Validation of the VirCapSeq-VERT system for differential diagnosis, detection, and surveillance of viral infections.

IMPORTANCE: Broad range assay for accurate and sensitive diagnostics.

Papillary Intralymphatic Angioendothelioma in a Child With PIK3CA-Related Overgrowth Spectrum: Implication of PI3K Pathway in the Vascular Tumorigenesis.

Papillary intralymphatic angioendothelioma (PILA) is an extremely rare vascular tumor and its pathogenesis is unknown. Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA)-related overgrowth spectrum (PROS) is a heterogeneous group of disorders caused by mosaicism for activating mutations of PIK3CA and characterized by asymmetric overgrowth, skeletal anomalies, skin lesions, and vascular malformations. An association between PILA and PROS has not been known. We report a case of PILA involving the spleen of a young girl with the clinical and molecular diagnosis of PROS. Sequencing of the patient's germ-line DNA detected a pathogenic PIK3CA variant c.1357G>A in 10.6% of alleles. Splenectomy revealed a 4-cm tumor composed of ectatic lymphatics with intraluminal papillary projections, consistent with PILA. The tumor cells showed immunohistochemical expression of CD31, CD34, ERG, FLI-1, PROX1, and caldesmon, while D2-40 was negative. The latter may suggest that the tumor derived from an endothelial precursor arrested in the final steps of lymphothelial differentiation, in keeping with the known role of the PIK3CA-governed molecular pathway in the progression of vascular progenitors to mature endothelial cells. The data implicates PIK3CA in the pathogenesis of PILA and broadens the spectrum of phenotypic expressions of PROS.

Phenogenomic heterogeneity of post-transplant plasmablastic lymphomas.

Plasmablastic lymphoma (PBL) is a rare and clinically aggressive neoplasm that typically occurs in immunocompromised individuals, including those with HIV infection and solid organ allograft recipients. Most prior studies have focused on delineating the clinicopathologic features and genetic attributes of HIV-related PBLs, where MYC deregulation and EBV infection, and more recently, mutations in JAK/STAT, MAP kinase, and NOTCH pathway genes have been implicated in disease pathogenesis. The phenotypic spectrum of post-transplant (PT)-PBLs is not well characterized and data on underlying genetic alterations are limited. Hence, we performed comprehensive histopathologic and immunophenotypic evaluation and targeted sequencing of 18 samples from 11 patients (8 males, 3 females, age range 12-76 years) with PT-PBL; 8 de novo and 3 preceded by other types of PTLDs. PT-PBLs displayed morphologic and immunophenotypic heterogeneity and some features overlapped those of plasmablastic myeloma. Six (55%) cases were EBV+ and 5 (45%) showed MYC rearrangement by fluorescence in situ hybridization. Recurrent mutations in epigenetic regulators (KMT2/MLL family, TET2) and DNA damage repair and response (TP53, mismatch repair genes, FANCA, ATRX), MAP kinase (KRAS, NRAS, HRAS, BRAF), JAK/STAT (STAT3, STAT6, SOCS1), NOTCH (NOTCH1, NOTCH3, SPEN), and immune surveillance (FAS, CD58) pathway genes were observed, with EBV+ and EBV- cases exhibiting similarities and differences in their mutational profiles. Clinical outcomes also varied, with survival ranging from 0-15.9 years postdiagnosis. Besides uncovering the biological heterogeneity of PT-PBL, our study highlights similarities and distinctions between PT-PBLs and PBLs occurring in other settings and reveals potentially targetable oncogenic pathways in disease subsets.

ZTTK syndrome: Clinical and molecular findings of 15 cases and a review of the literature.

Zhu-Tokita-Takenouchi-Kim (ZTTK) syndrome is caused by de novo loss-of-function variants in the SON gene (MIM #617140). This multisystemic disorder is characterized by intellectual disability, seizures, abnormal brain imaging, variable dysmorphic features, and various congenital anomalies. The wide application and increasing accessibility of whole exome sequencing (WES) has helped to identify new cases of ZTTK syndrome over the last few years. To date, there have been approximately 45 cases reported in the literature. Here, we describe 15 additional individuals with variants in the SON gene, including those with missense variants bringing the total number of known cases to 60. We have reviewed the clinical and molecular data of these new cases and all previously reported cases to further delineate the most common as well as emerging clinical findings related to this syndrome. Furthermore, we aim to delineate any genotype-phenotype correlations specifically for a recurring pathogenic four base pair deletion (c.5753_5756del) along with discussing the impact of missense variants seen in the SON gene.

