Unraveling the clinicopathological and molecular changes induced by neoadjuvant chemotherapy and endocrine therapy in hormone receptor-positive/HER2-low and HER2-0 breast cancer.

BACKGROUND: The characterization and comparison of gene expression and intrinsic subtype (IS) changes induced by neoadjuvant chemotherapy (NACT) and endocrine therapy in hormone receptor-positive (HR+)/human epidermal growth factor receptor 2 (HER2)-low versus HR+/HER2-0 breast cancer (BC) has not been conducted so far. Most evidence on the association of HER2 status with pathologic responses and prognosis in HR+/HER2-negative BC is controversial and restricted to NACT-treated disease. Similarly, a temporal heterogeneity in HER2 status has been described only with NACT. METHODS: We retrospectively recruited a consecutive cohort of 186 patients with stage I-IIIB HR+/HER2-negative BC treated with neoadjuvant therapy (NAT). Available diagnostic biopsies and surgical samples were characterized for main pathological features, PAM50 IS and ROR-P score, and gene expression. Associations with pathologic complete response, residual cancer burden-0/I, event-free survival (EFS) and overall survival (OS) based on HER2 status were assessed. Pre/post pathologic/molecular changes were analyzed in matched samples. RESULTS: The HER2-low (62.9%) and HER2-0 (37.1%) cohorts did not differ significantly in main baseline features, treatments administered, breast-conserving surgery, pathologic complete response and residual cancer burden-0/I rates, EFS, and OS. NAT induced, regardless of HER2 status, a significant reduction of estrogen receptor/progesterone receptor and Ki67 levels, a down-regulation of PAM50 proliferation- and luminal-related genes/signatures, an up-regulation of selected immune genes, and a shift towards less aggressive IS and lower ROR-P. Moreover, 25% of HER2-0 changed to HER2-low and 34% HER2-low became HER2-0. HER2 shifts were significant after NACT (P < 0.001), not neoadjuvant endocrine therapy (P = 0.063), with consistent ERBB2 mRNA level dynamics. HER2 changes were not associated with EFS/OS. CONCLUSIONS: HER2-low and HER2-0 status change after NAT in ∼30% of cases, mostly after NACT. Targeted adjuvant strategies should be investigated accordingly. Molecular downstaging with current chemo/endocrine agents and immunotherapy should not rely on HER2 immunohistochemical levels in HR+/HER2-negative BC. Instead, HER2-low-targeted approaches should be explored to pursue more effective and/or less toxic dimensional downstaging.

Prognostic value of HER2-low status in breast cancer: a systematic review and meta-analysis.

BACKGROUND: Human epidermal growth factor receptor 2 (HER2)-low expression in breast cancer has been recently identified as a new therapeutic target. However, it is unclear if HER2-low status has an independent impact on prognosis. MATERIALS AND METHODS: A systematic literature research was carried out to identify studies comparing survival outcomes of patients affected by HER2-low versus HER2-zero breast cancer. Using random-effects models, pooled hazard ratios (HRs) and odds ratios (ORs) with 95% confidence intervals (CIs) were calculated for progression-free survival (PFS) and overall survival (OS) in the metastatic setting as well as disease-free survival (DFS), OS and pathological complete response (pCR) in the early setting. Subgroup analyses by hormone receptor (HoR) status were carried out. The study protocol is registered on PROSPERO (n.CRD42023390777). RESULTS: Among 1916 identified records, 42 studies including 1 797 175 patients were eligible. In the early setting, HER2-low status was associated with significant improved DFS (HR 0.86, 95% CI 0.79-0.92, P < 0.001) and OS (HR 0.90, 95% CI 0.85-0.95, P < 0.001) when compared to HER2-zero status. Improved OS was observed for both HoR-positive and HoR-negative HER2-low populations, while DFS improvement was observed only in the HoR-positive subgroup. HER2-low status was significantly associated with a lower rate of pCR as compared to HER2-zero status both in the overall population (OR 0.74, 95% CI 0.62-0.88, P = 0.001) and in the HoR-positive subgroup (OR 0.77, 95% CI 0.65-0.90, P = 0.001). In the metastatic setting, patients with HER2-low breast cancers showed better OS when compared with those with HER2-zero tumours in the overall population (HR 0.94, 95% CI 0.89-0.98, P = 0.008), regardless of HoR status. No significant PFS differences were found. CONCLUSIONS: Compared with HER2-zero status, HER2-low status appears to be associated with a slightly increased OS both in the advanced and early settings, regardless of HoR expression. In the early setting, HER2-low tumours seem to be associated to lower pCR rates, especially if HoR-positive.

