likely to be detected in the early in situ or microinvasive stage (4 cases vs none, P =.008) and were more frequently amelanotic (14 vs 12, P =.049) than sinonasal melanomas. The sinonasal melanomas were frequently thicker (median thickness, 9 vs 2.6 mm), polypoid (29 vs none), ulcerated (57 vs 20), and necrotic (57 vs 14) than oral melanoma (P <.001). Pseudopapillary architecture was more frequent in sinonasal melanomas (16 tumors vs none, P <.001), and desmoplastic melanomas were more frequent in the oral mucosa (6 vs 1, P =.005). Sinonasal melanoma showed vascular and deep tissue invasion more frequently than oral melanoma; however, no significant difference in disease-specific survival was noted (median survival, 2.8 years vs 3.0 years; 5-year survival, 37% vs 35%, respectively). CONCLUSION Sinonasal melanomas demonstrated aggressive morphologic features significantly more frequently than oral melanomas; however, prognosis remained similar in both groups.

Prognostic factors for malignant melanoma of the squamous mucosa of the head and neck.

Primary malignant melanomas of the squamous mucosa of the head and neck are rare. To learn more about the prognostic significance of various histologic parameters we examined the pathologic features of squamous mucosa from 40 patients seen at a single institution and correlated them with clinical outcome. Follow-up information was available on 37 patients. Thirty-five were treated with surgical resection and two were treated with radiotherapy. Twenty-six were dead at follow-up. Twenty-one of them died of disease. The interval between diagnosis and death ranged from 1 month to 16.5 years (median survival, 2.4 years). Eleven patients were alive at 4 months to 19.5 years after the diagnosis: six of them with disease and five of them free of disease (mean follow-up, 3.5 years). Predictors of poor survival by univariate analysis were the presence of vascular invasion (overall survival, p = 0.007; disease-specific survival, p = 0.01), a polymorphous tumor cell population (overall survival, p = 0.007; disease-specific survival, p = 0.008), and necrosis (overall survival, p = 0.007; disease-specific survival, p = 0.056). However, because these three parameters were associated with each other, none of them remained of independent predictive value for outcome by multivariate analysis. No prognostic significance was found for tumor thickness, level of invasion, ulceration, mitotic index, or nerve/nerve sheath involvement. Thus, the histologic parameters relevant for the prognosis of squamous mucosa differ significantly from those of cutaneous melanomas.

Thyroid follicular lesion of undetermined significance: Evaluation of the risk of malignancy using the two-tier sub-classification

The Bethesda 2007 Thyroid Cytology Classification defines follicular lesion of undetermined significance as a heterogeneous category of cases that are not convincingly benign nor sufficiently atypical for a diagnosis of follicular neoplasm or suspicious for malignancy. In our institution, we refer to these cases as indeterminate, and they are further sub‐classified into two: (1) low cellularity with predominant microfollicular architecture and absence of colloid (INa) and (2) nuclear features not characteristic of benign lesions (nuclear atypia) (INb). We reviewed these indeterminate cases to document the follow‐up trend using this two‐tier classification. A search of the cytology records was performed for the period between January 2008 and June 2009. All thyroid fine‐needle aspiration (FNA) cases were reviewed and the ones diagnosed as indeterminate were identified. Correlating follow‐up FNA and/or surgical pathology reports were reviewed. The percentage of cases showing a malignancy was calculated. One hundred and seventy‐one indeterminate cases were identified, representing 2.8% of the 6,205 thyroid FNA cases examined during the time under review (107 INa, 64 INb). Records of follow‐up procedures were available in 106 (61%) cases. Malignancy was identified in 27% of all indeterminate cases. This was disproportionately more in the INb (56%) compared to the INa (7%) cases. A diagnosis of “INa” does not carry the same implication as that of “INb”. The INb category needs a more aggressive follow‐up than the INa category and may justify an immediate referral for lobectomy. Despite the vague morphologic criteria for this diagnostic category, the indeterminate rate remains relatively low and falls within the NCI recommendation (<7%). Diagn. Cytopathol. 2011;.

