Molecular profiling of pre- and post-treatment pediatric high-grade astrocytomas reveals acquired increased tumor mutation burden in a subset of recurrences.

Diffuse gliomas are a heterogeneous category of primary central nervous system tumors. Due to their infiltrative growth precluding complete surgical resection, most diffuse high-grade gliomas are treated with adjuvant chemotherapy and radiation. Recurrent/progressive diffuse gliomas may show genetic differences when compared to the primary tumors, giving insight into their molecular evolution and mechanisms of treatment resistance. In adult-type diffuse gliomas with or without isocitrate dehydrogenase gene mutations, tumor recurrence/progression can be associated with mutations in genes encoding DNA mismatch repair proteins, leading to a dramatic increase in tumor mutation burden. This phenomenon is closely linked to treatment with the DNA alkylating agent temozolomide, a mainstay of adult diffuse glioma chemotherapeutic management. Post-treatment mismatch repair deficiency and acquired high tumor mutation burden is relatively unexplored in pediatric patients who have recurrent high-grade gliomas. Here, we report a molecular and histological analysis of an institutional cohort of eleven pediatric patients with paired initial and recurrent high-grade astrocytoma samples with intervening temozolomide treatment. We identified three cases with evidence for increased tumor mutation burden at recurrence, including two cases of diffuse hemispheric glioma H3 G34-mutant (one previously reported). We also show that molecular analysis by next-generation DNA sequencing and DNA methylation-based profiling enabled an integrated diagnosis per 2021 World Health Organization criteria in 10 of 11 cases (91%). Our findings indicate that increased tumor mutation burden at post-treatment recurrence is relevant in pediatric-type diffuse high-grade gliomas. Diffuse hemispheric glioma H3 G34-mutant may be particularly susceptible to this phenomenon.

Mechanisms of targeted therapy resistance in a pediatric glioma driven by ETV6-NTRK3 fusion.

Chromosomal rearrangements of the NTRK genes generate kinase fusions that are targetable oncogenic drivers in diverse adult and pediatric malignancies. Despite robust clinical response to targeted NTRK inhibition, the emergence of therapeutic resistance poses a formidable clinical challenge. Here we report the characterization of an ETV6-NTRK3 fusion-driven pediatric glioma that progressed through NTRK-targeted treatments with entrectinib and selitrectinib. Genetic analysis of multifocal recurrent/resistant lesions identified a previously uncharacterized NTRK3 p.G623A and a known p.G623E resistance mutation, in addition to other alterations of potential pathogenic impact. Functional studies using heterologous reconstitution model systems and patient-derived tumor cell lines establish that NTRK3G623A and NTRK3G623E mutated kinases exhibit reduced sensitivity to entrectinib and selitrectinib, as well as other NTRK inhibitors tested herein. In summary, this genetic analysis of multifocal recurrent/resistant glioma driven by ETV6-NTRK3 fusion captured a cross section of resistance-associated alterations that, based on in vitro analysis, likely contributed to resistance to targeted therapy and disease progression.

GAB1-ABL1 fusions in tumors that have histologic overlap with NTRK-rearranged spindle cell tumors.

Fibroblastic spindle cell tumors are a heterogeneous group of rare soft tissue tumors that are increasingly recognized as associated with a variety of kinase gene fusions. We report two cases of GAB1-ABL1 fusions in spindle cell tumors that histologically overlap with neurotrophic tyrosine receptor kinase (NTRK)-rearranged spindle cell tumors. The first case occurred in a 76-year-old female who had a large deep-seated spindle cell tumor composed of monotonous ovoid to spindle cells in a background of thick stromal collagen bands with prominent hyalinized vessels and inconspicuous mitoses (<1/10 HPF). Immunohistochemical stains showed co-expression of S100 and CD34. A GAB1-ABL1 fusion was detected by whole transcriptome RNA sequencing. The patient had a partial response to imatinib. The second case was previously described as a solitary fibrous tumor, occurring in a 9-year-old female with a cellular spindle cell tumor with patchy CD34 immunoexpression but no expression of S100. Upon clinicopathologic re-review, including anchored multiplex next-generation sequencing, a GAB1-ABL1 fusion was identified. In summary, we report the first two cases of spindle cell tumors with variable expression of CD34 and/or S100, driven by GAB1-ABL1 gene fusions with histologic overlap with NTRK-rearranged spindle cell tumors, suggesting that ABL-fusions may also be oncogenic drivers within this spectrum of tumors. These cases highlight the evolving understanding of fibroblastic spindle cell tumor biology and the utility of sequencing in identifying a targetable alteration.

