Isolated Light Chain-restricted Germinal Centers are Common in Follicular Hyperplasia by Ultrasensitive In Situ Hybridization.

Ultrasensitive bright-field RNA in situ hybridization (BRISH) can be used to detect lower levels of light chain expression than immunohistochemical stains or conventional colorimetric RNA in situ hybridization. In this study, we retrospectively reviewed 77 lymph node specimens with follicular hyperplasia and kappa/lambda BRISH performed as part of the diagnostic evaluation. Thirty-two of the specimens had ≥1 germinal center(s) (GC) showing light chain restriction (14 specimens with lambda-restricted GC, 9 with kappa-restricted GC, and 9 with separate kappa-restricted or lambda-restricted GC). In all but 1 specimen, the light chain-restricted GC represented a minority of the total GC (average: 10%, range: 0.2% to 60%). There was no significant difference in age, sex, type of biopsy (core vs. excision), number of GCs, proportion of cases with a light chain-restricted B-cell population by flow cytometry, or proportion of cases with a positive IgH gene rearrangement study between the specimens with and without restricted GCs. In our cohort of follicular hyperplasia cases, BRISH identified light chain-restricted GC more frequently than flow cytometry identified a monotypic B-cell population. Our findings highlight the potential for overinterpretation of light chain restriction in limited samplings such as fine needle aspiration cell blocks or core needle sampling and reinforce that interpretation of BRISH staining needs to occur in the context of the morphologic features including tissue architecture and results of additional immunohistochemical stains.

Unraveling unique features of plasma cell clones in POEMS syndrome with single-cell analysis.

POEMS syndrome is a rare monoclonal plasma cell disorder, with unique symptoms distinct from those of other plasma cell neoplasms, including high serum VEGF levels. Because the prospective isolation of POEMS clones has not yet been successful, their real nature remains unclear. Herein, we performed single-cell RNA-Seq of BM plasma cells from patients with POEMS syndrome and identified POEMS clones that had Ig λ light chain (IGL) sequences (IGLV1-36, -40, -44, and -47) with amino acid changes specific to POEMS syndrome. The proportions of POEMS clones in plasma cells were markedly smaller than in patients with multiple myeloma (MM) and patients with monoclonal gammopathy of undetermined significance (MGUS). Single-cell transcriptomes revealed that POEMS clones were CD19+, CD138+, and MHC class IIlo, which allowed for their prospective isolation. POEMS clones expressed significantly lower levels of c-MYC and CCND1 than MM clones, accounting for their small size. VEGF mRNA was not upregulated in POEMS clones, directly indicating that VEGF is not produced by POEMS clones. These results reveal unique features of POEMS clones and enhance our understanding of the pathogenesis of POEMS syndrome.

Analysis of the complete lambda light chain germline usage in patients with AL amyloidosis and dominant heart or kidney involvement.

Light chain amyloidosis is one of the most common forms of systemic amyloidosis. The disease is caused by the misfolding and aggregation of immunoglobulin light chains to insoluble fibrils. These fibrils can deposit in different tissues and organs such as heart and kidney and cause organ impairments that define the clinical presentation. In this study, we present an overview of IGLV-IGLJ and IGLC germline utilization in 85 patients classified in three clinically important subgroups with dominant cardiac, renal as well as cardiac and renal involvement. We found that IGLV3 was the most frequently detected IGLV-family in patients with dominant cardiac involvement, whereas in renal patients IGLV1 were most frequently identified. For patients with dominant heart and kidney involvement IGLV6 was the most frequently detected IGLV-family. In more detailed analysis IGLV3-21 was observed as the most dominant IGLV-subfamily for patients with dominant heart involvement and IGLV1-44 as the most frequent IGLV-subfamily in the group of patients with dominant kidney involvement. For patients with dominant heart and kidney involvement IGLV6-57 was the most frequently detected IGLV-subfamily. Additionally, we were able to show an exclusive linkage between IGLJ1 and IGLC1 as well as between IGLJ2 and IGLC2 in the fully assembled IGL mRNA.

Ultrasensitive RNA In Situ Hybridization for Kappa and Lambda Light Chains Assists in the Differential Diagnosis of Nodular Lymphocyte-predominant Hodgkin Lymphoma.