Cytogenetic analysis of 130 renal oncocytomas identify three distinct and mutually exclusive diagnostic classes of chromosome aberrations.

The cytogenetic alterations in renal oncocytoma (RO) are poorly understood. We analyzed 130 consecutive RO for karyotypic alterations. Clonal chromosome abnormalities were identified in 63 (49%) cases, which could be categorized into three classes of mutually exclusive cytogenetic categories. Class 1 (N = 20) RO had diploid karyotypes with characteristic 11q13 rearrangement in balanced translocations with 10 or more different chromosome partners in all cases. We identified recurrent translocation partners at 5q35, 6p21, 9p24, 11p13-14, and 11q23, and confirmed that CCND1 gene rearrangement at 11q13 utilizing fluorescence in situ hybridization (FISH). Class 2 RO (N = 25) exhibited hypodiploid karyotypes with loss of chromosome 1 and/or losses of Y in males and X in females in all cases. The class 3 tumors comprising of 18 cases showed diverse types of abnormalities with the involvement of two or more chromosomes exclusive of abnormalities seen in classes 1 and 2 tumors. Furthermore, karyotypically uninformative cases were subjected to FISH analysis to identify classes 1 and 2 abnormalities. In this group, we found similar frequencies of CCND1 rearrangement, loss of chromosome 1 or Y as with karyotypically abnormal cases. We validated our results against 91 tumors from the Mitelman database. Correlation of clinical data with all the three classes of ROs showed no clear evidence of overall patient survival. Our findings support the hypothesis that RO exhibit three principal cytogenetic categories, which may have different roles in initiation and/or progression. These cytogenetic markers provide a key tool in the diagnostic evaluation of RO.

Genetic and phenotypic characterization of indolent T-cell lymphoproliferative disorders of the gastrointestinal tract.

Indolent T-cell lymphoproliferative disorders of the gastrointestinal tract are rare clonal T-cell diseases that more commonly occur in the intestines and have a protracted clinical course. Different immunophenotypic subsets have been described, but the molecular pathogenesis and cell of origin of these lymphocytic proliferations is poorly understood. Hence, we performed targeted next-generation sequencing and comprehensive immunophenotypic analysis of ten indolent T-cell lymphoproliferative disorders of the gastrointestinal tract, which comprised CD4+ (n=4), CD8+ (n=4), CD4+/CD8+ (n=1) and CD4-/CD8- (n=1) cases. Genetic alterations, including recurrent mutations and novel rearrangements, were identified in 8/10 (80%) of these lymphoproliferative disorders. The CD4+, CD4+/CD8+, and CD4-/CD8- cases harbored frequent alterations of JAK-STAT pathway genes (5/6, 82%); STAT3 mutations (n=3), SOCS1 deletion (n=1) and STAT3-JAK2 rearrangement (n=1), and 4/6 (67%) had concomitant mutations in epigenetic modifier genes (TET2, DNMT3A, KMT2D). Conversely, 2/4 (50%) of the CD8+ cases exhibited structural alterations involving the 3' untranslated region of the IL2 gene. Longitudinal genetic analysis revealed stable mutational profiles in 4/5 (80%) cases and acquisition of mutations in one case was a harbinger of disease transformation. The CD4+ and CD4+/CD8+ lymphoproliferative disorders displayed heterogeneous Th1 (T-bet+), Th2 (GATA3+) or hybrid Th1/Th2 (T-bet+/GATA3+) profiles, while the majority of CD8+ disorders and the CD4-/CD8- disease showed a type-2 polarized (GATA3+) effector T-cell (Tc2) phenotype. Additionally, CD103 expression was noted in 2/4 CD8+ cases. Our findings provide insights into the pathogenetic bases of indolent T-cell lymphoproliferative disorders of the gastrointestinal tract and confirm the heterogeneous nature of these diseases. Detection of shared and distinct genetic alterations of the JAK-STAT pathway in certain immunophenotypic subsets warrants further mechanistic studies to determine whether therapeutic targeting of this signaling cascade is efficacious for a proportion of patients with these recalcitrant diseases.

