FOXO1 Suppression is a Determinant of Acquired Lapatinib-Resistance in HER2-Positive Gastric Cancer Cells Through MET Upregulation / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: Lapatinib is a candidate drug for treatment of trastuzumab-resistant, human epidermal growth factor receptor 2 (HER2)–positive gastric cancer (GC). Unfortunately, lapatinib resistance renders this drug ineffective. The present study investigated the implication of forkhead box O1 (FOXO1) signaling in the acquired lapatinib resistance in HER2-positive GC cells. MATERIALS AND METHODS: Lapatinib-resistant GC cell lines (SNU-216 LR2-8) were generated in vitro by chronic exposure of lapatinib-sensitive, HER2-positive SNU-216 cells to lapatinib. SNU-216 LR cells with FOXO1 overexpression were generated by stable transfection of a constitutively active FOXO1 mutant (FOXO1A3). HER2 and MET in SNU-216 LR cells were downregulated using RNA interference. The sensitivity of GC cells to lapatinib and/or cisplatin was determined by crystal violet assay. In addition, Western blot analysis, luciferase reporter assay and reverse transcription–polymerase chain reaction were performed. RESULTS: SNU-216 LR cells showed upregulations of HER2 and MET, but downregulation of FOXO1 compared to parental SNU-216 cells. FOXO1 overexpression in SNU-216 LR cells significantly suppressed resistance to lapatinib and/or cisplatin. In addition, FOXO1 negatively controlled HER2 and MET at the transcriptional level and was negatively controlled by these molecules at the post-transcriptional level. A positive crosstalk was shown between HER2 and MET, each of which increased resistance to lapatinib and/or cisplatin. CONCLUSION: FOXO1 serves as an important linker between HER2 and MET signaling pathways through negative crosstalks and is a key regulator of the acquired lapatinib resistance in HER2-positive GC cells. These findings provide a rationale for establishing a novel treatment strategy to overcome lapatinib resistance in a subtype of GC patients.

Clinical Implications According to Diagnostic Methods of Human Epidermal Growth Factor Receptor 2 Positivity in Breast Cancer: A Retrospective Study

PURPOSE: According to American Society of Clinical Oncology/College of American Pathologists guidelines, breast cancer is human epidermal growth factor receptor 2 (HER2) positive if there is HER2 protein overexpression at a 3+ level on immunohistochemistry (IHC 3+) or gene amplification (more than six copies per nucleus) on fluorescence in situ hybridization (FISH+). However, there have been few reports on whether outcomes differ based on diagnosis by these two techniques. In this study, we compared outcomes based on the two methods in patients with HER2-positive breast cancer. METHODS: This study was a retrospective analysis of HER2-positive breast cancer in 18,304 patients, including 14,652 IHC 3+ patients and 3,652 FISH+ patients from the Korean Breast Cancer Society Registry. We compared breast cancer-specific survival and overall survival based on IHC 3+ and FISH+ status with or without trastuzumab. RESULTS: Breast cancer-specific survival was significantly different between the IHC 3+ and FISH+ groups, with 5-year cumulative survival rates of 95.0% for IHC 3+ and 98.5% for FISH+ patients who did not receive trastuzumab (p=0.001) in Kaplan-Meier methods. However, there were no significant differences in breast cancer-specific survival and overall survival between IHC 3+ and FISH+ groups regardless of trastuzumab treatment in Cox proportional hazards models. CONCLUSION: The survival outcomes were not affected by the different two diagnostic methods of HER2-positive breast cancer. Further research to evaluate differences in prognosis and other characteristics according to the diagnostic methods of HER2 positivity is needed in the future.