Prognostic value of intrinsic subtypes in hormone-receptor-positive metastatic breast cancer: systematic review and meta-analysis.

BACKGROUND: In hormone receptor-positive (HoR+) breast cancer (BC), gene expression analysis identifies luminal A (LumA), luminal B (LumB), human epidermal growth factor receptor 2 (HER2)-enriched (HER2-E), basal-like (BL) intrinsic subtypes and a normal-like group. This classification has an established prognostic value in early-stage HoR+ BC. Here, we carried out a trial-level meta-analysis to determine the prognostic ability of subtypes in metastatic BC (MBC). MATERIALS AND METHODS: We systematically reviewed all the available prospective phase II/III trials in HoR+ MBC where subtype was assessed. The primary endpoint was progression-free survival (PFS)/time to progression (TTP) of the LumA subtype compared to non-LumA. Secondary endpoints were PFS/TTP of each individual subtype, according to treatment, menopausal and HER2 status and overall survival (OS). The random-effect model was applied, and heterogeneity assessed through Cochran's Q and I2. Threshold for significance was set at P < 0.05. The study was registered in PROSPERO (ID: CRD42021255769). RESULTS: Seven studies were included (2536 patients). Non-LumA represented 55.2% and was associated with worse PFS/TTP than LumA [hazard ratio (HR) 1.77, P < 0.001, I2 = 61%], independently of clinical HER2 status [Psubgroup difference (Psub) = 0.16], systemic treatment (Psub = 0.96) and menopausal status (Psub = 0.12). Non-LumA tumors also showed worse OS (HR 2.00, P < 0.001, I2 = 65%), with significantly different outcomes for LumB (PFS/TTP HR 1.46; OS HR 1.41), HER2-E (PFS/TTP HR 2.39; OS HR 2.08) and BL (PFS/TTP HR 2.67; OS HR 3.26), separately (PFS/TTP Psub = 0.01; OS Psub = 0.005). Sensitivity analyses supported the main result. No publication bias was observed. CONCLUSIONS: In HoR+ MBC, non-LumA disease is associated with poorer PFS/TTP and OS than LumA, independently of HER2, treatment and menopausal status. Future trials in HoR+ MBC should consider this clinically relevant biological classification.

Identification of cell surface targets for CAR-T cell therapies and antibody-drug conjugates in breast cancer.

BACKGROUND: Two promising therapeutic strategies in oncology are chimeric antigen receptor-T cell (CAR-T) therapies and antibody-drug conjugates (ADCs). To be effective and safe, these immunotherapies require surface antigens to be sufficiently expressed in tumors and less or not expressed in normal tissues. To identify new targets for ADCs and CAR-T specifically targeting breast cancer (BC) molecular and pathology-based subtypes, we propose a novel in silico strategy based on multiple publicly available datasets and provide a comprehensive explanation of the workflow for a further implementation. METHODS: We carried out differential gene expression analyses on The Cancer Genome Atlas BC RNA-sequencing data to identify BC subtype-specific upregulated genes. To fully explain the proposed target-discovering methodology, as proof of concept, we selected the 200 most upregulated genes for each subtype and undertook a comprehensive analysis of their protein expression in BC and normal tissues through several publicly available databases to identify the potentially safest and viable targets. RESULTS: We identified 36 potentially suitable and subtype-specific tumor surface antigens (TSAs), including fibroblast growth factor receptor-4 (FGFR4), carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6), GDNF family receptor alpha 1 (GFRA1), integrin beta-6 (ITGB6) and ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1). We also identified 63 potential TSA pairs that might be appropriate for co-targeting strategies. Finally, we validated subtype specificity in a cohort of our patients, multiple BC cell lines and the METABRIC database. CONCLUSIONS: Overall, our in silico analysis provides a framework to identify novel and specific TSAs for the development of new CAR-T and antibody-based therapies in BC.

T-DM1 versus pertuzumab, trastuzumab and a taxane as first-line therapy of early-relapsed HER2-positive metastatic breast cancer: an Italian multicenter observational study.