Novel BRAF gene fusions and activating point mutations in spindle cell sarcomas with histologic overlap with infantile fibrosarcoma.

Infantile fibrosarcoma (IFS)/cellular congenital mesoblastic nephroma (cCMN) commonly harbors the classic ETV6-NTRK3 translocation. However, there are recent reports of mesenchymal tumors with IFS-like morphology harboring fusions of other receptor tyrosine kinases or downstream effectors, including NTRK1/2/3, MET, RET, and RAF1 fusions as well as one prior series with BRAF fusions. Discovery of these additional molecular drivers contributes to a more integrated diagnostic approach and presents important targets for therapy. Here we report the clinicopathologic and molecular features of 14 BRAF-altered tumors, of which 5 had BRAF point mutations and 10 harbored one or more BRAF fusions. Of the BRAF fusion-positive tumors, one harbored two BRAF fusions (FOXN3-BRAF, TRIP11-BRAF) and another harbored three unique alternative splice variants of EPB41L2-BRAF. Tumors occurred in ten males and four females, aged from birth to 32 years (median 6 months). Twelve were soft tissue based; two were visceral including one located in the kidney (cCMN). All neoplasms demonstrated ovoid to short spindle cells most frequently arranged haphazardly or in intersecting fascicles, often with collagenized stroma and a chronic inflammatory infiltrate. No specific immunophenotype was observed; expression of CD34, S100, and SMA was variable. To date, this is the largest cohort of BRAF-altered spindle cell neoplasms with IFS-like morphology, including not only seven novel BRAF fusion partners but also the first description of oncogenic BRAF point mutations in these tumors.

A Multiplexed Amplicon Approach for Detecting Gene Fusions by Next-Generation Sequencing.

Chromosomal rearrangements that result in oncogenic gene fusions are clinically important drivers of many cancer types. Rapid and sensitive methods are therefore needed to detect a broad range of gene fusions in clinical specimens that are often of limited quantity and quality. We describe a next-generation sequencing approach that uses a multiplex PCR-based amplicon panel to interrogate fusion transcripts that involve 19 driver genes and 94 partners implicated in solid tumors. The panel also includes control assays that evaluate the 3'/5' expression ratios of 12 oncogenic kinases, which might be used to infer gene fusion events when the partner is unknown or not included on the panel. There was good concordance between the solid tumor fusion gene panel and other methods, including fluorescence in situ hybridization, real-time PCR, Sanger sequencing, and other next-generation sequencing panels, because 40 specimens known to harbor gene fusions were correctly identified. No specific fusion reads were observed in 59 fusion-negative specimens. The 3'/5' expression ratio was informative for fusions that involved ALK, RET, and NTRK1 but not for BRAF or ROS1 fusions. However, among 37 ALK or RET fusion-negative specimens, four exhibited elevated 3'/5' expression ratios, indicating that fusions predicted solely by 3'/5' read ratios require confirmatory testing.

Novel mutations in neuroendocrine carcinoma of the breast: possible therapeutic targets.

Primary neuroendocrine carcinoma of the breast is a rare variant, accounting for only 2% to 5% of diagnosed breast cancers, and may have relatively aggressive behavior. Mutational profiling of invasive ductal breast cancers has yielded potential targets for directed cancer therapy, yet most studies have not included neuroendocrine carcinomas. In a tissue microarray screen, we found a 2.4% prevalence (9/372) of neuroendocrine breast carcinoma, including several with lobular morphology. We then screened primary or metastatic neuroendocrine breast carcinomas (excluding papillary and mucinous) for mutations in common cancer genes using polymerase chain reaction-mass spectroscopy (643 hotspot mutations across 53 genes), or semiconductor-based next-generation sequencing analysis (37 genes). Mutations were identified in 5 of 15 tumors, including 3 with PIK3CA exon 9 E542K mutations, 2 of which also harbored point mutations in FGFR family members (FGFR1 P126S, FGFR4 V550M). Single mutations were found in each of KDR (A1065T) and HRAS (G12A). PIK3CA mutations are common in other types of breast carcinoma. However, FGFR and RAS family mutations are exceedingly rare in the breast cancer literature. Likewise, activating mutations in the receptor tyrosine kinase KDR (VEGFR2) have been reported in angiosarcomas and non-small cell lung cancers; the KDR A1065T mutation is reported to be sensitive to VEGFR kinase inhibitors, and fibroblast growth factor receptor inhibitors are in trials. Our findings demonstrate the utility of broad-based genotyping in the study of rare tumors such as neuroendocrine breast cancer.