PIK3CA Mutations and Neoadjuvant Therapy Outcome in Patients with Human Epidermal Growth Factor Receptor 2-Positive Breast Cancer: A Sequential Analysis / 한국유방암학회지

PURPOSE: PIK3CA mutation is considered to be a possible cause for resistance to neoadjuvant chemotherapy (NAC) in human epidermal growth factor receptor 2 (HER2)-positive breast cancer. We investigated the association between PIK3CA mutations and the outcome of NAC in HER2-positive breast cancers. METHODS: A total of 100 HER2-positive breast cancer patients who had undergone NAC and surgery between 2004 and 2016 were examined. Mutation status was sequentially assessed in pre-NAC, post-NAC, and recurrent specimens taken from these patients. RESULTS: PIK3CA mutations were identified in the sequential specimens of 17 patients (17.0%). These 17 patients experienced shorter disease-free survival (DFS) than the rest of the patients (58.3 months vs. 119.3 months, p=0.020); however, there was no significant difference in pathologic complete response (pCR) and overall survival (OS) (pCR, 17.6% vs. 33.7%, p=0.191; OS, 84.5 months vs. 118.0 months, p=0.984). While there was no difference in pCR between the wild-type and mutant PIK3CA groups in pre-NAC specimens (25.0% vs. 31.8%, p=0.199), PIK3CA mutations correlated with lower pCR in post-NAC specimens (0.0% vs. 24.3%, p < 0.001). Multivariate analysis revealed significantly worse DFS in the mutant PIK3CA group than in the wild-type group (hazard ratio, 3.540; 95% confidence interval, 1.001–12.589; p=0.050). Moreover, the DFS curves of the change of PIK3CA mutation status in sequential specimens were significantly different (p=0.016). CONCLUSION: PIK3CA mutation in HER2-positive breast cancer was correlated with a lower pCR rate and shorter DFS. These results suggest that PIK3CA mutation is a prognostic marker for NAC in HER2-positive breast cancer, especially in post-NAC specimens.

Clinical Differences in Triple-Positive Operable Breast Cancer Subtypes in Korean Patients: An Analysis of Korean Breast Cancer Registry Data / 한국유방암학회지

PURPOSE: Triple-positive breast cancer is defined by estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 (HER2) positivity. Several systemic breast cancer therapies target hormonal and HER2 responsiveness. We compared clinical outcomes of triple-positive disease with those of HER2-enriched and luminal HER2-negative disease and investigated the clinical efficacy of anti-HER2 therapy for triple-positive disease. METHODS: We retrospectively compared overall and recurrence-free survival among cases included in the Korean Breast Cancer Society (KBCS) and Seoul St. Mary's Hospital breast cancer registries and the therapeutic efficacy of trastuzumab for triple-positive and HER2-enriched cases. RESULTS: KBCS registry data (2006–2010; median follow-up, 76 months) indicated that patients with triple-positive breast cancer had intermediate survival between those with luminal A and HER2-enriched subtypes (p < 0.001). Trastuzumab did not improve overall survival among patients with triple-positive breast cancer (p=0.899) in contrast to the HER2-enriched subtype (p=0.018). Seoul St. Mary's Hospital registry data indicated similar recurrence-free survival outcomes (p < 0.001) and a lack of improvement with trastuzumab among patients with triple-positive breast cancer (median follow-up, 33 months; p=0.800). Multivariate analysis revealed that patients with triple-positive breast cancer had better overall survival than those with HER2-enriched disease and similar survival as those with the luminal A subtype (triple-positive: hazard ratio, 1.258, p=0.118; HER2-enriched: hazard ratio, 2.377, p < 0.001). CONCLUSION: Our findings showed that anti-HER2 therapy was less beneficial for treatment of triple-positive breast cancer than for HER2-enriched subtypes of breast cancer, and the triple-positive subtype had a distinct prognosis.

FcrR3A-158 Polymorphism and Stromal Tumor-Infiltrating Lymphocytes and Survival among Patients with Metastatic HER2-Positive Breast Cancer Receiving Trastuzumab-Based Treatment / 한국유방암학회지