In vivo tissue responses to thermal-responsive shape memory polymer nanocomposites.

To explore the safe use of thermal-responsive shape memory polymers (SMPs) as minimally invasive tissue scaffolds, we recently developed a class of biodegradable POSS-SMP nanocomposites exhibiting stable temporary shape fixing and facile shape recovery within a narrow window of physiological temperatures. The materials were covalently crosslinked from star-branched building blocks consisting a bioinert polyhedral oligomeric silsesquioxane (POSS) core and 8 degradable poly(D,L-lactide) (PLA) arms. Here we examine the degradation profiles and immunogenicity of POSS-SMPs as a function of the PLA arm lengths using a rat subcutaneous implantation model. We show that POSS-SMPs elicited a mild foreign body type immune response upon implantation. The degradation rates of POSS-SMPs, both in vitro and in vivo, inversely correlated with the length of the PLA chains within the crosslinked amorphous network. Upon in vivo degradation of POSS-SMPs, a second acute inflammatory response was elicited locally, and the inflammation was able to resolve over time without medical interventions. One year after the implantation of POSS-SMPs, no pathologic abnormalities were detected from the vital/scavenger organs examined. These minimally immunogenic and biodegradable SMPs are promising candidates for scaffold-assisted tissue repair where both facile surgical delivery and controlled degradation of the scaffold are desired for achieving optimal short-term and long-term clinical outcomes.

BRAFV600E mutation in papillary thyroid microcarcinoma: a genotype-phenotype correlation.

BRAFV600E mutation has emerged as a marker of aggressive behavior in papillary thyroid carcinoma but its significance in microcarcinoma is not entirely clear. One-hundred and twenty-nine papillary thyroid microcarcinomas were tested for BRAFV600E mutation by single-strand conformation polymorphism, and their clinicopathologic features (age, sex, tumor size, multifocality, nodal metastases, histologic subtype, tumor cell morphology, architecture, tumor-associated stromal reaction, tumor interface to non-neoplastic thyroid (well circumscribed vs infiltrative), extrathyroidal extension, lymphovascular invasion, intratumoral multinucleated giant cells, and adjacent non-neoplastic thyroid pathology) were examined. Compared with tumors without the mutation (39/129, 30%), the mutated microcarcinomas (90/129, 70%) showed significantly higher prevalence of infiltrative tumor borders (78/90 vs 23/39, P=0.001), tumor-associated stromal desmoplasia/fibrosis and/or sclerosis (80/90 vs 25/39, P=0.002), classic nuclear features of papillary thyroid carcinoma (90/90 vs 35/39, P=0.008) and cystic change (43/90 vs 11/39, P=0.05). BRAFV600E mutation was more frequent in classic (75%), tall cell (91%), and other variants (>70%) than in follicular variant (21%) of papillary thyroid microcarcinoma. Tumors without the mutation were significantly more likely to be solid, well circumscribed, and lacked desmoplasia/fibrosis or sclerosis. However, on multivariate analysis, only the follicular variant of papillary microcarcinoma was significantly associated with the absence of mutation (odds ratio (95% confidence interval): 0.09 (0.01–0.54)). Lymph node metastases (n=24) were more frequent in microcarcinomas with mutation than without (21/24 vs 3/24, P=0.02). All patients with lateral cervical node metastasis (n=9), and all but one tumor with extrathyroidal extension (n=17/18) showed BRAFV600E mutation. No significant differences were noted in age, sex, tumor size, multifocality, lymphovascular invasion, psammoma bodies, stromal calcification, intratumoral multinucleated osteoclastic-type giant cells, and lymphocytic infiltration between the two groups of tumors. BRAFV600E mutation is an early event in thyroid carcinogenesis, and is associated with distinctive morphology and aggressive features even in papillary thyroid microcarcinomas.