Assessing copy number alterations in targeted, amplicon-based next-generation sequencing data.

Changes in gene copy number are important in the setting of precision medicine. Recent studies have established that copy number alterations (CNAs) can be detected in sequencing libraries prepared by hybridization-capture, but there has been comparatively little attention given to CNA assessment in amplicon-based libraries prepared by PCR. In this study, we developed an algorithm for detecting CNAs in amplicon-based sequencing data. CNAs determined from the algorithm mirrored those from a hybridization-capture library. In addition, analysis of 14 pairs of matched normal and breast carcinoma tissues revealed that sequence data pooled from normal samples could be substituted for a matched normal tissue without affecting the detection of clinically relevant CNAs (>|2| copies). Comparison of CNAs identified by array comparative genomic hybridization and amplicon-based libraries across 10 breast carcinoma samples showed an excellent correlation. The CNA algorithm also compared favorably with fluorescence in situ hybridization, with agreement in 33 of 38 assessments across four different genes. Factors that influenced the detection of CNAs included the number of amplicons per gene, the average read depth, and, most important, the proportion of tumor within the sample. Our results show that CNAs can be identified in amplicon-based targeted sequencing data, and that their detection can be optimized by ensuring adequate tumor content and read coverage.

Genetic profiling to determine risk of relapse-free survival in high-risk localized prostate cancer.

PURPOSE: The characterization of actionable mutations in human tumors is a prerequisite for the development of individualized, targeted therapy. We examined the prevalence of potentially therapeutically actionable mutations in patients with high-risk clinically localized prostate cancer. EXPERIMENTAL DESIGN: Forty-eight samples of formalin-fixed paraffin-embedded prostatectomy tissue from a neoadjuvant chemotherapy trial were analyzed. DNA extracted from microdissected tumor was analyzed for 643 common solid tumor mutations in 53 genes using mass spectroscopy-based sequencing. In addition, PTEN loss and erythroblast transformation-specific-related gene (ERC) translocations were examined using immunohistochemistry (IHC) in associated tissue microarrays. Association with relapse during 5 years of follow-up was examined in exploratory analyses of the potential clinical relevance of the genetic alterations. RESULTS: Of the 40 tumors evaluable for mutations, 10% had point mutations in potentially actionable cancer genes. Of the 47 tumors evaluable for IHC, 36% had PTEN loss and 40% had ERG rearrangement. Individual mutations were not frequent enough to determine associations with relapse. Using Kaplan-Meier analysis with a log-rank test, the 16 patients who had PTEN loss had a significantly shorter median relapse-free survival, 19 versus 106 months (P = 0.01). CONCLUSIONS: This study confirms that point mutations in the most common cancer regulatory genes in prostate cancer are rare. However, the PIK3CA/AKT pathway was mutated in 10% of our samples. Although point mutations alone did not have a statistically significant association with relapse, PTEN loss was associated with an increased relapse in high-risk prostate cancer treated with chemotherapy followed by surgery.

Combining highly multiplexed PCR with semiconductor-based sequencing for rapid cancer genotyping.

There is growing demand for routine identification of actionable mutations in clinical cancer specimens. Genotyping platforms must provide rapid turnaround times and work effectively with limited amounts of formalin-fixed, paraffin-embedded (FFPE) tissue specimens that often yield poor quality DNA. We describe semiconductor-based sequencing of DNA from FFPE specimens using a single-tube, multiplexed panel of 190 amplicons targeting 46 cancer genes. With just 10 ng of input DNA, average read depths of 2000× can be obtained in 48 hours, with >95% of the reads on target. A validation set of 45 FFPE tumor specimens containing 53 point mutations previously identified with a mass spectrometry-based genotyping platform, along with 19 indels ranging from 4 to 63 bp, was used to evaluate assay performance. With a mutant allele ratio cutoff of 8%, we were able to achieve 100% sensitivity (95% CI = 97.3% to 100.0%) and 95.1% specificity (95% CI = 91.8% to 98.0%) of point mutation detection. All indels were visible by manual inspection of aligned reads; 6/9 indels ≤12 bp long were detected by the variant caller software either exactly or as mismatched nucleotides within the indel region. The rapid turnaround time and low input DNA requirements make the multiplex PCR and semiconductor-based sequencing approach a viable option for mutation detection in a clinical laboratory.

Mucinous breast carcinomas lack PIK3CA and AKT1 mutations.