PURPOSE: The prognosis of human epidermal growth factor receptor 2 (HER2)-positive breast cancer has markedly improved since the introduction of trastuzumab. We aimed to evaluate the association between stromal tumor-infiltrating lymphocyte (sTIL) or FcrR polymorphisms and survival among patients with metastatic HER2-positive breast cancer who were treated with trastuzumab. METHODS: A total of 56 women with recurrent or metastatic HER2-positive breast cancer who received the trastuzumab-taxane combination as first-line treatment were included in this retrospective analysis. The single-step multiplex allele-specific real-time polymerase chain reaction technique was employed for FcrR3A genotyping. sTILs were identified via immunohistochemical analysis of surgical (n=34, 60.7%) or biopsy specimens of metastatic lesions (n=22, 39.3%). RESULTS: We classified patients based on the sTIL level (≤10% [n=44] or >10% [n=12]); high sTIL counts were more commonly observed in patients with hormone receptor-negative tumors than in those with hormone receptor-positive tumors (34.8% vs. 12.1%, p=0.02). There was a significant association between high sTIL levels and longer progression-free survival in comparison to low sTIL levels (median, 28.4 months vs. 16.8 months; p=0.03). With regard to the FcrR3A-158 genotype, patients were classified into the Phenylalanine/Phenylalanine group (23 patients, 41.1%), Phenylalanine/Valine group (23 patients, 41,1%), or Valine/Valine group (10 patients, 17.9%); these classifications were not associated with clinical outcomes. CONCLUSION: High sTIL expression may be associated with better efficacy of trastuzumab-containing therapy in patients with metastatic HER2-positive breast cancer. However, this finding warrants further evaluation in the larger population.

A Retrospective Analysis for Patients with HER2-Positive Gastric Cancer Who Were Treated with Trastuzumab-Based Chemotherapy: In the Perspectives of Ethnicity and Histology / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: While the Trastuzumab for Gastric Cancer (ToGA) trial demonstrated the efficacy and safety of trastuzumab-based chemotherapy in HER2-positive metastatic gastric cancer, the overall survival (OS) benefit was not found in Asian and diffuse-type cancer patients. The aim of the study is to investigate predictive markers for trastuzumab-based chemotherapy. MATERIALS AND METHODS: Data of patients with HER2-positive gastric cancer treated with trastuzumab-based chemotherapy were analyzed retrospectively. RESULTS: A total of 168 Asian patients were included. The median age was 60 years (range, 27 to 85 years) and the male:female ratio was 118 (70.2%):50 (29.8%). Fourteen (8.3%), 63 (37.5%), 75 (44.6%), and 11 (6.5%) patients had well, moderately, poorly-differentiated tubular adenocarcinoma and signet ring cell carcinoma, respectively. With 14 complete responses and 73 partial responses, the response rate was 50.6%. The median progression-free survival (PFS) was 10.2 months (95% confidence interval [CI], 8.7 to 11.7), and the median OS was 18.5 months (95% CI, 16.4 to 50.6). Next, we investigated the effect of poorly-differentiated histology (PDH, poorly-differentiated tubular adenocarcinoma+signet ring cell carcinoma) on clinical outcomes. The median PFS (8.9 months vs. 11.5 months, p=0.16) was slightly inferior in PDH patients, and the median OS was significantly shorter in PDH patients (14.6 months vs. 19.0 months, p=0.025). CONCLUSION: While subset analysis of the ToGA trial demonstrated that trastuzumab-based chemotherapy may not be beneficial for Asians and patients with PDH, our data may suggest that even in Asian patients and patients with PDH, trastuzumab-based chemotherapy could be associated with improved clinical outcomes in patients with HER2-positive gastric cancer.

Brain metastasis in human epidermal growth factor receptor 2-positive breast cancer: from biology to treatment

Overexpression of human epidermal growth factor receptor 2 (HER2) is found in about 20% of breast cancer patients. With treatment using trastuzumab, an anti-HER2 monoclonal antibody, systemic control is improved. Nonetheless, the incidence of brain metastasis does not be improved, rather seems to be increased in HER2-positive breast cancer. The mainstay treatment for brain metastases is radiotherapy. According to the number of metastatic lesions and performance status of patients, radiosurgery or whole brain radiotherapy can be performed. The concurrent use of a radiosensitizer further improves intracranial control. Due to its large molecular weight, trastuzumab has a limited ability to cross the blood-brain barrier. However, small tyrosine kinase inhibitors such as lapatinib, has been noted to be a promising agent that can be used as a radiosensitizer to affect HER2-positive breast cancer. This review will outline general management of brain metastases and will focus on preclinical findings regarding the radiosensitizing effect of small molecule HER2 targeting agents.