Refractory celiac disease type II: An atypical case highlighting limitations of the current classification system.

Refractory celiac disease (RCD) is a rare condition associated with high morbidity that develops in individuals with celiac disease. It is known to be biologically heterogeneous, and currently two types are recognized based on immunophenotypic and molecular features, type I (RCD I) and type II (RCD II). Differentiating between RCD I and RCD II is critical, as patients with RCD II have substantially worse outcomes and a high risk of developing enteropathy-associated T-cell lymphoma. However, the current RCD classification is limited in scope, and atypical presentations and immunophenotypes are not recognized at present. Herein, we describe a unique case of RCD II with atypical clinical (primarily neurologic manifestations and lack of significant gastrointestinal symptoms), histopathologic (no villous atrophy), immunophenotypic (virtual absence of cytoplasmic CD3 expression), and molecular features (absence of clonal TR rearrangement and identification of pathogenic STAT3 and KMT2D mutations). This case highlights limitations of the current RCD classification system and the utility of next generation sequencing (NGS) studies in the diagnostic workup of RCD. Future algorithms need to recognize extraintestinal manifestations and incorporate atypical histopathologic and immunophenotypic features, as well as results of NGS analysis for RCD II classification.

Predictive testing for neurodegenerative diseases in the age of next-generation sequencing.

The availability and cost of next-generation sequencing (NSG) now allow testing large numbers of genes simultaneously. However, the gold standard for predictive testing has been to test only for a known family mutation or confirmed family disease. The goal of this study was to investigate the psychological impact of predictive testing for autosomal dominant neurodegenerative diseases without a known family mutation using next-generation sequencing panels compared to single-gene testing of a known family mutation. Fourteen individuals from families with a known mutation and 10 individuals with unknown family mutations participated. Participants completed questionnaires on demographics, genetic knowledge, and psychological measures of anxiety, depression, perceived personal control, rumination, and intolerance to uncertainty at baseline and 1 and 6 months after receiving results. Decision regret was measured 1 and 6 months after receiving results. Participants completed a modified Huntington disease genetic testing protocol with genetic counseling and neurological and psychological evaluation. Genetic testing of either the known family mutation or an NGS panel of neurodegenerative disease genes was performed. Semi-structured interviews were performed at 6 months post-results about their experience. Two-sample t tests were performed on data collected at each time point to identify significant between-group differences in demographic variables, baseline psychological scores, and baseline genetic knowledge scores. Within-group change over time was assessed by a mixed-effects model. Results of this study indicate that NGS panels for predictive testing for neurodegenerative disease are safe and beneficial to participants when performed within a modified HD protocol. Though significant differences in psychological outcomes were found, these differences may have been driven by genetic results and baseline psychological differences between individuals within the groups. Participants did not regret their decision to test and were largely pleased with the testing protocol.

Pathway analysis of genomic pathology tests for prognostic cancer subtyping.

Genomic test results collected during the provision of medical care and stored in Electronic Health Record (EHR) systems represent an opportunity for clinical research into disease heterogeneity and clinical outcomes. In this paper, we evaluate the use of genomic test reports ordered for cancer patients in order to derive cancer subtypes and to identify biological pathways predictive of poor survival outcomes. A novel method is proposed to calculate patient similarity based on affected biological pathways rather than gene mutations. We demonstrate that this approach identifies subtypes of prognostic value and biological pathways linked to survival, with implications for precision treatment selection and a better understanding of the underlying disease. We also share lessons learned regarding the opportunities and challenges of secondary use of observational genomic data to conduct such research.

Identification of recurrent mutational events in anorectal melanoma.

Anorectal melanoma is a rare disease that carries a poor prognosis. To date, limited genetic analyses confirmed KIT mutations as a recurrent genetic event similar to other mucosal melanomas, occurring in up to 30% of anorectal melanomas. Importantly, a subset of tumors harboring activating KIT mutations have been found to respond to c-Kit inhibitor-based therapy, with improved patient survival at advanced tumor stages. We performed comprehensive targeted exon sequencing analysis of 467 cancer-related genes in a larger series of 15 anorectal melanomas, focusing on potentially actionable variants based on gain- and loss-of-function mutations. We report the identification of oncogenic driver events in the majority (93%) of anorectal melanomas. These included variants in canonical MAPK pathway effectors rarely observed in cutaneous melanomas (including an HRAS mutation, as well as a BRAF mutation resulting in duplication of threonine 599), and recurrent mutations in the tumor suppressor NF1 in 20% of cases, which represented the second-most frequently mutated gene after KIT in our series. Furthermore, we identify SF3B1 mutations as a recurrent genetic event in mucosal melanomas. Our findings provide an insight into the genetic diversity of anorectal melanomas, and suggest significant potential for alternative targeted therapeutics in addition to c-Kit inhibitors for this melanoma subtype.

