Patricia A. Spears

Recommendations for human epidermal growth factor receptor 2 testing in breast cancer: American Society of Clinical Oncology/College of American Pathologists clinical practice guideline update.

PURPOSE To update the American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guideline recommendations for human epidermal growth factor receptor 2 (HER2) testing in breast cancer to improve the accuracy of HER2 testing and its utility as a predictive marker in invasive breast cancer. METHODS ASCO/CAP convened an Update Committee that included coauthors of the 2007 guideline to conduct a systematic literature review and update recommendations for optimal HER2 testing. RESULTS The Update Committee identified criteria and areas requiring clarification to improve the accuracy of HER2 testing by immunohistochemistry (IHC) or in situ hybridization (ISH). The guideline was reviewed and approved by both organizations. RECOMMENDATIONS The Update Committee recommends that HER2 status (HER2 negative or positive) be determined in all patients with invasive (early stage or recurrence) breast cancer on the basis of one or more HER2 test results (negative, equivocal, or positive). Testing criteria define HER2-positive status when (on observing within an area of tumor that amounts to > 10% of contiguous and homogeneous tumor cells) there is evidence of protein overexpression (IHC) or gene amplification (HER2 copy number or HER2/CEP17 ratio by ISH based on counting at least 20 cells within the area). If results are equivocal (revised criteria), reflex testing should be performed using an alternative assay (IHC or ISH). Repeat testing should be considered if results seem discordant with other histopathologic findings. Laboratories should demonstrate high concordance with a validated HER2 test on a sufficiently large and representative set of specimens. Testing must be performed in a laboratory accredited by CAP or another accrediting entity. The Update Committee urges providers and health systems to cooperate to ensure the highest quality testing. This guideline was developed through a collaboration between the American Society of Clinical Oncology and the College of American Pathologists and has been published jointly by invitation and consent in both Journal of Clinical Oncology and the Archives of Pathology & Laboratory Medicine.

Recommendations for Human Epidermal Growth Factor Receptor 2 Testing in Breast Cancer: American Society of Clinical Oncology/College of American Pathologists Clinical Practice Guideline Update

PURPOSE To update the American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guideline recommendations for human epidermal growth factor receptor 2 (HER2) testing in breast cancer to improve the accuracy of HER2 testing and its utility as a predictive marker in invasive breast cancer. METHODS ASCO/CAP convened an Update Committee that included coauthors of the 2007 guideline to conduct a systematic literature review and update recommendations for optimal HER2 testing. RESULTS The Update Committee identified criteria and areas requiring clarification to improve the accuracy of HER2 testing by immunohistochemistry (IHC) or in situ hybridization (ISH). The guideline was reviewed and approved by both organizations. RECOMMENDATIONS The Update Committee recommends that HER2 status (HER2 negative or positive) be determined in all patients with invasive (early stage or recurrence) breast cancer on the basis of one or more HER2 test results (negative, equivocal, or positive). Testing criteria define HER2-positive status when (on observing within an area of tumor that amounts to >10% of contiguous and homogeneous tumor cells) there is evidence of protein overexpression (IHC) or gene amplification (HER2 copy number or HER2/CEP17 ratio by ISH based on counting at least 20 cells within the area). If results are equivocal (revised criteria), reflex testing should be performed using an alternative assay (IHC or ISH). Repeat testing should be considered if results seem discordant with other histopathologic findings. Laboratories should demonstrate high concordance with a validated HER2 test on a sufficiently large and representative set of specimens. Testing must be performed in a laboratory accredited by CAP or another accrediting entity. The Update Committee urges providers and health systems to cooperate to ensure the highest quality testing.

Postmastectomy Radiotherapy: An American Society of Clinical Oncology, American Society for Radiation Oncology, and Society of Surgical Oncology Focused Guideline Update.