Examining ERBB2 as a candidate gene for susceptibility to leprosy (Hansen’s disease) in Brazil

Leprosy remains prevalent in Brazil. ErbB2 is a receptor for leprosy bacilli entering Schwann cells, which mediates Mycobacterium leprae-induced demyelination and the ERBB2 gene lies within a leprosy susceptibility locus on chromosome 17q11-q21. To determine whether polymorphisms at the ERBB2 locus contribute to this linkage peak, three haplotype tagging single nucleotide polymorphisms (tag-SNPs) (rs2517956, rs2952156, rs1058808) were genotyped in 72 families (208 cases; 372 individuals) from the state of Pará (PA). All three tag-SNPs were associated with leprosy per se [best SNP rs2517959 odds ratio (OR) = 2.22; 95% confidence interval (CI) 1.37-3.59; p = 0.001]. Lepromatous (LL) (OR = 3.25; 95% CI 1.37-7.70; p = 0.007) and tuberculoid (TT) (OR = 1.79; 95% CI 1.04-3.05; p = 0.034) leprosy both contributed to the association, which is consistent with the previous linkage to chromosome 17q11-q21 in the population from PA and supports the functional role of ErbB2 in disease pathogenesis. To attempt to replicate these findings, six SNPs (rs2517955, rs2517956, rs1810132, rs2952156, rs1801200, rs1058808) were genotyped in a population-based sample of 570 leprosy cases and 370 controls from the state of Rio Grande do Norte (RN) and the results were analysed using logistic regression analysis. However, none of the associations were replicated in the RN sample, whether analysed for leprosy per se, LL leprosy, TT leprosy, erythema nodosum leprosum or reversal reaction conditions. The role of polymorphisms at ERBB2 in controlling susceptibility to leprosy in Brazil therefore remains unclear.

Compensatory activation of estrogen receptor α signaling in acquired resistance to lapatinib of HER2-overexpressing/ERα-positive breast cancer cells / 第二军医大学学报

Objective To investigate the role of compensatory activation of estrogen receptor α (ERα) signaling in acquired resistance to lapatinib of breast cancer cells BT474 and the related mechanism. Methods Real-time PCR and Western blotting analysis were used to determine the changes of HER2 and ERα pathways in BT474 cells treated by lapatinib. Acquired resistant model of rBT474 cellswas induced with increasing concentrations of lapatinib (from 0. 25 μmol/L to 5 μmol/L); the apoptosis in rBT474 cells was determined by flow cytometry. Western blotting analysis was used to evaluate the differences between BT474 and rBT474 in the HER2 and ER pathways. The growth of rBT474 cells treated by lapatinib and/or fulvestrant was detected by MTT assay, andcolony formation was used to observe the possibility of preventing acquired resistance to lapatinib in BT474 cells by double targets therapy. Results The results of real-time PCR and Western blotting analysis showed that Lapatinib inhibited phosphorylation of HER2 and induced expression of forkhead-box protein O 3A (F0X03a) and progesterone receptor (PR) in BT474 cells. Acquired resistance cell model of rBT474 was established in a 5 μmol/L lapatinib condition. Western blotting analysis showed that the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) pathway was inhibited in rBT474 cells compared with that in the BT474 cells, while the mitogen-activated protein kinase (MAPK) pathways, especially the ER pathway, were activated in the BT474 cells. MTT results showed that, Lapatinib combined with fulvestrant had a significantly greater inhibition on rBT474 cell vitality compared with lapatinib alone (P<0. 01). Colony formation results also showed that combination of lapatinib and fulvestrant had a significantly greater inhibition effect against colon formation of BT474 cells compared with each drug alone and DMSO (P<0. 01), showing a possible prevention ability against acquired resistance. Conclusion Compensatory activation of estrogen receptor a signaling might be one of the mechanisms of acquired resistance to lapatinib in HER2-overexpressing/ERcrpositive breast cancer cells, and inhibition of PI3K/AKT and activation of MAPK might be the main reason for compensatory activation of ERα.

