Macrocephaly and developmental delay caused by missense variants in RAB5C.

Rab GTPases are important regulators of intracellular vesicular trafficking. RAB5C is a member of the Rab GTPase family that plays an important role in the endocytic pathway, membrane protein recycling and signaling. Here we report on 12 individuals with nine different heterozygous de novo variants in RAB5C. All but one patient with missense variants (n = 9) exhibited macrocephaly, combined with mild-to-moderate developmental delay. Patients with loss of function variants (n = 2) had an apparently more severe clinical phenotype with refractory epilepsy and intellectual disability but a normal head circumference. Four missense variants were investigated experimentally. In vitro biochemical studies revealed that all four variants were damaging, resulting in increased nucleotide exchange rate, attenuated responsivity to guanine exchange factors and heterogeneous effects on interactions with effector proteins. Studies in C. elegans confirmed that all four variants were damaging in vivo and showed defects in endocytic pathway function. The variant heterozygotes displayed phenotypes that were not observed in null heterozygotes, with two shown to be through a dominant negative mechanism. Expression of the human RAB5C variants in zebrafish embryos resulted in defective development, further underscoring the damaging effects of the RAB5C variants. Our combined bioinformatic, in vitro and in vivo experimental studies and clinical data support the association of RAB5C missense variants with a neurodevelopmental disorder characterized by macrocephaly and mild-to-moderate developmental delay through disruption of the endocytic pathway.

Iatrogenic Kaposi's sarcoma from induction therapy for myeloma: to transplant or not to transplant?

We present the case of an HIV-negative man in his 50s who developed a generalised nodular rash while having first-line bortezomib-cyclophosphamide-dexamethasone chemotherapy for multiple myeloma. The rash was biopsied and proven to be Kaposi's sarcoma. The patient's treatment was interrupted at the sixth cycle of chemotherapy, by which time the rash had also spread to the oral mucosa and eyelid. The rash regressed spontaneously on stopping treatment. We were reluctant to restart myeloma treatment, but on the other hand, we wished to consolidate the very good partial response achieved. An autologous marrow transplant was done months later without any recurrence of his Kaposi's with the initiation of bortezomib maintenance. Bortezomib has putative activity against Kaposi's. The patient could benefit from imid-based (thalidomide, lenalidomide, pomalidomide) combination chemotherapy once his myeloma progresses or if there is a recurrence of Kaposi's sarcoma.

Acquired angioedema: an unusual presentation of haematological malignancy.

A previously healthy man in his 60s being worked up for splenomegaly presented to the emergency department with recurrent episodes of angioedema. Each episode was attributed to a precipitating cause, and consequently, the predisposing C1 esterase inhibitor (C1-INH) deficiency remained undiagnosed until the third presentation. The aetiology of acquired C1-INH deficiency would be primarily obscure and require further investigations to identify. A clonal B cell population was finally isolated by flow cytometry after multiple repeat marrow samples, and a diagnosis of splenic marginal zone lymphoma was subsequently reached. Response to single-agent rituximab was observed with resolution of splenomegaly, disappearance of the antibody and restoration of C1-INH levels.

Genomic and phenotypic analyses of diverse non-clinical Acinetobacter baumannii strains reveals strain-specific virulence and resistance capacity.

Acinetobacter baumannii is a critically important pathogen known for its widespread antibiotic resistance and ability to persist in hospital-associated environments. Whilst the majority of A. baumannii infections are hospital-acquired, infections from outside the hospital have been reported with high mortality. Despite this, little is known about the natural environmental reservoir(s) of A. baumannii and the virulence potential underlying non-clinical strains. Here, we report the complete genome sequences of six diverse strains isolated from environments such as river, soil, and industrial sites around the world. Phylogenetic analyses showed that four of these strains were unrelated to representative nosocomial strains and do not share a monophyletic origin, whereas two had sequence types belonging to the global clone lineages GC1 and GC2. Further, the majority of these strains harboured genes linked to virulence and stress protection in nosocomial strains. These genotypic properties correlated well with in vitro virulence phenotypic assays testing resistance to abiotic stresses, serum survival, and capsule formation. Virulence potential was confirmed in vivo, with most environmental strains able to effectively kill Galleria mellonella greater wax moth larvae. Using phenomic arrays and antibiotic resistance profiling, environmental and nosocomial strains were shown to have similar substrate utilisation patterns although environmental strains were distinctly more sensitive to antibiotics. Taken together, these features of environmental A. baumannii strains suggest the existence of a strain-specific distinct gene pools for niche specific adaptation. Furthermore, environmental strains appear to be equally virulent as contemporary nosocomial strains but remain largely antibiotic sensitive.