Purpose A joint American Society of Clinical Oncology, American Society for Radiation Oncology, and Society of Surgical Oncology panel convened to develop a focused update of the American Society of Clinical Oncology guideline concerning use of postmastectomy radiotherapy (PMRT). Methods A recent systematic literature review by Cancer Care Ontario provided the primary evidentiary basis. The joint panel also reviewed targeted literature searches to identify new, potentially practice-changing data. Recommendations The panel unanimously agreed that available evidence shows that PMRT reduces the risks of locoregional failure (LRF), any recurrence, and breast cancer mortality for patients with T1-2 breast cancer with one to three positive axillary nodes. However, some subsets of these patients are likely to have such a low risk of LRF that the absolute benefit of PMRT is outweighed by its potential toxicities. In addition, the acceptable ratio of benefit to toxicity varies among patients and physicians. Thus, the decision to recommend PMRT requires a great deal of clinical judgment. The panel agreed clinicians making such recommendations for individual patients should consider factors that may decrease the risk of LRF, attenuate the benefit of reduced breast cancer-specific mortality, and/or increase risk of complications resulting from PMRT. When clinicians and patients elect to omit axillary dissection after a positive sentinel node biopsy, the panel recommends that these patients receive PMRT only if there is already sufficient information to justify its use without needing to know additional axillary nodes are involved. Patients with axillary nodal involvement after neoadjuvant systemic therapy should receive PMRT. The panel recommends treatment generally be administered to both the internal mammary nodes and the supraclavicular-axillary apical nodes in addition to the chest wall or reconstructed breast.

Characterization of Escherichia coli Type 1 Pilus Mutants with Altered Binding Specificities

PCR mutagenesis and a unique enrichment scheme were used to obtain two mutants, each with a single lesion in fimH, the chromosomal gene that encodes the adhesin protein (FimH) of Escherichia coli type 1 pili. These mutants were noteworthy in part because both were altered in the normal range of cell types bound by FimH. One mutation altered an amino acid at a site previously shown to be involved in temperature-dependent binding, and the other altered an amino acid lining the predicted FimH binding pocket.

Unexpected Similarities between Bordetella avium and Other Pathogenic Bordetellae

ABSTRACT Bordetella avium causes an upper respiratory tract disease (bordetellosis) in avian species. Commercially raised turkeys are particularly susceptible. Like other pathogenic members of the genus Bordetella (B. pertussis and B. bronchiseptica) that infect mammals, B. avium binds preferentially to ciliated tracheal epithelial cells and produces similar signs of disease. These similarities prompted us to study bordetellosis in turkeys as a possible nonmammalian model for whooping cough, the exclusively human childhood disease caused by B. pertussis. One impediment to accepting such a host-pathogen model as relevant to the human situation is evidence suggesting that B. avium does not express a number of the factors known to be associated with virulence in the other two Bordetella species. Nevertheless, with signature-tagged mutagenesis, four avirulent mutants that had lesions in genes orthologous to those associated with virulence in B. pertussis and B. bronchiseptica (bvgS, fhaB, fhaC, and fimC) were identified. None of the four B. avium genes had been previously identified as encoding factors associated with virulence, and three of the insertions (in fhaB, bvgS, and fimC) were in genes or gene clusters inferred as being absent or incomplete in B. avium, based upon the lack of DNA sequence similarities in hybridization studies and/or the lack of immunological cross-reactivity of the putative products. We further found that the genotypic arrangements of most of the B. avium orthologues were very similar in all three Bordetella species. In vitro tests, including hemagglutination, tracheal ring binding, and serum sensitivity, helped further define the phenotypes conferred by the mutations. Our findings strengthen the connection between the causative agents and the pathogenesis of bordetellosis in all hosts and may help explain the striking similarities of the histopathologic characteristics of this upper airway disease in avian and mammalian species.