Activating point mutations in the phosphatidylinositol-3-kinase catalytic subunit (PIK3CA) are among the most common molecular defects in invasive breast cancer. Point mutations in the downstream kinase AKT1 are seen in a minority of carcinomas. These mutations are found preferentially in estrogen receptor-positive and Her2-positive breast carcinomas; however, special morphologic types of breast cancer have not been well studied. Twenty-nine cases of pure invasive mucinous carcinoma and 9 cases of ductal carcinoma with mucinous differentiation were screened for a panel of point mutations (>321 mutations in 30 genes) using a multiplex polymerase chain reaction panel with mass spectroscopy readout. In addition, associated ductal carcinoma in situ, hyperplasia, or columnar cell lesions were separately tested where available (25 lesions). In 3 invasive cases and 15 ductal carcinoma in situ/proliferative lesions, PIK3CA hotspot mutations were, instead, tested by direct sequencing. No point mutations were identified in invasive mucinous breast carcinoma. This contrasts with the 35% frequency of PIK3CA mutations in a comparative group of invasive ductal carcinomas of no special type. Interestingly, PIK3CA hotspot point mutations were identified in associated ductal carcinoma in situ (3/14) and hyperplasia (atypical ductal hyperplasia [2/3], usual ductal hyperplasia [2/3], columnar cell change [1/5]), suggesting that PIK3CA mutations may play a role in breast epithelial proliferation. This series represents the largest study, to date, of PIK3CA genotyping in mucinous carcinoma and supports the unique pathogenetics of invasive mucinous breast carcinoma.

Multiplex high-throughput gene mutation analysis in acute myeloid leukemia.

Classification of acute myeloid leukemia increasingly depends on genetic analysis. However, the number of known mutations in acute myeloid leukemia is expanding rapidly. Therefore, we tested a high-throughput screening method for acute myeloid leukemia mutation analysis using a multiplex mass spectrometry-based approach. To our knowledge, this is the first reported application of this approach to genotype leukemias in a clinical setting. One hundred seven acute myeloid leukemia cases were screened for mutations using a panel that covers 344 point mutations across 31 genes known to be associated with leukemia. The analysis was performed by multiplex polymerase chain reaction for mutations in genes of interest followed by primer extension reactions. Products were analyzed on a Sequenom MassARRAY system (San Diego, CA). The multiplex panel yielded mutations in 58% of acute myeloid leukemia cases with normal cytogenetics and 21% of cases with abnormal cytogenetics. Cytogenetics and routine polymerase chain reaction-based screening of NPM1, CEBPA, FLT3-ITD, and KIT was also performed on a subset of cases. When combined with the results of these standard polymerase chain reaction-based tests, the mutation frequency reached 78% in cases with normal cytogenetics. Of these, 42% harbored multiple mutations primarily involving NPM1 with NRAS, KRAS, CEBPA, PTPN11, IDH1, or FLT3. In contrast, cases with abnormal cytogenetics rarely harbored more than 1 mutation (1.5%), suggesting different underlying biology. This study demonstrates the feasibility and utility of broad-based mutation profiling of acute myeloid leukemia in a clinical setting. This approach will be helpful in defining prognostic subgroups of acute myeloid leukemia and contribute to the selection of patients for enrollment into trials with novel inhibitors.

Phosphatidylinositol-3-kinase pathway mutations are common in breast columnar cell lesions.

The phosphatidylinositol-3-kinase pathway is one of the most commonly mutated pathways in invasive breast carcinoma, with PIK3CA mutations in ∼25% of invasive carcinomas, and AKT1 mutations in up to 5%. Ductal carcinoma in situ, and benign papillomas harbor similar mutations. However, activating point mutations in breast columnar cell lesions have been infrequently studied. Twenty-three breast resection specimens containing columnar cell lesions were identified; 14 with associated invasive carcinoma or carcinoma in situ. DNA extracts were prepared from formalin-fixed paraffin-embedded tissue and screened for a panel of point mutations (321 mutations in 30 genes) using a multiplex PCR panel with mass-spectroscopy readout. PIK3CA mutations were identified in 13/24 columnar cell lesions (54%) and 3/8 invasive carcinomas (37%). The mutation status of columnar cell lesions and associated carcinoma was frequently discordant. Of the 14 cases, only 5 demonstrated the same genotype in matched samples of columnar cell lesions and carcinoma (4 wild type, 1 PIK3CA H1047R). Interestingly, five patients had mutations in columnar cell lesions with wild-type carcinoma; two patients had different point mutations in columnar cell lesions and carcinoma. Only three cases had wild-type columnar cell lesion and mutated carcinoma. The 50% PIK3CA mutation prevalence in columnar cell lesions is greater than reported in most studies of invasive breast cancer. Further, columnar cell lesions and carcinoma were frequently discordant for PIK3CA/AKT1 mutation status. These findings raise interesting questions about the role of PIK3CA/AKT pathway in breast carcinogenesis, and the biologic/precursor potential of columnar cell lesions.