Establishing a diagnosis of nodular lymphocyte-predominant Hodgkin lymphoma (nLPHL) is often challenging as the differential diagnosis is broad, including classic Hodgkin lymphoma (cHL), progressive transformation of germinal centers (PTGC), and other lymphoproliferative disorders. In this study, we investigate the utility of a recently described ultrasensitive in situ hybridization assay for kappa and lambda immunoglobulin light chains in distinguishing nLPHL, cHL, and PTGC. A total of 72 cases were examined (21 nLPHL, 33 cHL, and 18 PTGC). In nLPHL, the large neoplastic cells were light chain restricted in 21/21 (100%) cases (16 kappa, 5 lambda). In contrast, Reed-Sternberg cells of cHL were negative for kappa and lambda in all cases (0/33, 0%; P <0.001). In PTGC, polytypic B cells were noted in mantle zones and germinal centers in all cases, with 1 case (5%) also showing focal collections of light chain restricted large B cells. Background monotypic small B cells were identified in 3 cases, including 1 nLPHL and 2 cHL (1 of which arose in chronic lymphocytic leukemia). Ultrasensitive in situ hybridization for kappa and lambda is a useful addition to a standard immunophenotyping panel for the evaluation of suspected nLPHL.

Compound screening identified gossypetin and isoquercitrin as novel inhibitors for amyloid fibril formations of Vλ6 proteins associated with AL amyloidosis.

AL amyloidosis is a life-threatening disease characterized by the deposition of amyloidogenic immunoglobulin light chain secreted from clonal plasma cells. Here we established an in-vitro screening system of amyloid inhibition of a variable domain in λ6 light chain mutant (Vλ6), Wil, and screened a food-additive compound library to identify compounds inhibiting the fibril formation. We found gossypetin and isoquercitrin as novel inhibitors. NMR analysis showed that both compounds directly interacted with natively-folded Wil, and proteolysis experiments demonstrated that these compounds conferred proteolytic resistance, suggesting that the compounds enhance the kinetic stability of Wil. Since gossypetin and isoquercitrin specifically interacted with the protein at micromolar concentrations, these compounds could be used as lead to further develop inhibitors against AL amyloidosis.

Identification of clonal immunoglobulin λ light-chain gene rearrangements in AL amyloidosis using next-generation sequencing.

Amyloid light-chain (AL) amyloidosis is caused by deposition of abnormally folded clonal immunoglobulin (Ig) light chains made by malignant plasma cells in the bone marrow (BM), leading to multiorgan dysfunction. However, little is known of the factors that regulate the organ tropism of amyloid deposition in this disease. We aimed to identify the clonal composition of Igλ light-chain variable region (IGLV) genes in BM cells in patients with AL amyloidosis using next-generation sequencing. Based on our definition of the clonal IGLV rearrangement (dominant clone >2.5%, dominant cluster >5%), we identified clonal IGLV in 33 of 38 patients with AL amyloidosis (86.8%), 6 of 9 with monoclonal gammopathy of undetermined significance (67%), and 7 of 7 with multiple myeloma (100%). The clones in AL amyloidosis were significantly smaller than those in multiple myeloma (p < 0.01) but comparable to those in monoclonal gammopathy of undetermined significance. Importantly, in patients with AL amyloidosis, the difference in involved and uninvolved free light chains was not correlated with the clonal size of BM plasma cells in our repertoire analysis using NGS. In summary, the clonal composition of IGLV genes in the BM was successfully identified in most patients with AL amyloidosis using NGS. The clonal size of plasma cells in the BM is small, and small malignant clones of plasma cells may secrete free light chi and cause light chain depositions in AL amyloidosis.

An expanded genetic code facilitates antibody chemical conjugation involving the lambda light chain.