An experimental chain of infection reveals that distinct Borrelia burgdorferi populations are selected in arthropod and mammalian hosts

The prokaryotic, spirochaetal microorganism Borrelia burgdorferi is the causative agent of Lyme disease, an arthropod‐borne disease of a variety of vertebrates and the most prevalent arthropod‐borne disease of humans in the United States. In order to understand better the normal life cycle of B. burgdorferi, an experimental chain of infection was devised that involved multiple sequential arthropod and mammalian passages. By examining populations of B. burgdorferi emerging from different points in this infectious chain, we demonstrate that selection of B. burgdorferi populations peculiar to arthropod or vertebrate hosts is a property of at least one of the two ecologically distinct strains we examined. Distinct B. burgdorferi populations were identified using an antigenic profile, defined by a set of monoclonal antibodies to eight B. burgdorferi antigens, and a plasmid profile, defined by the naturally occurring plasmids in the starting clonal populations. These two profiles constituted the phenotypical signature of the population. In the strain exhibiting selection in the different hosts, transition from one host to another produced a striking series of alternating phenotypical signatures down the chain of infection. At the molecular level, the alternating signatures were manifested as a reciprocal relationship between the expression of certain antigenic forms of outer surface protein (Osp) B and OspC. In the case of OspC, the antigenic changes could be correlated to the presence of one of two distinctly different alleles of the ospC gene in a full‐length and presumably transcriptionally active state. In the case of OspB, two alleles were again identified. However, their differences were minor and their relationship to OspB antigenic variation more complicated. In addition to the reciprocating changes in the antigenic profile, a reciprocating change in the size (probably the multimeric state) of a 9.0 kbp supercoiled plasmid was also noted. Selection of distinct populations in the tick may be responsible for the microorganisms ability to infect a wide range of vertebrate hosts efficiently, in that the tick might provide selective pressure for the elimination of the population selected in the previous host.

Influence of Pregnancy on the Pathogenesis of Listeriosis in Mice Inoculated Intragastrically

ABSTRACT Pregnancy increases the risk of listeriosis, a systemic disease caused by Listeria monocytogenes. However, there is incomplete agreement on the reasons for this increased risk. We examined two features of listeriosis in gravid and nongravid female mice following intragastric (gavage) inoculation, namely, (i) disease severity (measured by lethality) and (ii) listerial infectivity (measured by liver and spleen colonization levels up to 120 h postinoculation). Two listerial strains of differing serotype (1/2a and 4nonb) were initially employed. Neither strain produced a lethal infection in nonpregnant female mice (dose range, 106 to 109 CFU/mouse), and only the 4nonb strain produced lethalities in pregnant mice (dose range, 106 to 108 CFU/mouse). The 4nonb strain also produced a higher level of liver and spleen colonization than the 1/2a strain following gavage administration. (The two strains showed similar levels of colonization if parenterally administered.) Both strains were equally capable of binding to and forming plaques upon cultured mouse enterocytes. The ability of the 4nonb strain to produce a lethal infection in pregnant animals did not correlate with an increased incidence or level of liver and spleen colonization over that in nonpregnant females. However, the lethality rate did correlate well with the rate at which embryos and their surrounding decidual covering became infected, suggesting that intrauterine infection could be responsible for the increased disease severity in the gravid females.

A Listeria monocytogenes Mutant Defective in Bacteriophage Attachment Is Attenuated in Orally Inoculated Mice and Impaired in Enterocyte Intracellular Growth

ABSTRACT A Listeria monocytogenes bacteriophage was used to identify a phage-resistant Tn917 insertion mutant of the mouse-virulent listerial strain F6214-1. The mutant was attenuated when it was inoculated orally into female A/J mice and failed to replicate efficiently in cultured mouse enterocytes. Phage binding studies indicated that the mutant had a cell surface alteration that precluded phage attachment. All phenotypes associated with the mutation could be complemented in trans by a single open reading frame (ORF) that corresponded to the ORF interrupted by the Tn917 insertion. The complementation effected was, in all cases, at a level indistinguishable from that of the parent. The Tn917 insertion interrupted a gene that is predicted to encode a group 2 glycosyl transferase (provisionally designated glcV). A similar glcV gene is present in Listeria welshimeri and Listeria innocua and in some serotypes of L. monocytogenes. We speculate that the loss of the glcV product results in a defective phage receptor and that this alteration coincidentally influences a feature of the normal host-pathogen interaction required for virulence. Interestingly, the glcV lesion, while preventing phage attachment, did not alter the mutants ability to bind to cultured mouse enterocyte monolayers. Rather, the mutation appeared to alter a subsequent step in intracellular replication measured by a reduction in plaque-forming efficiency and plaque size. In vivo, the mutant was undetectable in the liver and spleen 48 h after oral inoculation. The mutation is significant in part because it is one of the few that produce attenuation when the mutant is delivered orally.