The Epidemiology and Outcomes of Septic Arthritis in the Maltese Islands: A Hospital-Based Retrospective Cohort Study.

OBJECTIVE/AIM: Septic arthritis is an uncommon but important disease with significant morbidity and mortality, especially if inadequately managed. The aim of this epidemiological study was to identify the characteristics and outcomes of patients treated for septic arthritis at Mater Dei Hospital, Malta, over a 10-year period. METHODS: This was a retrospective observational study. Patients diagnosed with septic arthritis between 2008 and 2018 were recruited. Cases were identified by reviewing all inhospital episodes of patients diagnosed with septic arthritis according to Newman criteria. RESULTS: There were 124 cases of native joint septic arthritis and 138 of prosthetic joint infection. Cases were present amongst all age groups, with the highest incidence amongst those aged 61-70 years for both native and prosthetic infections. Fever was present in around 40% of cases. Raised white cell count was prevalent in 66.9% of native joint infections and 52.9% of prosthetic joints. Elevated C-reactive protein was overwhelmingly seen in most cases, present in 93.5% (median=159.5 mg/L; IQR=85.8-291) of native joints and 92.0% of prosthetic joint infections (median=68.7 mg/L; IQR=20.5-186). Over 55% of patients had one or more risk factors for joint sepsis, diabetes mellitus being the most prevalent clinical comorbidity (22.6% and 24.6% for native and prosthetic joint infections respectively). Synovial cultures were positive in 66% and 82% of native and prosthetic joint aspirates respectively. Staphylococcus aureus was the most commonly isolated organism from both native and prosthetic joint infection, followed by streptococcal infections in native joints and coagulase negative staphylococci and gram-negative infections in prosthetic joints. Fifteen deaths were directly attributed to joint sepsis. CONCLUSION: Absence of fever and elevated white cell count does not exclude the diagnosis. The mortality rate due to septic arthritis in this cohort of patients was found to be 5.7%. All deaths occurred in elderly patients with clinical comorbidities suggesting that this group is at highest risk.

POEMS syndrome: a unique presentation and a diagnostic challenge.

We present the case of a previously well, 70-year-old lady who presented with gait deterioration and a clinicoradiological picture of myositis with T2-enhancement on MRI of the distal lower limb muscles. A muscle biopsy pointed towards neurogenic amyotrophy and a mixed demyelinating and axonal sensorimotor polyneuropathy was confirmed on nerve conduction studies. This was initially thought to be secondary to a myeloproliferative disorder but a positron emission tomography scan showed uptake in the left hip which corresponded to what was previously reported as a subchondral cyst on CT. A biopsy showed this to be a plasmacytoma in the context of a normal serum protein electrophoresis and a polyclonal increase in light chains on serum-free light chain estimation. The patient was started on treatment 6 months after the presentation for a diagnosis of POEMS syndrome, by which time, the patient had become bed-bound and fully dependent.

Is completion axillary lymph node dissection necessary in patients who are underrepresented in the ACOSOG Z0011 trial?