Most of the currently approved therapeutic antibodies are of the immunoglobulin gamma (IgG) κ isotype, leaving a vast opportunity for the use of IgGλ in medical treatments. The incorporation of designer amino acids into antibodies enables efficient and precise manufacturing of antibody chemical conjugates. Useful conjugation sites have been explored in the constant domain of the human κ-light chain (LCκ), which is no more than 38% identical to its LCλ counterpart in amino acid sequence. In the present study, we used an expanded genetic code for site-specifically incorporating Nε-(o-azidobenzyloxycarbonyl)-l-lysine (o-Az-Z-Lys) into the antigen-binding fragment (Fab) of an IgGλ, cixutumumab. Ten sites in the LCλ constant domain were found to support efficient chemical conjugation exploiting the bio-orthogonal azido chemistry. Most of the identified positions are located in regions that differ between the two light chain isotypes, thus being specific to the λ isotype. Finally, o-Az-Z-Lys was incorporated into the Fab fragments of cixutumumab and trastuzumab to chemically combine them; the resulting bispecific Fab-dimers showed a strong antagonistic activity against a cancer cell line. The present results expand the utility of the chemical conjugation method to the whole spectrum of humanized antibodies, including the λ isotype.

Effect of O-glycosylation on amyloid fibril formation of the variable domain in the Vλ6 light chain mutant Wil.

Glycosylation is one of the major post-translational modifications in eukaryotic cells and has been reported to affect the amyloid fibril formation in several amyloidogenic proteins and peptides. In this study, we expressed a Vλ6 light chain mutant, Wil, which is an amyloidogenic mutant in AL amyloidosis, by the yeast Pichia pastoris. After separation by cation exchange chromatography, we obtained the O-glycosylated and non-glycosylated Wil mutants in high yield. The structures of these Wil mutants were identical except with respect to glycosylation, and the stabilities were also identical. On the other hand, the O-glycosylation retarded the amyloid fibril formation in a sugar size-dependent manner. From these results, we discussed the role of covalently attached glycan in the retardation of amyloid fibril formation.

Reporting Apparent Biclonal Immunoglobulin-A Monoclonal Proteins with Identical Light Chains.

It is generally not well appreciated that Immunoglobulin A (IgA) can present as apparent biclonal monoclonal protein (M-protein) comprising monomers and polymers, despite the fact that Kyle pointed this out in 1999. In this clinical communication, we report on 3 patients with Ig-A multiple myeloma (MM) who displayed this phenomenon. Of these 3 patients, 2 had identical kappa light chains suggesting biclonality, while the other had 2 lambda light chains. Because the Ig-A M-proteins are clustered in the beta-area, accurate densitometric quantification is made difficult. Hence, we suggest assaying Ig-A levels and free light chains in concert with the M-protein levels to monitor these patients on therapy. In conclusion, when encountering biclonal Ig-A M-proteins with identical light chains, the laboratorian should add the 2 values and present one composite number to the clinician.

Analytical evaluation of the clonoSEQ Assay for establishing measurable (minimal) residual disease in acute lymphoblastic leukemia, chronic lymphocytic leukemia, and multiple myeloma.

BACKGROUND: The clonoSEQ® Assay (Adaptive Biotechnologies Corporation, Seattle, USA) identifies and tracks unique disease-associated immunoglobulin (Ig) sequences by next-generation sequencing of IgH, IgK, and IgL rearrangements and IgH-BCL1/2 translocations in malignant B cells. Here, we describe studies to validate the analytical performance of the assay using patient samples and cell lines. METHODS: Sensitivity and specificity were established by defining the limit of detection (LoD), limit of quantitation (LoQ) and limit of blank (LoB) in genomic DNA (gDNA) from 66 patients with multiple myeloma (MM), acute lymphoblastic leukemia (ALL), or chronic lymphocytic leukemia (CLL), and three cell lines. Healthy donor gDNA was used as a diluent to contrive samples with specific DNA masses and malignant-cell frequencies. Precision was validated using a range of samples contrived from patient gDNA, healthy donor gDNA, and 9 cell lines to generate measurable residual disease (MRD) frequencies spanning clinically relevant thresholds. Linearity was determined using samples contrived from cell line gDNA spiked into healthy gDNA to generate 11 MRD frequencies for each DNA input, then confirmed using clinical samples. Quantitation accuracy was assessed by (1) comparing clonoSEQ and multiparametric flow cytometry (mpFC) measurements of ALL and MM cell lines diluted in healthy mononuclear cells, and (2) analyzing precision study data for bias between clonoSEQ MRD results in diluted gDNA and those expected from mpFC based on original, undiluted samples. Repeatability of nucleotide base calls was assessed via the assay's ability to recover malignant clonotype sequences across several replicates, process features, and MRD levels. RESULTS: LoD and LoQ were estimated at 1.903 cells and 2.390 malignant cells, respectively. LoB was zero in healthy donor gDNA. Precision ranged from 18% CV (coefficient of variation) at higher DNA inputs to 68% CV near the LoD. Variance component analysis showed MRD results were robust, with expected laboratory process variations contributing ≤3% CV. Linearity and accuracy were demonstrated for each disease across orders of magnitude of clonal frequencies. Nucleotide sequence error rates were extremely low. CONCLUSIONS: These studies validate the analytical performance of the clonoSEQ Assay and demonstrate its potential as a highly sensitive diagnostic tool for selected lymphoid malignancies.