Perfil imuno-histoquímico e variáveis clinicopatológicas no câncer de mama / Immunohistochemical profile and clinical-pathological variables in breast cancer

OBJETIVO: Descrever as principais características em mulheres com câncer de mama, de acordo com o perfil imuno-histoquímico. MÉTODOS: A população foi composta a partir de coorte hospitalar formada por mulheres com diagnóstico de câncer de mama efetuado entre 2003 e 2005 (n = 601) e atendidas em centro de referência em assistência oncológica de Juiz de Fora-MG. Foram selecionadas apenas 397 mulheres que possuíam imunohistoquímica completa. Para definição dos grupos segundo perfil imuno-histoquímico, optou-se por classificação baseada na avaliação dos receptores de estrógeno e de progesterona, índice de proliferação celular Ki67 e superexpressão de HER2. De acordo com os diferentes fenótipos, foram definidos cinco subtipos: luminal A, luminal B-HER2 negativo, luminal B-HER2 positivo, triplo negativo e HER2 superexpresso. RESULTADOS: A maioria dos pacientes tinha pele branca (80,7%) e era pós-menopausada (64,9%), com idade média de 57,4 anos (±13,5). Ao diagnóstico, 57,5% delas tinha tumor de tamanho > 2,0 cm, e 41,7% exibiam comprometimento linfonodal. Os subtipos mais frequentes foram luminal B-HER2 negativo (41,8%) e triplo negativo (24,2%). No subtipo luminal A, 72,1% das pacientes eram pós-menopausadas, enquanto que os maiores percentuais na pré-menopausa foram observados nos subtipos luminal B-HER2 positivo e triplo negativo (45,2% e 44,2%, respectivamente). Verificou-se maior frequência de tumores > 2,0 cm e com linfonodos comprometidos nos subtipos triplo negativo e HER2 positivo. CONCLUSÃO: Esta pesquisa possibilitou avaliar a distribuição das principais características clinicopatológicas e relacionadas aos serviços de saúde em coorte de mulheres brasileiras com câncer de mama, segundo os subtipos tumorais imuno-histoquímicos.

OBJECTIVE: To describe the main characteristics of women with breast cancer, according to the immunohistochemical profile. METHODS: The population comprised a hospital cohort, consisting of women diagnosed with breast cancer between 2003 and 2005 (n = 601) and treated at a referral center for cancer care in Juiz de Fora, MG, Brazil. Only 397 women who had complete immunohistochemistry analysis were selected. To define the groups according to the immunohistochemical profile, the assessment of estrogen and progesterone receptors, Ki-67 cell proliferation index, and overexpression of human epidermal growth factor receptor 2 (HER2) was chosen. According to the different phenotypes, five subtypes were defined: luminal A, luminal B HER2 negative, luminal B HER2 positive, triple negative, and HER2 overexpression. RESULTS: Most patients were white (80.7%) and post-menopausal (64.9%), with a mean age of 57.4 years (± 13.5). At diagnosis, 57.5% had tumor size > 2.0 cm, and 41.7% had lymph node involvement. The most common subtypes were luminal B - HER2 negative (41.8%) and triple negative (24.2%). In the luminal A subtype, 72.1% of patients were post-menopausal, while the highest percentage of premenopausal women were observed in the luminal B - HER2 positive and triple negative subtypes (45.2% and 44.2%, respectively). A higher frequency of tumors > 2.0 cm and lymph node involvement was observed in triple negative and HER2 positive subtypes. CONCLUSION: This study allowed the distribution assessment of the main clinical and pathological characteristics and those related to health services in a cohort of Brazilian women with breast cancer, according to the immunohistochemical tumor subtypes.

Patterns of Recurrence after Breast-Conserving Treatment for Early Stage Breast Cancer by Molecular Subtype / 한국유방암학회지

PURPOSE: To study clinical features and patterns of recurrence after breast-conserving treatment (BCT) for three molecular subtypes of early stage breast cancer. METHODS: The sample studied included 596 patients with T1-2N0-1 breast cancer who received BCT. Three groups were defined by receptor status. Luminal: estrogen receptor (ER) or progesterone receptor (PR) positive; triple negative (TN): ER, PR, and epidermal growth factor receptor-2 (HER2) receptor negative; and HER2 overexpressing: ER and PR negative but HER2 receptor positive. RESULTS: The number of patients in each group was 408 (68.5%), 105 (17.6%), and 83 (13.9%), respectively. The median follow-up period was 79 months. The TN and HER2 subtypes occurred in younger patients (p=0.0007) and had higher nuclear grade and poorer histologic grade (p<0.0001 and 0.0071, respectively). During the follow-up period, locoregional recurrence was detected as the first site of recurrence in 26 (6.4%), 11 (10.5%), and 9 (10.8%) patients in the luminal, TN, and HER2 subtypes, respectively (p=0.1924). Thirty-one (7.6%), 7 (6.7%), and 7 (8.4%) patients in each group had distant metastases as the first sign of recurrence (p=0.8996). Median time to locoregional and distant recurrence was shorter in the HER2 subtype (p=0.0889 and 0.0780, respectively), and the HER2 subtype was significantly associated with poor overall survival (p=0.0009). CONCLUSION: After BCT in Korean women with early stage breast cancer, the patterns of recurrence were not different among the molecular subtypes, although the TN and HER2 subtypes were associated with younger age, higher nuclear grade, and poorer histologic grade.