PURPOSE: The American College of Surgeons Oncology Group trial Z0011 demonstrated that axillary node dissection (ALND) can be omitted in patients managed with breast conserving surgery and 1 to 2 positive sentinel lymph nodes (SLNs) without adverse effects on locoregional recurrence or disease-free survival (DFS). We investigated patients with breast cancer for whom clinicopathologic features were underrepresented in the Z0011 trial and analyzed radiation therapy treatment patterns and clinical outcomes. METHODS AND MATERIALS: We retrospectively reviewed records of patients who underwent a lumpectomy and SLN biopsy with positive SLNs but not an ALND and completed adjuvant radiation therapy. Eligible patients had T3 tumors, >2 positive SLNs, invasive lobular carcinoma, estrogen receptor negative status, extranodal extension, Nottingham Grade 3, or were age <50 years. RESULTS: We identified 105 women treated between July 2011 and July 2016 with a median follow-up time of 48.5 months (Range, 11-83 months). There were 40 women with an extranodal extension (38.9%) and 42 women with grade 3 disease (40.0%). Nineteen patients received whole breast irradiation alone (18.1%) and 86 patients were treated with modified tangent fields including the superior axilla level I/II (81.9%). Thirty-three patients (31.4%) also received a 3rd supraclavicular, nodal-directed field. Among the 86 patients who received axillary nodal irradiation, nodal volume contouring was performed in 77 patients (89.5%). Fifty-one patients (48.6%) also received adjuvant chemotherapy. The overall rates of 4-year DFS and locoregional control (LRC) were 94.3% and 98.1%, respectively. Off all patients, 1 patient experienced an internal mammary nodal recurrence, another patient a contralateral breast tumor, and two patients distant metastases. There were no axillary or ipsilateral breast tumor recurrences. CONCLUSIONS: This retrospective analysis of women who were underrepresented or excluded from the Z11 trial and underwent a lumpectomy and SLN biopsy with positive SLNs demonstrated comparable rates of LRC and DFS. The high rates of LRC and DFS suggest that completion ALND may be safely omitted in this patient population but larger data sets and longer follow-up times are needed to confirm this finding.

Low Estrogen Receptor (ER)-Positive Breast Cancer and Neoadjuvant Systemic Chemotherapy: Is Response Similar to Typical ER-Positive or ER-Negative Disease?

OBJECTIVES: Pathologic complete response (pCR) rate after neoadjuvant chemotherapy was compared between 141 estrogen receptor (ER)-negative (43%), 41 low ER+ (13%), 47 moderate ER+ (14%), and 98 high ER+ (30%) tumors. METHODS: Human epidermal growth factor receptor 2-positive cases, cases without semiquantitative ER score, and patients treated with neoadjuvant endocrine therapy alone were excluded. RESULTS: The pCR rate of low ER+ tumors was similar to the pCR rate of ER- tumors (37% and 26% for low ER and ER- respectively, P = .1722) but significantly different from the pCR rate of moderately ER+ (11%, P = .0049) and high ER+ tumors (4%, P < .0001). Patients with pCR had an excellent prognosis regardless of the ER status. In patients with residual disease (no pCR), the recurrence and death rate were higher in ER- and low ER+ cases compared with moderate and high ER+ cases. CONCLUSIONS: Low ER+ breast cancers are biologically similar to ER- tumors. Semiquantitative ER H-score is an important determinant of response to neoadjuvant chemotherapy.

Prognostic Significance of Modified Residual Disease in Breast and Nodes (mRDBN) Algorithm After Neoadjuvant Chemotherapy for Breast Cancer.

OBJECTIVES: We hypothesized that prognostic accuracy of the residual disease in breast and lymph nodes (RDBN) method, which is calculated using residual tumor size, nodal involvement, and tumor grade, may be improved by incorporating residual tumor cellularity. METHODS: Cases included 614 patients who underwent neoadjuvant therapy for breast cancer. Tumor size was adjusted for residual cellularity of invasive carcinoma and used to calculate modified RDBN (mRDBN) and compared with unmodified gross tumor size (gRDBN). RESULTS: RDBN could be calculated in 428 cases. Relative risks of recurrence and death were significantly higher for RDBN-3 and RDBN-4 compared with RDBN-1. Kaplan-Meier analysis showed significant differences in disease-free survival and overall survival for estrogen receptor (ER)-negative/human epidermal growth factor receptor 2 (HER2)-negative and ER-positive/HER2-negative subgroups (P < .0001). CONCLUSIONS: Both mRDBN and gRDBN provide prognostic information, particularly in HER2-negative carcinoma; however, mRDBN showed better stratification of RDBN-3 and RDBN-4 patients.