Murine gammaherpesvirus infection is skewed toward Igλ+ B cells expressing a specific heavy chain V-segment.

One of the defining characteristics of the B cell receptor (BCR) is the extensive diversity in the repertoire of immunoglobulin genes that make up the BCR, resulting in broad range of specificity. Gammaherpesviruses are B lymphotropic viruses that establish life-long infection in B cells, and although the B cell receptor plays a central role in B cell biology, very little is known about the immunoglobulin repertoire of gammaherpesvirus infected cells. To begin to characterize the Ig genes expressed by murine gammaherpesvirus 68 (MHV68) infected cells, we utilized single cell sorting to sequence and clone the Ig variable regions of infected germinal center (GC) B cells and plasma cells. We show that MHV68 infection is biased towards cells that express the Igλ light chain along with a single heavy chain variable gene, IGHV10-1*01. This population arises through clonal expansion but is not viral antigen specific. Furthermore, we show that class-switching in MHV68 infected cells differs from that of uninfected cells. Fewer infected GC B cells are class-switched compared to uninfected GC B cells, while more infected plasma cells are class-switched compared to uninfected plasma cells. Additionally, although they are germinal center derived, the majority of class switched plasma cells display no somatic hypermutation regardless of infection status. Taken together, these data indicate that selection of infected B cells with a specific BCR, as well as virus mediated manipulation of class switching and somatic hypermutation, are critical aspects in establishing life-long gammaherpesvirus infection.

Immunoglobulin variable domain high-throughput sequencing reveals specific novel mutational patterns in POEMS syndrome.

Polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes (POEMS) syndrome is a rare multisystem disease resulting from an underlying plasma cell (PC) dyscrasia. The pathophysiology of the disease remains unclear, but the role of the monoclonal immunoglobulin (Ig) light chain (LC) is strongly suspected because of the highly restrictive usage of 2 λ variable (V) domains (IGLV1-40 and IGLV1-44) and the general improvement of clinical manifestations after PC clone-targeted treatment. However, the diagnostic value of Ig LC sequencing, especially in the case of incomplete forms of the disease, remains to be determined. Using a sensitive high-throughput Ig repertoire sequencing on RNA (rapid amplification of cDNA ends-based repertoire sequencing [RACE-RepSeq]), we detected a λ LC monoclonal expansion in the bone marrow (BM) of 83% of patients with POEMS syndrome, including some in whom BM tests routinely performed to diagnose plasma cell dyscrasia failed to detect λ+ monoclonal PCs. Twenty-four (83%) of the 29 LC clonal sequences found were derived from the IGLV1-40 and IGLV1-44 germline genes, as well as 2 from the closely related IGLV1-36 gene, and all were associated with an IGLJ3*02 junction (J) gene, confirming the high restriction of VJ region usage in POEMS syndrome. RACE-RepSeq VJ full-length sequencing additionally revealed original mutational patterns, the strong specificity of which might crucially help establish or eliminate the diagnosis of POEMS syndrome in uncertain cases. Thus, RACE-RepSeq appears as a sensitive, rapid, and specific tool to detect low-abundance PC clones in BM and assign them to POEMS syndrome, with all the consequences for therapeutic options.

IGLV3-21*01 is an inherited risk factor for CLL through the acquisition of a single-point mutation enabling autonomous BCR signaling.