BACKGROUND: The current standard first-line treatment of human epidermal growth factor receptor 2 (HER2)-positive (+) metastatic breast cancer is the combination of pertuzumab, trastuzumab and a taxane (P + T + taxane), while standard second-line is ado-trastuzumab-emtansine (T-DM1). The registration trial of pertuzumab, however, did not include early-relapsing patients, defined as patients experiencing tumor relapse ≤12 months from the end of (neo)adjuvant anti-HER2 therapy. Conversely, the pivotal trial of T-DM1 included some patients relapsing ≤6 months after the end of (neo)adjuvant trastuzumab. Thus, a proportion of early-relapsing patients are currently eligible to receive T-DM1 as first-line treatment. Nevertheless, no direct comparison exists between the two regimens in this clinical setting. PATIENTS AND METHODS: We retrospectively compared T-DM1 versus P + T + taxane as first-line treatment in two cohorts of early-relapsing patients in an Italian 'real-world' setting, involving 14 public health care institutions. The primary endpoint was progression-free survival. Secondary endpoints included patients' characterization, overall survival and post-progression survival. Univariate and multivariate analyses were carried out. All tests were two-sided and a P ≤ 0.05 was considered statistically significant. RESULTS: Among 1252 screened patients, 75 met the inclusion criteria. Forty-four (58.7%) received P + T + taxane and 31 (41.3%) received T-DM1. The two cohorts showed similar characteristics of aggressiveness and no significant differences in treatment history. T-DM1, compared with P + T + taxane was associated with worse progression-free survival (adjusted hazard ratio: 2.26, 95% confidence interval: 1.13-4.52, P = 0.021) and overall survival (adjusted hazard ratio: 3.95, 95% confidence interval: 1.38-11.32, P = 0.010), irrespective of previous (neo)adjuvant treatment, age, hormone receptors status, time-to-relapse (≤6 months or within 6-12 months) and presence of visceral/brain metastases. No differences were observed in post-progression survival (P = 0.095). CONCLUSIONS: Our study suggests superiority for P + T + taxane over T-DM1 as up-front treatment of early-relapsing HER2+ metastatic breast cancer, which merits further assessment in larger and prospective trials.

Thrombin generation in children with acute lymphoblastic leukemia: effect of leukemia immunophenotypic subgroups.

Elevated plasma concentrations of endogenous thrombin generation markers and thrombotic events have been reported in children with leukemia. The aim of this study was to evaluate the effects of cancer and its treatment on thrombin generation (TAT levels) in children with acute lymphoblastic leukemia (ALL). The authors evaluated 32 children (23 M, 9 F) aged between 1 and 15 years (mean 6) affected by ALL (immunophenotypic subgroups: 16 common, 7 T, and 9 pre-B type). In all patients TAT levels at onset and after 5-6 doses of L-asparaginase were evaluated. TAT levels were higher in patients both at onset (13.04 +/- 10.90 ng/L) and after the 5-6 doses of L-asp (19.41 +/- 11.05 ng/L) with respect to controls (4 +/- 1 ng/L) (p < .001 and p < .001). TAT levels after 5-6 doses of L-asp were higher than those at onset (p < .001). Factorial ANOVA showed that at onset there was a significant effect of leukemia immunophenotypic subgroups upon TAT levels (p < .05) and no effect of inherited thrombotic risk factors. These results indicate that in children with ALL an important role is played by acquired thrombotic risk factors, among which the indirect cancer procoagulant activity has its importance.

Malignancy: X-Linked Cytochrome B Positive Chronic Granulomatous Disease treated by Bone Marrow Transplantation.

Chronic granulomatous disease is caused by a genetic defect in the oxidase of phagocytic cells which results in increased susceptibility to recurrent infections. Conventional treatment includes the use of antimicrobials and interpheron-gamma. This study was performed to assess the clinical efficacy of allogeneic bone marrow transplantation in definitively correcting the functional underlying defect of chronic granulomatous disease. An 8-year-old boy with a rare type X-linked cytochrome b positive chronic granulomatous disease underwent allogeneic bone marrow transplantation after conditioning with busulfan and cyclophosphamide. The patient's HLA identical sister was marrow donor. The post-transplant outcome was uneventful. During the 9 year follow-up period the patient has been constantly free of infections, maintained an excellent clinical performance with full correction of the granulocyte functional defect. This case confirms that allogeneic bone marrow transplantation is the only treatment capable to cure chronic granulomatous disease to those patients who cannot be optimally treated with conventional therapy.

rhG-CSF in severe chronic neutropenia: successful intermittent treatment in three infants.