New diagnosis of atypical ataxia-telangiectasia in a 17-year-old boy with T-cell acute lymphoblastic leukemia and a novel ATM mutation.

Ataxia-telangiectasia (A-T) is an autosomal recessive chromosome breakage disorder caused by mutations in the ATM gene. Typically, it presents in early childhood with progressive cerebellar dysfunction along with immunodeficiency and oculocutaneous telangiectasia. An increased risk of malignancy is also associated with the syndrome and, rarely, may be the presenting feature in small children. We describe a 17-year-old boy with slurred speech, mild motor delays and learning disability diagnosed with atypical A-T in the setting of T-cell acute lymphoblastic leukemia. Suspicion for A-T was raised after review of a peripheral blood karyotype demonstrating rearrangements involving chromosomes 7 and/or 14. The diagnosis was confirmed after molecular testing identified a novel homozygous missense variant in ATM (c.5585T>A; p.Leu1862His) that resulted in protein instability and abolished serine/threonine protein kinase activity. To our knowledge, this is the first report of concurrent A-T and lymphoid malignancy diagnoses in an older child or adult with only mild neurological disease. Our experience suggests that screening for the disorder should be considered in any individual with lymphoid malignancy and neurological findings, especially as radiation and certain chemotherapy protocols are contraindicated in A-T.

Epigenetic inactivation of TRAIL decoy receptors at 8p12-21.3 commonly deleted region confers sensitivity to Apo2L/trail-Cisplatin combination therapy in cervical cancer.

Multiple chromosomal regions are affected by deletions in cervical cancer (CC) genomes, but their consequence and target gene involvement remains unknown. Our single nucleotide polymorphism (SNP) array identified 8p copy number losses localized to an 8.4 Mb minimal deleted region (MDR) in 36% of CC. The 8p MDR was associated with tumor size, treatment outcome, and with multiple HPV infections. Genetic, epigenetic, and expression analyses of candidate genes at MDR identified promoter hypermethylation and/or inactivation of decoy receptors TNFRSF10C and TNFRSF10D in the majority of CC patients. TNFRSF10C methylation was also detected in precancerous lesions suggesting that this change is an early event in cervical tumorigenesis. We further demonstrate here that CC cell lines exhibiting downregulated expression of TNFRSF10C and/or TNFRSF10D effectively respond to TRAIL-induced apoptosis and this affect was synergistic in combination with DNA damaging chemotherapeutic drugs. We show that the CC cell lines harboring epigenetic inactivation of TRAIL decoy receptors effectively activate downstream caspases suggesting a critical role of inactivation of these genes in efficient execution of extrinsic apoptotic pathway and therapy response. Therefore, these findings shed new light on the role of genetic/epigenetic defects in TRAIL decoy receptor genes in the pathogenesis of CC and provide an opportunity to explore strategies to test decoy receptor gene inactivation as a biomarker of response to Apo2L/TRAIL-combination therapy.

Impact of the Arg 16 allele of the B2AR gene on the effect of withdrawal of LABA in patients with moderate to severe asthma.