The prognosis of chronic lymphocytic leukemia (CLL) depends on different markers, including cytogenetic aberrations, oncogenic mutations, and mutational status of the immunoglobulin (Ig) heavy-chain variable (IGHV) gene. The number of IGHV mutations distinguishes mutated (M) CLL with a markedly superior prognosis from unmutated (UM) CLL cases. In addition, B cell antigen receptor (BCR) stereotypes as defined by IGHV usage and complementarity-determining regions (CDRs) classify ∼30% of CLL cases into prognostically important subsets. Subset 2 expresses a BCR with the combination of IGHV3-21-derived heavy chains (HCs) with IGLV3-21-derived light chains (LCs), and is associated with an unfavorable prognosis. Importantly, the subset 2 LC carries a single-point mutation, termed R110, at the junction between the variable and constant LC regions. By analyzing 4 independent clinical cohorts through BCR sequencing and by immunophenotyping with antibodies specifically recognizing wild-type IGLV3-21 and R110-mutated IGLV3-21 (IGLV3-21R110), we show that IGLV3-21R110-expressing CLL represents a distinct subset with poor prognosis independent of IGHV mutations. Compared with other alleles, only IGLV3-21*01 facilitates effective homotypic BCR-BCR interaction that results in autonomous, oncogenic BCR signaling after acquiring R110 as a single-point mutation. Presumably, this mutation acts as a standalone driver that transforms IGLV3-21*01-expressing B cells to develop CLL. Thus, we propose to expand the conventional definition of CLL subset 2 to subset 2L by including all IGLV3-21R110-expressing CLL cases regardless of IGHV mutational status. Moreover, the generation of monoclonal antibodies recognizing IGLV3-21 or mutated IGLV3-21R110 facilitates the recognition of B cells carrying this mutation in CLL patients or healthy donors.

Concurrent immunoglobulin G-lambda type multiple myeloma and mixed cellularity classical Hodgkin lymphoma: A case report.

A 66-year-old man with a swollen right inguinal lymph node (LN) had pain on the lower side of the back. Computed tomography revealed bone disease in the back and swollen right inguinal LNs. Laboratory studies showed anemia and serum immunoglobulin G-lambda (IgG-λ) type monoclonal protein. The bone marrow contained 39.6% plasma cells. He was diagnosed with IgG-λ type multiple myeloma (MM). However, the pathological findings of the right inguinal LN were mixed cellular classical Hodgkin lymphoma (HL). The administration of melphalan, prednisone, and bortezomib (MPB) was started for MM; however, swelling in the right inguinal LN increased. After three cycles of MPB, the administration of doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD) was started for HL. However, HL was refractory to ABVD. Pancytopenia subsequently progressed and rapid swelling occurred in his LNs. He died 7 months after diagnosis. Multiple myeloma was diagnosed, based on the typical symptoms, although the pathological findings of the LN indicated a diagnosis of HL. We analyzed the molecular relationship between MM and HL cells using a direct sequencing method. The sequencing results demonstrated that the variable-diversity-joining (VDJ) region of the IgH gene was identified with 94.4% of IGLV3-32*01 in the bone marrow sample at diagnosis. Furthermore, clonotypic IgH sequence was identified in CD30-positive cells from the LN. These results suggested that the clonal HL cells were derived from the same source as the clonal MM cells and demonstrated that MM and HL in this patient may have originated from the same B cell progenitor.

T-cell large granular lymphocytic (LGL) leukemia consists of CD4+/CD8dim and CD4-/CD8+ LGL populations in association with immune thrombocytopenia, autoimmune neutropenia, and monoclonal B-cell lymphocytosis.

CD3+/CD57+ T-cell large granular lymphocyte leukemia (T-LGLL) is an indolent neoplasm, exhibiting mostly CD8+, less frequently CD4+ phenotypes, and T-LGLL consisting of 2 populations with CD8+ and CD4+ phenotypes is markedly rare. An 87-year-old female was admitted under a diagnosis of immune thrombocytopenia (ITP) with a platelet count of 5.0×109/L and increased number of LGL with unknown etiology. Her neutrophil count also decreased to 0.27×109/L and she was positive for antineutrophil antibody. The WBC count was 2.7×109/L with 34.7% LGL and flow cytometry (FCM) analysis revealed 16% CD3+/CD4+/CD8dim/CD57+ and 20.9% CD3+/CD8+/CD57+ populations. These populations also expressed granzyme B and perforin. Circulating mononuclear cells were found to be clonal by PCR analysis of T-cell receptor ß-chain gene. Serum immunofixation and bone marrow FCM analyses demonstrated 2 clonal B-cells producing IgG-λ and IgA-λ. Deep amplicon sequencing of STAT3 and STAT5B genes revealed a STAT3 R302G mutation with an allele burden of 2.6%. The thrombocytopenia and neutropenia were successfully treated by prednisolone and romiplostim with negative conversion of antineutrophil antibody. This is the first reported case of T-LGLL with dual components of CD4+/CD8dim and CD4-/CD8+ populations in terms of multiple comorbidities related to the respective CD8+ and CD4+ T-LGLLs.