We report three cases of infants affected by severe chronic neutropenia (SCN). All the patients were treated with recombinant human granulocyte colony-stimulating factor (rhG-CSF) to avoid or reduce recurrent fevers and severe infections. In order to obtain better compliance and reduce the costs of long-term therapy while achieving the same effectiveness of therapy, we decided to evaluate intermittent treatment in patients with SCN. With a single dose of 3-10 micrograms/kg/d subcutaneously every 3-7 days, the patient attained an absolute neutrophil count (ANC) of 0.5-1.5 x 10(9)/L, a reduction of infections, and no notable side effects.

P16INK4A gene homozygous deletions in human acute leukaemias with alterations of chromosome 9.

Acute leukaemias are characterized by nonrandom chromosomal aberrations which are often strictly related to the inactivation of tumour suppressor genes (TSGs). Alterations at the short arm of chromosome 9 have been reported in a remarkable percentage of acute lymphoblastic leukaemias (ALL) and have been suggested to cause the loss of activity of the putative TSG, p16INK4A (MTS1/CDKN2) gene. In order to evaluate the correlation between this gene inactivation and visible cytogenetic abnormalities, we have investigated p16INK4A homozygous gene deletions in 10 paediatric acute leukaemias of different cell lineages which demonstrated karyotype aberrations involving chromosome 9. Moreover, the dimension of the genetic alteration was evaluated by studying the loss of heterozygosity of two highly polymorphic markers of chromosome 9p, namely alpha-interferon (IFNA) and D9S104, and the deletion of 5'-methylthioadenosine phosphorylase (MTAPase) gene. Finally, the deletion of a gene belonging to p16INK4A family, the p18 gene, was analysed in these acute leukaemias. Our results demonstrated that: (1) the biallelic loss of p16INK4A gene is strictly related to a specific immunophenotype, namely ALL of T-cell lineage; (ii) no significant correlation exists between alterations at chromosome 9p level and the homozygous deletions of p16INK4A gene; and (iii) p18 gene was not deleted in the examined cases. These findings suggest a possible correlation between the T-lymphocyte phenotype and the expression of p16INK4A gene. Moreover, the absence of MTAPase activity seems to be a valuable marker of p16INK4A gene inactivation, thus indicating that the deleted chromosomal area on 9p21 very frequently involves the MTAPase gene.

Analysis of cyclin-dependent kinase inhibitor genes (CDKN2A, CDKN2B, and CDKN2C) in childhood rhabdomyosarcoma.

p16INK4A, p15INK4B, and p18 proteins are highly specific inhibitors of cyclin-dependent serine/threonine kinase (CDK) activities required for GI-S transition in the eukaryotic cell division cycle. Mutations, mainly homozygous deletions, of the CDKN2A (p16INK4A/MTSI) gene have been recently found in tumor cell lines and in many primary tumors. We looked for homozygous deletions of CDKN2A, CDKN2B (p15INK4B), and CDKN2C (p18) in 12 primary rhabdomyosarcoma (RMS) specimens and in five cell lines established from this cancer type. By means of polymerase chain reaction (PCR) and PCR-single strand conformation polymorphism (PCR-SSCP), we analyzed the presence of biallelic gene deletion or point mutation causing gene function loss. All the examined tumor cell lines (100%) and three of 12 (25%) primary tumors showed homozygous deletion of CDKN2A. Furthermore, no aberrant bands in primary tumors were detected via SSCP, suggesting the absence of mutations in the coding region. In all cases the deleted area at 9p21 also involved the CDKN2B gene. Conversely, no homozygous deletion or point mutations were detected when CDKN2C was analyzed. Our results strongly indicate that the p16INK4A (and/or p15INK4B) protein plays a key role in the development and/or progression of childhood rhabdomyosarcoma and suggest that this CDK-inhibitor protein might control proliferation and/or differentiation of human muscle cells. Moreover, alteration of CDKN2C does not appear to be involved in the genesis of rhabdomyosarcoma.