Crystal structure of a UDP-GlcNAc epimerase for surface polysaccharide biosynthesis in Acinetobacter baumannii.

With new strains of Acinetobacter baumannii undergoing genomic analysis, it has been possible to define regions of genomic plasticity (RGPs), encoding specific adaptive elements. For a selected RGP from a community-derived isolate of A. baumannii, we outline sequences compatible with biosynthetic machinery of surface polysaccharides, specifically enzymes utilized in the dehydration and conversion of UDP-N-acetyl-D-glucosamine (UDP-D-GlcNAc). We have determined the crystal structure of one of these, the epimerase Ab-WbjB. This dehydratase belongs to the 'extended' short-chain dehydrogenase/reductase (SDR) family, related in fold to previously characterised enzymes CapE and FlaA1. Our 2.65Å resolution structure of Ab-WbjB shows a hexamer, organised into a trimer of chain pairs, with coenzyme NADP+ occupying each chain. Specific active-site interactions between each coenzyme and a lysine quaternary group of a neighbouring chain interconnect adjacent dimers, so stabilising the hexameric form. We show UDP-GlcNAc to be a specific substrate for Ab-WbjB, with binding evident by ITC (Ka = 0.23 µmol-1). The sequence of Ab-WbjB shows variation from the consensus active-site motifs of many SDR enzymes, demonstrating a likely catalytic role for a specific threonine sidechain (as an alternative to tyrosine) in the canonical active site chemistry of these epimerases.

Magee Equation 3 predicts pathologic response to neoadjuvant systemic chemotherapy in estrogen receptor positive, HER2 negative/equivocal breast tumors.

Magee Equations were derived as an inexpensive, rapid alternative to Oncotype DX. The Magee Equation 3 utilizes immunohistochemical and FISH data for estrogen receptor (ER), progesterone receptor (PR), HER2 and Ki-67 for its calculation (24.30812+ERIHC × (-0.02177)+PRIHC × (-0.02884)+(0 for HER2 negative, 1.46495 for equivocal, 12.75525 for HER2 positive)+Ki-67 × 0.18649). We hypothesize that Magee Equation 3 scores from pre-therapy core biopsy can predict response to neoadjuvant systemic chemotherapy. A prospectively-maintained database of patients who received neoadjuvant systemic therapy from 2010 to 2014 at a single institution was retrospectively reviewed. Pathologic complete response was defined as absence of invasive tumor in the breast and regional lymph nodes. Of the 614 cases, tumors with missing immunohistochemical results and those that were ER negative or HER2 positive were excluded. This resulted in 237 ER positive, HER2 negative/equivocal tumors that formed the basis of this study. Magee Equation 3 scores were divided into 3 categories similar to Oncotype DX, ie, 0 to <18 (low), 18 to <31 (intermediate), and 31 or higher (high) scores. The pathologic complete response rate for low, intermediate and high Magee Equation 3 scores was 0%, 4%, and 36%, respectively. Patients with high Magee Equation 3 scores were 13 times more likely to achieve pathologic complete response compared to those with Magee Equation 3 scores less than 31 (95% CI 5.09-32.87, P<0.0001). For patients that did not achieve pathologic complete response, high Magee Equation 3 correlated with higher recurrence rate, with the majority occurring in patients with positive lymph nodes in the resection specimen. Magee Equation 3 score ≥31 predicts pathologic complete response in the neoadjuvant setting and for tumor recurrence, when pathologic complete response is not achieved. These results show the utility of Magee Equation 3 in predicting patients who will benefit from chemotherapy but warrant prospective multi-institutional validation.