Hypothalamic EAP1 (enhanced at puberty 1) is required for menstrual cyclicity in nonhuman primates.

Mammalian reproductive cyclicity requires the periodic discharge of GnRH from hypothalamic neurons into the portal vessels connecting the neuroendocrine brain to the pituitary gland. GnRH secretion is, in turn, controlled by changes in neuronal and glial inputs to GnRH-producing neurons. The transcriptional control of this process is not well understood, but it appears to involve several genes. One of them, termed enhanced at puberty 1 (EAP1), has been postulated to function in the female hypothalamus as an upstream regulator of neuroendocrine reproductive function. RNA interference-mediated inhibition of EAP1 expression, targeted to the preoptic region, delays puberty and disrupts estrous cyclicity in rodents, suggesting that EAP1 is required for the normalcy of these events. Here, we show that knocking down EAP1 expression in a region of the medial basal hypothalamus that includes the arcuate nucleus, via lentiviral-mediated delivery of RNA interference, results in cessation of menstrual cyclicity in female rhesus monkeys undergoing regular menstrual cycles. Neither lentiviruses encoding an unrelated small interfering RNA nor the placement of viral particles carrying EAP1 small interfering RNA outside the medial basal hypothalamus-arcuate nucleus region affected menstrual cycles, indicating that region-specific expression of EAP1 in the hypothalamus is required for menstrual cyclicity in higher primates. The cellular mechanism by which EAP1 exerts this function is unknown, but the recent finding that EAP1 is an integral component of a powerful transcriptional-repressive complex suggests that EAP1 may control reproductive cyclicity by inhibiting downstream repressor genes involved in the neuroendocrine control of reproductive function.

Multiplex mutation screening by mass spectrometry evaluation of 820 cases from a personalized cancer medicine registry.

There is an immediate and critical need for a rapid, broad-based genotyping method that can evaluate multiple mutations simultaneously in clinical cancer specimens and identify patients most likely to benefit from targeted agents now in use or in late-stage clinical development. We have implemented a prospective genotyping approach to characterize the frequency and spectrum of mutations amenable to drug targeting present in urothelial, colorectal, endometrioid, and thyroid carcinomas and in melanoma. Cancer patients were enrolled in a Personalized Cancer Medicine Registry that houses both clinical information and genotyping data, and mutation screening was performed using a multiplexed assay panel with mass spectrometry-based analysis to detect 390 mutations across 30 cancer genes. Formalin fixed, paraffin-embedded specimens were evaluated from 820 Registry patients. The genes most frequently mutated across multiple cancer types were BRAF, PIK3CA, KRAS, and NRAS. Less common mutations were also observed in AKT1, CTNNB1, FGFR2, FGFR3, GNAQ, HRAS, and MAP2K1. Notably, 48 of 77 PIK3CA-mutant cases (62%) harbored at least one additional mutation in another gene, most often KRAS. Among melanomas, only 54 of 73 BRAF mutations (74%) were the V600E substitution. These findings demonstrate the diversity and complexity of mutations in druggable targets among the different cancer types and underscore the need for a broad-spectrum, prospective genotyping approach to personalized cancer medicine.

In vivo manipulation of gene expression in non-human primates using lentiviral vectors as delivery vehicles.

Non-human primates (NHPs) are an invaluable resource for the study of genetic regulation of disease mechanisms. The main disadvantage of using NHPs as a preclinical model of human disease is the difficulty of manipulating the monkey genome using conventional gene modifying strategies. Lentiviruses offer the possibility of circumventing this difficulty because they can infect and transduce either dividing or nondividing cells, without producing an immune response. In addition, lentiviruses can permanently integrate into the genome of host cells, and are able to maintain long-term expression. In this article we describe the lentiviral vectors that we use to both express transgenes and suppress expression of endogenous genes via RNA interference (RNAi) in NHPs. We also discuss the safety features of currently available vectors that are especially important when lentiviral vectors are used in a species as closely related to humans as NHPs. Finally, we describe in detail the lentiviral vector production protocol we use and provide examples of how the vector can be employed to target peripheral tissues and the brain.