A role for lipopolysaccharide in turkey tracheal colonization by Bordetella avium as demonstrated in vivo and in vitro.

We isolated two insertion mutants of Bordetella avium that exhibited a peculiar clumped‐growth phenotype and found them to be attenuated in turkey tracheal colonization. The mutants contained transposon insertions in homologues of the wlbA and wlbL genes of Bordetella pertussis. The wlb genetic locus of B. pertussis has been previously described as containing 12 genes involved in lipopolysaccharide (LPS) biosynthesis. Polyacrylamide gel analysis of LPS from B. avium wlbA and wlbL insertion mutants confirmed an alteration in the LPS profile. Subsequent cloning and complementation of the wlbA and wlbL mutants in trans with a recombinant plasmid containing the homologous wlb locus from B. avium eliminated the clumped‐growth phenotype and restored the LPS profile to that of wild‐type B. avium. Also, a parental level of tracheal colonization was restored to both mutants by the recombinant plasmid. Interestingly, complementation of the wlbA and wlbL mutants with a recombinant plasmid containing the heterologous wlb locus from B. pertussis, B. bronchiseptica, or Bordetella parapertussis eliminated the clumped‐growth phenotype and resulted in a change in the LPS profile, although not to that of wild‐type B. avium. The mutants also acquired resistance to a newly identified B. avium‐specific bacteriophage, Ba1. Complementation of both wlbA and wlbL mutants with the homologous wlb locus of B. avium, but not the heterologous B. pertussis locus, restored sensitivity to Ba1. Complementation of the wlbL mutant, but not the wlbA mutant, with the heterologous wlb locus of Bordetella bronchiseptica or B. parapertussis restored partial sensitivity to Ba1. Comparisons of the LPS profile and phage sensitivity of the mutants upon complementation by wlb loci from the heterologous species and by B. avium suggested that phage sensitivity required the presence of O‐antigen. At the mechanistic level, both mutants showed a dramatic decrease in serum resistance and a decrease in binding to turkey tracheal rings in vitro. In the case of serum resistance, complementation of both mutants with the homologous wlb locus of B. avium restored serum resistance to wild‐type levels. However, in the case of epithelial cell binding, only complementation of the wlbA mutant completely restored binding to wild‐type levels (binding was only partially restored in the wlbL mutant). This is the first characterization of LPS mutants of B. avium at the genetic level and the first report of virulence changes by both in vivo and in vitro measurements