Improved Breast Cancer Care Quality Metrics After Implementation of a Standardized Tumor Board Documentation Template.

PURPOSE: Cancer treatment requires a coordinated multidisciplinary treatment approach, which led to the development of the Rapid Quality Reporting System by the Commission on Cancer. However, the lack of immediate availability of documented treatment plans and the inefficiency of global medical record reviews represent significant barriers to adherence reporting and the timely implementation of quality improvement measures. METHODS: Adherence to national guidelines in the areas of radiation treatment, chemotherapy, and hormone therapy was assessed after breast conservation surgery (BCS). Adherence rates within 1 year of BCS were analyzed 10 weeks before and after the implementation of a standardized documentation template at weekly multidisciplinary breast cancer conferences. RESULTS: Documented adherence rates increased postimplementation in patients undergoing consideration for both radiation treatment and hormone therapy within 1 year of BCS (89% v 65%; P = .045% and 85% v 62%; P = .002, respectively). No change was observed in patients undergoing evaluation for cytotoxic chemotherapy (80% v 85%; P = 1.00). CONCLUSION: The addition of a documentation template to multidisciplinary breast cancer conferences resulted in increased recorded adherence rates to national guidelines. This template provided a means of both accurate and efficient documentation of evidence-based practice, which represents a concept with broad application in quality improvement. Although evaluation of the project was not continued beyond the pilot stage, current quality measure scores remain within the same range.

A novel family of integrases associated with prophages and genomic islands integrated within the tRNA-dihydrouridine synthase A (dusA) gene.

Genomic islands play a key role in prokaryotic genome plasticity. Genomic islands integrate into chromosomal loci such as transfer RNA genes and protein coding genes, whilst retaining various cargo genes that potentially bestow novel functions on the host organism. A gene encoding a putative integrase was identified at a single site within the 5' end of the dusA gene in the genomes of over 200 bacteria. This integrase was discovered to be a component of numerous genomic islands, which appear to share a target site within the dusA gene. dusA encodes the tRNA-dihydrouridine synthase A enzyme, which catalyses the post-transcriptional reduction of uridine to dihydrouridine in tRNA. Genomic islands encoding homologous dusA-associated integrases were found at a much lower frequency within the related dusB and dusC genes, and non-dus genes. Excision of these dusA-associated islands from the chromosome as circularized intermediates was confirmed by polymerase chain reaction. Analysis of the dusA-associated islands indicated that they were highly diverse, with the integrase gene representing the only universal common feature.

The complete genome and phenome of a community-acquired Acinetobacter baumannii.

Many sequenced strains of Acinetobacter baumannii are established nosocomial pathogens capable of resistance to multiple antimicrobials. Community-acquired A. baumannii in contrast, comprise a minor proportion of all A. baumannii infections and are highly susceptible to antimicrobial treatment. However, these infections also present acute clinical manifestations associated with high reported rates of mortality. We report the complete 3.70 Mbp genome of A. baumannii D1279779, previously isolated from the bacteraemic infection of an Indigenous Australian; this strain represents the first community-acquired A. baumannii to be sequenced. Comparative analysis of currently published A. baumannii genomes identified twenty-four accessory gene clusters present in D1279779. These accessory elements were predicted to encode a range of functions including polysaccharide biosynthesis, type I DNA restriction-modification, and the metabolism of novel carbonaceous and nitrogenous compounds. Conversely, twenty genomic regions present in previously sequenced A. baumannii strains were absent in D1279779, including gene clusters involved in the catabolism of 4-hydroxybenzoate and glucarate, and the A. baumannii antibiotic resistance island, known to bestow resistance to multiple antimicrobials in nosocomial strains. Phenomic analysis utilising the Biolog Phenotype Microarray system indicated that A. baumannii D1279779 can utilise a broader range of carbon and nitrogen sources than international clone I and clone II nosocomial isolates. However, D1279779 was more sensitive to antimicrobial compounds, particularly beta-lactams, tetracyclines and sulphonamides. The combined genomic and phenomic analyses have provided insight into the features distinguishing A. baumannii isolated from community-acquired and nosocomial infections.