Homozygous deletions of cyclin-dependent kinase inhibitor genes, p16(INK4A) and p18, in childhood T cell lineage acute lymphoblastic leukemias.

p16(INK4A) and p18 proteins are highly specific inhibitors of cyclin-dependent serine/threonine kinase activities required for the overcoming of the G1 checkpoint in the eukaryotic cell division cycle. The frequent cytogenetic aberrations occurring in several human neoplasms at the level of their codifying genes along with their molecular function strongly suggest that they might be important tumor suppressor genes. We looked for homozygous deletions of p16(INK4A) and p18 genes in 21 cases of childhood T cell lineage acute lymphoblastic leukemia (ALL). Twenty of 21 patients (95%) had homozygous deletions of p16(INK4A) gene while three out of 21 (14%) showed p18 gene biallelic deletion. Loss of heterozygosity studies were performed in 18 of the T cell ALL investigated by means of two highly polymorphic 9p21 markers. The results obtained demonstrated that genetic deletions of different extension occur on the short arms of the 9 chromosome pair. Karyotypic analyses, performed in 13 cases, failed to demonstrate 9p alterations in 12 samples, (92%) thus demonstrating that p16(INK4A) gene homozygous deletions are not restricted to cases with cytogenetically detectable 9p aberrations. The high incidence of p16(INK4A) gene deletions in pediatric T cell lineage ALL suggests that this genetic alteration could represent an early and key event in the development of such a malignancy but it should not have any prognostic value. Conversely, the inactivation of p18 gene, observed in a lower but significant number of cases, could participate in the progression of acute leukemias towards a more aggressive disease. Finally, our results may suggest that p16(INK4A) protein plays a key role in the control of proliferation and/or differentiation of human T lymphocytes.

High frequency of homozygous deletions of CDK4I gene in childhood acute lymphoblastic leukaemia.

To determine the incidence of homozygous deletions of the newly identified tumour suppressor gene, CDK4I, molecular genomic DNA analyses by PCR technique were performed on primary neoplastic cells from 22 childhood acute leukaemias obtained at presentation. The blast cells derived in all the analysed cases from bone marrow. We found that none of acute myeloblastic leukaemias (four cases) showed the CDK4I alteration, whereas 6/13 (46%) common acute lymphoblastic leukaemias (ALLs) displayed homozygous deletions. Moreover, and even more important, all the blasts purified from ALLs derived from early lymphoid precursors (three early-T ALLs and two pre-B ALLs) showed the absence of CDK4I gene. When the entire coding sequence of the CDK4I gene from samples without homozygous deletions was analysed by the single-strand conformational polymorphism method, no point mutations were identified. These results demonstrate that CDK4I gene deletions are very frequent and probably early events in childhood acute leukaemias of lymphoid origin and especially in early-T and pre-B ALLs. Moreover, the molecular mechanism of the loss of function of the gene is correlated, at least in childhood ALLs, almost exclusively to deletions and not to point mutations.

I-131 MIBG scintigraphy of neuroectodermal tumors. Comparison between I-131 MIBG and In-111 DTPA-octreotide.

An account is given of the results observed with I-131 MIBG scintigraphy in four patients (1 bladder pheochromocytoma, 3 neuroblastomas) chosen on account of their particular clinical and diagnostic interest from a series of 41 apudoma patients examined by means of this technique. In the first patient, the unusual site of the tumor in the posterior wall of the bladder meant that its detection by I-131 MIBG was only possible after catheterization of the bladder. In the second patient, uptake in the metastasis was only evident after removal of the primary tumor. In the third patient, the scintiscan revealed several metastases (some in bone) not detected by CT. In the fourth patient (congenital neuroblastoma), enhanced uptake accompanied the appearance of high plasma catecholamine and urinary vanillylhandelic acid values, suggesting a functional switch from a nonsecreting to a secreting form. a supplementary In-111 DTPA-Octreotide (OCT) scintiscan of this patient demonstrated the presence of somatostatin receptors on the neuroblasts. Thus, this examination would seem particularly useful for the differentiation of nonsecreting neuroblastomas. Its employment in assessment of the therapeutic capacity of OCT itself is also suggested.