INTRODUCTION: Long-acting beta agonists (LABAs) are effective for controlling asthma, however questions about their safety have led to concerns over use. Genetic polymorphisms at the 16 amino acid position of the beta-2 adrenergic receptor gene (B2AR) may be associated with increased risk. METHODS: A randomized, double blind study was conducted in patients with moderate to severe asthma being treated with combined inhaled corticosteroids/LABA (ICS/LABA), comparing the effect of LABA continuation versus withdrawal on asthma outcomes among patients stratified by B2AR genotype (Arg/Arg vs. Gly/Gly at the 16th amino acid position). RESULTS: 67 participants (31 Arg/Arg, 36 Gly/Gly) were randomized to receive fluticasone alone (F) or continue combined fluticasone/salmeterol (F/S) after a run-in period on F/S. Among Gly/Gly subjects, those in the F/S treatment group showed improvement in AM PEFR (+ 8.4 L/s) whereas those receiving F alone experienced a reduction in AM PEFR over the study period (-14.4 L/s), (p = 0.06). There was no significant difference in morning peak expiratory flow rate (AM PEFR) in Arg/Arg participants randomized to receive F/S (-15.7L) vs F alone (-5.6 L/s) (p = 0.61). There was no significant difference in exacerbations in the Arg/Arg subjects treated with F/S compared with those treated with F (p = 0.65). CONCLUSIONS: Withdrawal of LABA therapy in asthmatics with the Arg/Arg genotype at the 16th amino acid position of B2AR did not lead to significant improvement in AM PEFR. LABA withdrawal in the Gly/Gly genotype however led to a borderline significant decline in AM PEFR.

Intraorbital granuloma annulare in an elderly patient.

Classically, granuloma annulare (GA) is a cutaneous disorder localized to the dorsum of the hands and/or feet in children and young adults. Very rarely it can present on the face and rarer still on periorbital structures such as the eyelid and orbital rim. Diagnosis hinges on clinical presentation and histological features, such as palisading granulomas with central destruction of collagen, presence of mucin and lymphohistiocytic infiltration. The etiology of this condition remains unknown, but may involve a delayed-type hypersensitivity reaction, malignancy and/or infection. Herein is the first reported case of an intraorbital GA in an 86-year-old male patient who presented with right eye proptosis.

Practical diagnostic approaches to composite plasma cell neoplasm and low grade B-cell lymphoma/clonal infiltrates in the bone marrow.

Composite plasma cell neoplasm (PCN) and low grade B-cell lymphoma (B-NHL) in the bone marrow are uncommon and raise the differential diagnosis of B-NHL with plasmacytic differentiation and PCN with lymphoplasmacytic morphology. This can be a challenging differential diagnosis, and the distinctions are important because of differences in management. We report five cases of composite PCN with B-NHL or clonal B-cell infiltrates involving the bone marrow. By using multiple different diagnostic modalities, including immunophenotyping by flow cytometry and immunohistochemistry, cytogenetic analysis and IGH gene rearrangement studies by polymerase chain reaction, we were able to distinguish two distinct clonally unrelated neoplasms in all cases. We describe the utility and pitfalls of these different diagnostic modalities. Flow cytometric analysis with a panel of antibodies that includes CD19, CD56, CD138, CD45 and other aberrant markers commonly expressed by PCN will allow identification of clonally unrelated PCN and B-NHL in a composite neoplasm, and distinguish them from B-NHL with plasmacytic differentiation and PCN with lymphoplasmacytic morphology. Cytogenetic and molecular analyses can give false-negative or false-positive results. In summary, a multimodal approach utilizing these different tools, including clinical data, should be used to arrive at the correct diagnosis.

Complete hydatidiform mole and live fetus in a singleton pregnancy with confined placental mosaicism and fetomaternal hemorrhage: a case report.

BACKGROUND: Coexistence of complete mole and a live fetus is uncommon (1:22,000-100,000), more so with euploidy. CASE: We present a case of a molar pregnancy with a euploid fetus who had close fetal evaluation for second trimester bleeding. The patient presented at 29 weeks' pregnancy with decreased fetal movements, a result of fetomaternal hemorrhage. She underwent cesarean section and delivered a live infant. By close follow-up and a multidisciplinary approach, the appropriate diagnosis and a favorable outcome were achieved. Both mother and the child at 5 years of age are doing well. CONCLUSION: Detailed anatomic and molecular studies demonstrated a complete mole resulting from confined placental mosaicism, with molar tissue showing a single paternal allele at 8/8 informative loci, all shared with the fetus, thus this coexistent molar pregnancy was not that of a separate conceptus.

Molecular and morphologic characterization of intraductal tubulopapillary neoplasms of pancreas with novel potentially targetable fusions.