A novel family of genomic resistance islands, AbGRI2, contributing to aminoglycoside resistance in Acinetobacter baumannii isolates belonging to global clone 2.

OBJECTIVES: To determine the context and location of antibiotic resistance genes in carbapenem- and aminoglycoside-resistant Acinetobacter baumannii global clone 2 (GC2) isolates carrying a class 1 integron. METHODS: Isolates were from Sydney hospitals. Resistance to antibiotics was determined by disc diffusion. BLAST searches identified relevant DNA fragments in a draft genome sequence. PCR was used to assemble fragments and map equivalent regions. RESULTS: In two isolates belonging to GC2, WM99c and A91, the bla(TEM) gene, the class 1 integron carrying the aacC1-orfP-orfP-orfQ-aadA1 cassette array and sul1 gene, and the aphA1b gene in Tn6020 were each in segments flanked by IS26. These, together with a fourth IS26-flanked segment, formed a 19.5 kb genomic resistance island (GRI), designated AbGRI2-1, containing five copies of IS26. Part of this island was identical to part of the multiple antibiotic resistance region of AbaR-type islands found in global clone 1 (GC1). AbGRI2-1 has replaced a 40.9 kb segment found in the AB0057 genome. Related GRIs were identified in the same location in published GC2 genomes and appear to have arisen from AbGRI2-1 via IS26-mediated deletions. Like A91, WM99c carries ISAba1 upstream of ampC and Tn6167, an AbGRI1-type island in the chromosomal comM gene containing sul2, tet(B), strA and strB genes and bla(OXA-23) in Tn2006. In WM99c, the chromosomal gene encoding OXA-Ab is interrupted by ISAba17. CONCLUSIONS: AbGRI2-1 is the largest so far of a new type of GRI designated AbGRI2 to distinguish them from the islands in comM in GC1 isolates (AbaR type) and in GC2 isolates (AbGRI1 type).

The mitochondrial genome of the soybean cyst nematode, Heterodera glycines.

We sequenced the entire coding region of the mitochondrial genome of Heterodera glycines. The sequence obtained comprised 14.9 kb, with PCR evidence indicating that the entire genome comprised a single, circular molecule of approximately 21-22 kb. The genome is the most T-rich nematode mitochondrial genome reported to date, with T representing over half of all nucleotides on the coding strand. The genome also contains the highest number of poly(T) tracts so far reported (to our knowledge), with 60 poly(T) tracts ≥ 12 Ts. All genes are transcribed from the same mitochondrial strand. The organization of the mitochondrial genome of H. glycines shows a number of similarities compared with Radopholus similis, but fewer similarities when compared with Meloidogyne javanica. Very few gene boundaries are shared with Globodera pallida or Globodera rostochiensis. Partial mitochondrial genome sequences were also obtained for Heterodera cardiolata (5.3 kb) and Punctodera chalcoensis (6.8 kb), and these had identical organizations compared with H. glycines. We found PCR evidence of a minicircular mitochondrial genome in P. chalcoensis, but at low levels and lacking a noncoding region. Such circularised genome fragments may be present at low levels in a range of nematodes, with multipartite mitochondrial genomes representing a shift to a condition in which these subgenomic circles predominate.

Detection of splicing aberrations caused by BRCA1 and BRCA2 sequence variants encoding missense substitutions: implications for prediction of pathogenicity.