Multiple myeloma immunoglobulin lambda translocations portend poor prognosis.

Multiple myeloma is a malignancy of antibody-secreting plasma cells. Most patients benefit from current therapies, however, 20% of patients relapse or die within two years and are deemed high risk. Here we analyze structural variants from 795 newly-diagnosed patients as part of the CoMMpass study. We report translocations involving the immunoglobulin lambda (IgL) locus are present in 10% of patients, and indicative of poor prognosis. This is particularly true for IgL-MYC translocations, which coincide with focal amplifications of enhancers at both loci. Importantly, 78% of IgL-MYC translocations co-occur with hyperdiploid disease, a marker of standard risk, suggesting that IgL-MYC-translocated myeloma is being misclassified. Patients with IgL-translocations fail to benefit from IMiDs, which target IKZF1, a transcription factor that binds the IgL enhancer at some of the highest levels in the myeloma epigenome. These data implicate IgL translocation as a driver of poor prognosis which may be due to IMiD resistance.

Zebrafish model of amyloid light chain cardiotoxicity: regeneration versus degeneration.

Cardiac dysfunction is the most frequent cause of morbidity and mortality in amyloid light chain (AL) amyloidosis caused by a clonal immunoglobulin light chain (LC). Previously published transgenic animal models of AL amyloidosis have not recapitulated the key phenotype of cardiac dysfunction seen in AL amyloidosis, which has limited our understanding of the disease mechanisms in vivo, as well as the development of targeted AL therapeutics. We have developed a transgenic zebrafish model in which a λ LC derived from a patient with AL amyloidosis is conditionally expressed in the liver under the control of the Gal4 upstream activation sequence enhancer system. Circulating LC levels of 125 µg/ml in these transgenic zebrafish are comparable to median pathological serum LC levels. Functional analysis links abnormal contractile function with evidence of cellular and molecular proteotoxicity in the heart, including increased cell death and autophagy. However, despite pathological and functional phenotypes analogous to human AL, the lifespan of the transgenic fish is comparable to control fish without the expressed AL-LC transgene. Nuclear labeling experiments suggest increased cardiac proliferation in the transgenic fish, which can be counteracted by treatment with a small molecule proliferation inhibitor leading to increased zebrafish mortality because of cardiac apoptosis and functional deterioration. This transgenic zebrafish model provides a platform to study underlying AL disease mechanisms in vivo further. NEW & NOTEWORTHY Heart failure is a major cause of mortality in amyloid light (AL) amyloidosis, yet it has been difficult to model in animals. We report the generation of a transgenic zebrafish model for AL amyloidosis with pathological concentration of circulating human light chain protein that results in cardiac dysfunction. The light chain toxicity triggers regeneration in the zebrafish heart resulting in functional compensation early in life, but with age develops into cardiac dysfunction.

Localized Lichen Myxedematosus With Plasma Cell Light Chain Restriction: Is It the Exception or the Rule?

Lichen myxedematosus is a chronic cutaneous mucinosis that can present on a spectrum from localized cutaneous lesions to systemic disease of scleromyxedema. The clinical presentation of localized cutaneous lichen myxedematosus is waxy lichenoid papules, nodules, and/or plaques that have histopathologic findings of mucin deposition and a variable degree of fibroblast proliferation. There is an absence of serum paraproteins, and there are no other systemic causes of cutaneous mucinosis such as thyroid disease. The pathogenesis of lichen myxedematosus is unknown. We report 3 cases of localized cutaneous lichen myxedematosus with a light chain-restricted plasmacytic component by in situ hybridization. Our findings deliver an insight for disease pathogenesis and highlight for the first time, the significance of plasma cells in lesions of localized cutaneous lichen myxedematosus. We suggest that plasma cell light chain restriction could represent a clue to distinguish localized cutaneous disease from systemic disease.