Reply to E.A. Rakha et al

We thank Rakha et al for their correspondence concerning the 2013 American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) human epidermal growth factor receptor 2 (HER2) testing guideline update for invasive breast cancer. We also appreciate their previous thoughtful commentary and concur that the recommendations of the ASCO/CAP Panel were aimed to “improve the analytic validity of HER2 testing, its clinical utility, and the communication among health-care providers.” Improvements in the analytic performance of HER2 testing in clinical practice since 2007 led the Panel to expand its focus beyond earlier concerns to reduce false-positive tests (and increase specificity). Longer-term confirmation of the survival benefit offered by HER2targeted therapy led some investigators to even consider retesting all invasive tumors that test as HER2 negative to reduce false-negative tests (and increase sensitivity). However, the 2013 Update Panel did not support such a general recommendation. Data on improved proficiency in HER2 testing since 2007 suggest that the true frequency of false-negative HER2 test results in clinical practice likely falls below the intrinsic variability of existing HER2 assays in clinical use (Data Supplement 2D, available at http://www.asco.org/quality-guidelines/ recommendations-human-epidermal-growth-factor-receptor-2testing-breast-cancer). In other words, a different HER2 test result could in some cases occur purely by chance. At the same time, the Panel chose to provide further guidance regarding less common clinical scenarios to allow greater discrimination between HER2-positive and -negative results. Since then, many pathologists and oncologists (including members of the Panel) have identified sections of the 2013 guideline document that could benefit from greater clarity. Our response to this correspondence from Rakha et al serves as an opportunity for us to immediately address two specific areas, and the Panel will be issuing a revision of Figure 1 and an update of Table 2 from the 2013 HER2 testing guideline. In Figure 1 (showing the algorithm for HER2 testing by immunohistochemistry [IHC]), IHC 2 (equivocal) was described as invasive breast cancer with “circumferential membrane staining that is incomplete and/or weak/moderate and within 10% of tumor cells.” Many pathologists expressed concern that the terms circumferential and incomplete cannot be reconciled when used together. Consequently, larger number of IHC 1 (HER2 negative) tumors risk being called IHC 2 (equivocal) and submitted for reflex testing. The statement, “circumferential membrane staining that is intense and within 10% of tumor cells,” was also believed to refer to an unusual pattern that did not need to be specified in the main portion of the figure. Therefore, the Panel will be publishing two revisions in Figure 1. First, the definition for IHC 2 in invasive breast cancer will now simply reflect the commonly accepted definition: “a weak to moderate complete membrane staining [that] is observed in 10% of tumor cells.” Second, discussions about possible uncommon IHC scenarios will be limited to the figure legend. The Figure 1 legend will be partly revised to read: “Unusual staining patterns of HER2 by IHC can be encountered that are not covered by these definitions. In practice, these patterns are rare and if encountered should be considered IHC 2 equivocal. As one example, some rare breast cancers (eg, micropapillary carcinomas) show IHC staining that is moderate to intense but incomplete (basolateral or lateral) and can be found to be HER2 amplified. Another example describes circumferential membrane IHC staining that is intense but within 10% of tumor cells.” Table 2 of the 2013 guideline document originally called the attention of the reader to “Histopathologic Features Suggestive of Possible HER2 Test Discordance.” Under “Criteria to Consider,” the word “must” was used to indicate that, on the basis of some criteria (including a tumor grade 3), “If the initial HER2 test result in a core needle biopsy specimen of a primary breast cancer is negative, a new HER2 test must be ordered on the excision specimen . . .” In fact, the Panel had already discussed in the 2013 guideline document (Data Supplement 2D of 2013 guideline) that “Smaller datasets from several investigators appeared to suggest that it might be possible to identify subsets where the level of suspicion of false negativity is markedly raised. However, many of these criteria are consistent with true triple-negative disease, and the Update Committee was unsure whether re-testing was indicated for all such cancers. Also, investigators at the Royal Marsden observed an excellent concordance among 336 patients with early stage breast cancer with paired samples (core biopsy and excision) when tested in both samples for ER [estrogen receptor], PgR [progesterone receptor] and HER2 (using IHC as initial test) and a discordance frequency of 1.8%, 15%, and 1.2%, respectively. Therefore, the Update Committee was unable to identify a specific subgroup that would benefit from mandatory reflex testing if IHC is less than 2 .” In their correspondence, Rakha et al describe their own small institutional experience, several references already mentioned in the 2013 guideline document, and a few other articles. In view of the greater clinical experience that confirms the high concordance in HER2 testing between core and excisional biopsies, the Panel will be publishing an update of Table 2 that will now allow the pathologistandoncologisttoexerciseclinical judgmentandwillnolonger indicate that grade 3 alone suffices as a criterion for mandatory retesting. The revised language will state: “If the initial HER2 test result in a core needle biopsy specimen of a primary breast cancer is negative, a new HER2 test ‘may’ be ordered on the excision specimen . . .” The Panel remains aware of the limitations of efforts to retrospectively assess clinical benefit of HER2-targeted therapy in various subsets within the prospective clinical trials. The Panel maintains its goal to provide physicians and patients with actionable guidance such as when to order a reflex test or a new test, and reaffirms its 2013 intent to err on the side of sensitivity rather than specificity in view of the known benefits and low risks associated with anti-HER2 therapy such as trastuzumab. The Panel continues to maintain that guidelines are living documents that must be evidence based, reflect clinical experience, and evolve as new data become available on clinical outcomes. The Panel JOURNAL OF CLINICAL ONCOLOGY C O R R E S P O N D E N C E VOLUME 33 NUMBER 11 APRIL 1