Missense substitutions in high-risk cancer susceptibility genes create clinical uncertainty in the genetic counseling process. Multifactorial likelihood classification approaches and in vitro assays are useful for the classification of exonic sequence variants in BRCA1 and BRCA2, but these currently rely on the assumption that changes in protein function are the major biological mechanism of pathogenicity. This study investigates the potentially pathogenic role of aberrant splicing for exonic variants predicted to encode missense substitutions using patient-derived RNA. No splicing aberrations were identified for BRCA1c.5054C>T and BRCA2c.7336A>G, c.8839G>A, and c.9154C>T. However, RT-PCR analysis identified a major splicing aberration for BRCA1c.4868C>G(p.Ala1623Gly), a variant encoding a missense substitution considered likely to be neutral. Splicing aberrations were also observed for BRCA2c.7988A>T(p.Glu2663Val) and c.8168A>G(p.Asp2723Gly), but both variant and wildtype alleles were shown to be present in full-length mRNA transcripts, suggesting that variant protein may be translated. BRCA2 protein function assays indicated that BRCA2p.Glu2663Val, p.Asp2723Gly and p.Arg3052Trp missense proteins have abrogated function consistent with pathogenicity. Multifactorial likelihood analysis provided evidence for pathogenicity for BRCA1 c.5054C>T(p.Thr1685Ile) and BRCA2c.7988A>T(p.Glu2663Val), c.8168A>G(p.Asp2723Gly) and c.9154C>T(p.Arg3052Trp), supporting experimentally derived evidence. These findings highlight the need for improved bioinformatic prediction of splicing aberrations and to refine multifactorial likelihood models used to assess clinical significance.

Functional assays for classification of BRCA2 variants of uncertain significance.

The assessment of the influence of many rare BRCA2 missense mutations on cancer risk has proved difficult. A multifactorial likelihood model that predicts the odds of cancer causality for missense variants is effective, but is limited by the availability of family data. As an alternative, we developed functional assays that measure the influence of missense mutations on the ability of BRCA2 to repair DNA damage by homologous recombination and to control centriole amplification. We evaluated 22 missense mutations from the BRCA2 DNA binding domain (DBD) that were identified in multiple breast cancer families using these assays and compared the results with those from the likelihood model. Thirteen variants inactivated BRCA2 function in at least one assay; two others truncated BRCA2 by aberrant splicing; and seven had no effect on BRCA2 function. Of 10 variants with odds in favor of causality in the likelihood model of 50:1 or more and a posterior probability of pathogenicity of 0.99, eight inactivated BRCA2 function and the other two caused splicing defects. Four variants and four controls displaying odds in favor of neutrality of 50:1 and posterior probabilities of pathogenicity of at least 1 x 10(-3) had no effect on function in either assay. The strong correlation between the functional assays and likelihood model data suggests that these functional assays are an excellent method for identifying inactivating missense mutations in the BRCA2 DBD and that the assays may be a useful addition to models that predict the likelihood of cancer in carriers of missense mutations.

Secondary mutations as a mechanism of cisplatin resistance in BRCA2-mutated cancers.

Ovarian carcinomas with mutations in the tumour suppressor BRCA2 are particularly sensitive to platinum compounds. However, such carcinomas ultimately develop cisplatin resistance. The mechanism of that resistance is largely unknown. Here we show that acquired resistance to cisplatin can be mediated by secondary intragenic mutations in BRCA2 that restore the wild-type BRCA2 reading frame. First, in a cisplatin-resistant BRCA2-mutated breast-cancer cell line, HCC1428, a secondary genetic change in BRCA2 rescued BRCA2 function. Second, cisplatin selection of a BRCA2-mutated pancreatic cancer cell line, Capan-1 (refs 3, 4), led to five different secondary mutations that restored the wild-type BRCA2 reading frame. All clones with secondary mutations were resistant both to cisplatin and to a poly(ADP-ribose) polymerase (PARP) inhibitor (AG14361). Finally, we evaluated recurrent cancers from patients whose primary BRCA2-mutated ovarian carcinomas were treated with cisplatin. The recurrent tumour that acquired cisplatin resistance had undergone reversion of its BRCA2 mutation. Our results suggest that secondary mutations that restore the wild-type BRCA2 reading frame may be a major clinical mediator of acquired resistance to platinum-based chemotherapy.