Intraductal tubulopapillary neoplasms (ITPNs) are rare pancreatic tumors with distinct histological and molecular features. Distinction of ITPN from other pancreatic neoplasms is crucial given the known favorable prognosis and the high frequency and diversity of potentially targetable fusions in ITPN. While the histological features of ITPN are well documented, there are few reports on the cytological features, and molecular characterization of ITPN. The authors reported three cases diagnosed in their laboratory between 2016 and 2021. Clinical data, cytomorphological and histological features, with immunophenotypic and molecular characterizations of these cases are described and compared with those reported in the literature. All 3 cases were diagnosed as ITPN based on the microscopic presence of intraductal nodules composed of tightly packed small tubular glands lined by cuboidal cells lacking apparent mucin. On molecular profiling KRAS and TP53 variants were found in Case 1, FGFR2-INA fusion in Case 2, and STARD3NL-BRAF fusion was detected in Case 3. Immunohistochemistry (IHC) revealed that the neoplastic cells in Case 1 were MUC2 positive and MUC6 negative, but in Cases 2 and 3, were negative for MUC2 and positive for MUC6. These results demonstrate the immunophenotypic and molecular variabilities of histologically similar pancreatic neoplasms. The absence of alterations characteristic of more common pancreatic neoplasms should prompt the consideration of fusion studies in morphologically relevant cases. The combination of morphological, IHC, and molecular analyses is important for reliable identification of ITPN given its potential clinical management implications.

Clinical Utility and Reimbursement of Next-Generation Sequencing-Based Testing for Myeloid Malignancies.

Next-generation sequencing is becoming increasingly important for the diagnosis, risk stratification, and management of patients with established or suspected myeloid malignancies. These tests are being incorporated into clinical practice guidelines and many genetic alterations now constitute disease classification criteria. However, the reimbursement for these tests is uncertain. This study analyzed the clinical impact, ordering practices, prior authorization, and reimbursement outcomes of 505 samples from 477 patients sequenced with a 50-gene myeloid next-generation sequencing panel or a 15-gene myeloproliferative neoplasm subpanel. Overall, 98% (496 of 505) of tests provided clinically useful data. Eighty-nine percent of test results, including negative findings, informed or clarified potential diagnoses, 94% of results informed potential prognoses, and 19% of tests identified a potential therapeutic target. Sequencing results helped risk-stratify patients whose bone marrow biopsy specimens were inconclusive for dysplasia, monitor genetic evolution associated with disease progression, and delineate patients with mutation-defined diagnoses. Despite the clinical value, prior authorization from commercial payors or managed government payors was approved for less than half (45%) of requests. Only 51% of all cases were reimbursed, with lack of medical necessity frequently cited as a reason for denial. This study demonstrates the existence of a substantial gap between clinical utility and payor policies on test reimbursement.

Thymidylate synthase expression and molecular alterations in adenosquamous carcinoma of the lung.

Thymidylate synthase expression is known to be higher in squamous cell carcinoma than in adenocarcinoma of the lung. It is thought that this is the reason for the poor efficacy of pemetrexed in squamous cell carcinoma. However, there is limited data on thymidylate synthase expression in adenosquamous carcinoma, a distinct subtype of lung cancer containing both squamous and glandular differentiation. Furthermore, molecular alterations like epidermal growth factor receptor and Kirsten rat sarcoma 2 viral oncogene homolog mutations, which are seen in adenocarcinomas, are not well understood in mixed histology tumors such as adenosquamous carcinoma. In our study, we sought to better characterize adenosquamous tumors of the lung. Using immunohistochemistry to evaluate thymidylate synthase protein levels, we found that the expression of thymidylate synthase in these mixed tumors roughly parallel that of squamous cell carcinoma, instead of falling in between squamous cell and adenocarcinoma. Of note, in adenosquamous samples, the expression of thymidylate synthase was more closely correlated within the two components than would be expected by random chance alone. Also, we had a relatively high rate of epidermal growth factor receptor (11%) and Kirsten rat sarcoma 2 viral oncogene homolog (33%) mutations in these specimens, with the mutations showing convergence in both the glandular and squamous components upon microdissection. Our results indicate that adenosquamous carcinomas are not simple mixtures of their two histological components; they rather behave as their own entity, and it is important to further understand their behavior. Given the similarity of thymidylate synthase expression between squamous cell and adenosquamous carcinoma, and that thymidylate synthase is the main target of pemetrexed, we extrapolate that pemetrexed may also have inferior clinical activity in adenosquamous carcinoma.