Branched-chain Assay for ER, PR, and HER2 RNA Levels is a Useful Adjunct in the Evaluation of ER, PR, and HER2 in Breast Cancer / 한국유방암학회지

PURPOSE: The status of estrogen receptor (ER), progesterone receptor (PR) and HER2 is critical in patients undergoing breast cancer treatment. We performed this study to evaluate the QuantiGene Reagent System as a clinical test for detecting ER, PR, and HER2 RNA levels in formalin-fixed, paraffin-embedded (FFPE) tissue. METHODS: The RNA levels of ER, PR and HER2 were measured using the QuantiGene2.0 assay in FFPE tissue from breast cancer patients (n=40) and compared with ER and PR immunohistochemistry results and HER2 fluorescence in situ bybridization (FISH) results. RESULTS: When the cut-off values for ER, PR, and HER2 RNA levels were 5.0, 7.2, and 50, respectively, the sensitivity, specificity, and positive and negative predictive value of the QuantiGene 2.0 Assay were 96.6%, 90%, 96.7%, and 90%, for ER; 89.7%, 81.8%, 92.9%, and 75% for PR; and 83.3%, 96.4%, 90.9%, and 93.1% for HER2, respectively. The Allred scores for ER and PR as well as the HER2 FISH ratio were correlated with RNA levels (p=0.046, r=0.32; p<0.001, r=0.61; p<0.001, r=0.75, respectively). CONCLUSION: We demonstrated that the ER, PR, and HER2 RNA levels as measured by the QuantiGene 2.0 assay were reproducible and correlated well with immunohistochemistry and FISH results. Measuring ER, PR, and HER2 RNA levels from formalin-fixed, paraffin-embedded tissue using the QuantiGene 2.0 assay, which was a relatively simple technique easily performed in a usual laboratory, appeared to a helpful adjunct in determining the status of ER, PR, and HER2 in breast cancer.

Comparison of Silver-Enhanced in situ Hybridization and Fluorescence in situ Hybridization for HER2 Gene Status in Breast Carcinomas / 한국유방암학회지

PURPOSE: Silver-enhanced in situ hybridization (SISH) is a newly developed method to evaluate HER2 gene amplification in invasive breast carcinomas. Most laboratories widely use fluorescence in situ hybridization (FISH) to evaluate the HER2 gene amplification status because FISH is a very sensitive and accurate technique. However, this technique is not the best because it requires specialized equipment and interpretation skills. We compared a new technique of SISH with FISH for assessing HER2 gene amplification in invasive breast carcinomas. METHODS: HER2 gene amplification was assessed in 165 cases of invasive breast carcinoma by FISH and SISH with constructing a tissue microarray. The tumors were assessed by the guidelines of the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP). Positivity was defined as a HER2/Chromosome 17 ratio greater than 2.2. Negativity was defined if the ratio was less than 1.8. The tumor was considered as equivocal for HER2 gene amplification if the ratio was between 1.8 and 2.2. The HER2 protein status was assessed. Immunostaining for HER2 protein was performed in a Benchmark automatic immunostaining device with using whole tissue sections. RESULTS: There was agreement of the HER2 gene amplification status by SISH and FISH in 162 of 165 cases, which is a concordance rate of 98.2% (kappa=0.94). There were three discrepant cases, with two of them being FISH positive and SISH negative (one case was IHC negative and one case was IHC positive) and one case was FISH negative and SISH equivocal. CONCLUSION: The 98.2% concordance between FISH and SISH meets the ASCO/CAP requirements for test validation of >95% concordance. These results indicate that SISH can be used as an alternative to FISH for assessing the HER2 gene amplification status in breast carcinomas.