[Nuclear magnetic resonance and iron overload in thalassemia]. / Risonanza magnetica nucleare e sovraccarico marziale nella talassemia.

We assessed the iron load content in 36 beta-thalassemia patients by NMR correlating the results with serum ferritin levels. 22 of them were affected by beta-thalassemia major on hyper-transfusional regimen (Group A), 4 by beta-thalassemia intermedia (Group B) and 10 by beta-thalassemia major, who had been previously bone marrow transplanted (Group C). In A and C Groups the liver showed the lowest signal intensity on spin echo images (p less than 0.01; p less than 0.06, respectively). A significant correlation between the summation of signals obtained from all the examined organs and serum ferritin levels was observed by evaluating both all the patients globally (r = 0.78; p less than 0.001) and the A and C Group patients. This correlation was confirmed only in the liver both in all the patients (r = 0.77; p less than 0.001) and in A and C Group patients, when the signals obtained from each organ were evaluated.

[Multicenter study of serum ferritin assay for the surveillance of subjects at risk of iron deficiency]. / Indagine multicentrica sul dosaggio della ferritina sierica per la sorveglianza dei soggetti a rischio di carenza marziale.

A multicentre study was carried out in a sample population of healthy volunteers in order to assess the usefulness of assaying serum ferritin to monitor the extent of reserves in subjects with a risk of iron deficiency. A total of 317 subjects were included in the study. Ninety-nine were children with a mean age of 19 months, 121 were adolescents with a mean age of 18 years and 7 months, and 97 were women with a mean age of 28 years and 9 months. Levels of serum ferritin below the normal minimum levels for each age bracket, an indication of the exhaustion of the body's reserves, were found in 29% of children, 32.2% of adolescents and 27.8% of women. This study therefore confirm the frequent onset, even in our modern society, of iron-deficient states at particular times of life and that these are easily overlooked. The measurements of serum ferritin levels, in addition to being a confirmatory test in cases of suspected sideropenic anemia, has the peculiar characteristic of being the only test able to identify risk subjects before they become symptomatic. This enables rapid treatment to be commenced or better, efficient prevention.

Unbalanced coagulation-fibrinolysis potential during L-asparaginase therapy in children with acute lymphoblastic leukaemia.

Treatment of acute lymphoblastic leukaemia (ALL) with L-asparaginase (L-asp) may be associated with thrombotic complications, but the pathogenetic mechanisms of thrombus formation and persistence remain unclear. We studied the procoagulant activity (PCA) of peripheral blood mononuclear cells and some components of the plasma fibrinolytic system in 10 children with ALL undergoing remission induction therapy which includes L-asp. Mononuclear cells obtained 14 days after starting L-asp treatment generated significantly higher amounts of PCA (identified as tissue factor) than cells isolated before the first dose of L-asp and 7 days after the cessation of L-asp administration (p less than 0.01). Augmented PCA coincided with an increase in the plasma D-dimer. The plasma levels of type 1 plasminogen activator inhibitor were found significantly elevated during L-asp therapy (p less than 0.05), whereas plasminogen levels were markedly decreased (p less than 0.05). These findings suggest that, during the course of L-asp treatment, the coagulation-fibrinolysis balance is shifted towards promotion of fibrin formation and deposition. Although it remains to be conclusively established whether L-asp per se or the concurrent administration of multiple chemotherapeutic agents is responsible for these changes, the latter could contribute to the thrombotic complications associated with remission induction therapy for ALL.

Reconstitution of normal neutrophil function in chronic granulomatous disease by bone marrow transplantation.

Allogeneic bone marrow transplantation was carried out on an 11-year-old boy with chronic granulomatous disease and severe chronic pulmonary insufficiency of restrictive type. After preparative regimen with busulfan (13 mg/kg) and cyclophosphamide (200 mg/kg), the patient received marrow cells from his HLA-identical and MLC-nonreactive sister. Durable sustained engraftment of donor hematopoietic and lymphoid populations occurred, as documented by analysis of genetic markers and complete reversal of the neutrophil function defect. No episode of infection occurred in the post-transplant course and, currently, 40 months after transplantation the patient is in excellent health and growing normally and showing an increasing improvement of his respiratory capacity. The successful outcome in this patient demonstrates that marrow transplantation is at present the only curative approach for this congenital disorder of neutrophil function.